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CHAPTER I

INTRODUCTION TO THE CELL

1.1. Historic of Cell Biology

Cell biology is a complex fundamental science that studies the functional structures and
general biological phenomena that take place at the cell’s level. The knowledge about the cell
was brought by the studies made by the light microscope.
In 1590, Jansen brothers put in a tube some glass lens. They used this apparatus for
navigation and astronomy. In 1665, Robert Hooke modified this apparatus and observed some
vegetal cells. The Dutch Leeuwenhoeck improved the microscope so that it magnified 300
times. He described some microorganisms and spermatozoa. He also described in the middle of
the cell the presence of a spherical corpuscle (the nucleus). Malpighi described the cells and
named them utricule and saccule. He also described in the kidney the Malpighi’s corpuscle.
Preformist theory has persisted in the eighteen centuries. It supposed that the beings exist in a
preformated state in the shape of miniature - beings (homunculus) into sexual cells sad they
develop by growing processes. Spallanzani refuted this theory through the following
experiment: he collected spermatic fluid from frog and filtered it. Then he mixed the residuum
with frog eggs and new beings resulted. The filterable liquid mixed with frog eggs does not form
anything. So that one has concluded the followings:
1) The spermatozoon is the fecundating agent;
2) The spermatozoon combined with the ovule forms a new being;
3) There are not performed beings.
In the XIX century there were three theories:
1) Cell Theory
2) Evolution theory
3) Heredity laws

Cell Theory
In 1905, Oken in his work named ”The Generation” assumed that all the beings had
developed from cells. Schleiden and Schwann formulated the cell theory and the following laws:
a) Vegetal and animal bodies are formed by cells; b) Each ceH has its individual cell c) The
individual life of each cell influences the life of’ the whole body and vice- versa. At cellular
level there are two phenomena: - metabolic phenomena - plastic phenomenon at cellular
structures’ level.
Virchow applies the cellular theory in embryology: the union of two gametes forms all the
cells. The progressive development of the egg cell builds up the organism.
The Theory of Evolution
Darwin and Wallace gave the first modern scientific response to three central questions, in
their theory of evolution:
- How did life originate?
- Why does “like begged like”?
- How does an individual animal or plant develop from a fertilized?
Heredity Laws
Gregor Mendel formulated the heredity laws. The characters of each being are written on a
material particle in the gametes. The particle is named cellular factor. The method he used is
named experimental hybridization.

The use of staining from textile industry guided to the discovery of new cellular
components:
- Brown discovered the nucleus, a constant component of the cell;
- Bovery described the cellular center;
- Waldayer described the chromosomes;
- Flemming and Strasburger described mitosis;
- Altman described the mitochondrion;
- Nissl described the Tigroid substance (Nissl corpuscle) that is the granular (rough)
endoplasmic reticulum from central nervous system;
- Berg described the Berg corpuscles from liver cells.
Cellular biology was considered as a proper science when cell culture technique,
differential ultracentrifugation technique and electron microscopy appeared.

Cell Culture Technique


Harisson and Carrel suggested this method in 1912. It allows the cells to develop
spontaneously when they are taken out from the body influence. This method gives data for
embryological experiments and other experiments.

The Differential Ultracentrifugation


Claude and Christian De Duve suggested this technique and they received the Nobel Prize
for it in 1974. This method separates in pure state different cellular components: nuclei, nuclear
cover, chromatin, nucleolus, DNA, and RNA molecules. The molecular structure of each
component can be defined.

The Electron Microscope


The electron microscope has the resolution power of 1 Å. It describes the cell’s
ultrastructure, the molecular organization of DNA and RNA. There are different kinds of
electron microscopes:
1) Ernst Ruska and Max Knoll set up the electron microscope in 1931. The
transmission electron microscope TEM uses ultrathin sections less than 1000 Å and the
acceleration tension being 80 –100 kV.
2) The high voltage electron microscope has an acceleration tension of 1 – 3 MV.
This method can be used of living cells because the electron s can pass through very big
thickness;
3) The scanning electron microscope studies the surface of the cell;
4) The electron microsonde that studies in the same time the morphological and
molecular structure.
George Palade discovered the ribosome, called also Palade’s corpuscle, and ribosome’s role
in protein synthesis, being awarded Nobel Prize in 1974. He also described the Golgi complex
and the endoplasmic reticulum together with Porter.
1.2. The Appearance of the Ancestral Cell

Cells make all living creatures. The cells are small membrane bounded compartments filled
with concentrated water solution of chemicals. Superior organisms, such as, are like cellular
cities in which groups of cells perform specialized functions and are halfway between molecules
and individual beings. We study them to learn how they are made by molecules and, on the
other hand, how they cooperate to make an organism as complex as the human being.
All organisms and all the cells that constitute them are assumed to descend from a common
ancestral cell by evolution. Evolution involves two essential processes:
1) The occurrence of random variations in the genetic information passed from an
individual to its descendants;
2) The selection of genetic information that helps its possessors to survive and
multiply.
Remarkable scientific progress in last 50 years made possible the pertinent but still
incomplete explanation concerning the appearance of the first living structure, the ancestral cell,
and at the same time its evolution in time up to the appearance of the impressive cell variety find
now on earth.
The conditions that existed on the earth in its first thousand million years are still a matter
of dispute. Everyone seems to agree, however, that the earth was a violent place with volcanic
eruptions, high temperatures and electrical discharges. There was little of any free oxygen and
no layer of ozone to absorb the harsh ultraviolet radiation from the sun. That atmosphere
contained N, H, NH 3 , CH 4 , CO 2 , and water vapors. From the moment when the planet
temperature downed, water vapors condensed, starting rains that involved the chemical elements
from atmosphere dissolving them and earthly. In this manner it comes into being the primitive
ocean, called the prebiotic soup. Simple organic molecules seem that are produced under such
conditions and this phenomenon was also proved experimentally. Thus, weather a mixture of
gases such as CH 4 , NH 3 , H 2 is warmed together with water into a closed tool and the mixture is
forced to electrical discharges it is observed the forming of small organic molecules as HCN and
HCHO that combine slowly in aqueous solution to form cyanhydrine. In the presence of
ammonia, this forms glycerol, the simplest amino acid. The formed simple organic molecules
polymerized to realize macromolecules that were accumulated in ocean to born organic soups.
The four major classes of small organic molecules formed in cells are aminoacids,
nucleotides, sugars, and fatty acids. Simple organic molecules such as aminoacids and
nucleotides can associate to form large polymers. One aminoacid can join with another by
forming a peptide bond, while two nucleotides can join by a phosphodiester bond. The repetition
of these reactions leads to linear polymers known as polypeptides and polynucleotides. In
present days, in the living organisms, the polypeptides (known as proteins), and polynucleotides
[as both ribonucleic acids (RNA) and deoxyribonucleic acids (DNA)] are commonly viewed as
the most important constituents.
Figure 1. Miller experience, simulating
conditions on primitive earth.

The formation of polynucleotides and proteins requires the presence of specific protein
catalysts or enzymes, which were not present in the “prebiotic soup”. However, less efficient
catalysts as minerals or metal ions would have been present.
The appearance of protein synthesis controlled by nucleic acids was one of the crucial
events leading to the formation of this first cell. Another important step must have been the
development of an external membrane. It was postulated that the first assembled into such
membranous structures, enclosing a self – replicating mixture of RNA and protein molecules. At
some later stages in evolutionary process, DNA replaced RNA as hereditary material.
So, the first living cells probably appeared on Earth 3.5 billions years ago as a result of
spontaneous reactions between the molecules of medium, which is in chemical unstable
equilibrium. The performed recently studies on old sedimentary rocks made evident the
presence of the unicellular organisms similar to blue-green algae on Earth 3.6 billions years ago
that lead to the hypothesis that life appeared during the first thousand billion year of the planet.
The research results done until now lead to the hypothesis that the first living organisms
developed trough autocatalytic mechanisms starting with the RNA molecular family evolution
that can catalyze their owner replication. In the process of time, some of these, probably,
developed their capacity to manage the polypeptide synthesis. As more protein catalysts
accumulated, they became more efficiently allowing the cells evolution, RNA being took by
double helix DNA that has molecules more stable and able to storage the complex genetic
information.
So, the hypothesis would be RNA preceded DNA in the evolution having genetic and
catalytic properties.
It is thought that all organisms living now on earth derive from one simple primordial cell
born several thousand million years ago. This cell, reproducing out its competitors, takes the
lead in the process of cell division and evolution that would eventually cover the earth in green,
changes the composition of the atmosphere, and makes it the human intelligent life.

1.3. Differences between Prokaryotes and Eukaryotes

One important landmark along this evolutionary road occurred about 1.5 billion years ago,
when there was a transition from small cells with a relatively simple internal structure (the
prokaryotes, that include the various types of bacteria and the blue-green alga) to the larger and
radically more complex eukaryotic cells such as those we found in superior animals and plants.
The eukaryotic cells can be unicellular or pluricellular and the prokaryotes are only
unicellular. Between eukaryotes and prokaryotes are the following differences:

No. STRUCTURES PROKARYOTES EUKARYOTES


1 Organisms Bacteria and cyanobacteria Protist, fungi, plants and
animals
2 Cell’s size Generally 1-10 μm in linear Generally 1-100 μm in linear
dimension dimension
3 Metabolism Anaerobic or aerobic Aerobic
4 Cellular organization Mainly unicellular Unicellular and multicellular,
with differentiation of cells
5 Membrane Cell wall (that contains a - Plasma membrane that is a
peptide-glycan with a N- continuos sheet of lipid
acetyl-muramic acid in its molecules about 4-5 nm
structure) and plasma thick in which are
membrane embedded various proteins;
- Plant cells are surrounded
by a rigid wall composed by
fibrils of cellulose laid
down in matrix of other
polysaccharides.
6 Cytoplasm No cytoskeleton, no - They have cytoskeleton
cytoplasmic streaming, composed by protein filaments
endocytosis, no mitochondria that help organize mitochondria
(the enzymes of the (the enzymes of respiratory
respiratory) chain are on the internal face of
the cell’s membrane)
7 Nucleus and nuclear - It does not exist the - They have a nucleus enclosed
cover nucleus; by a double layer of membrane
- Exists a nuclear equivalent
or nucleoid.
8 DNA and - A single circular uncombined - Very long DNA containing
chromosomes molecule of DNA in cytoplasm many noncoding regions
that represents the single and combined with histone
chromosome proteins;
- The nuclear material
condenses in the time of
division in more
chromosomes
9 RNA and protein - RNA and protein synthesized - RNA synthesized and
in same compartment processed in nucleus
- Proteins synthesized in
cytoplasm
10 Cell division - Amitosis - By mitosis (or meiosis)

Table 1. Comparison of prokaryotic and eukaryotic organisms.

1.4. Similarities between Prokaryotes and Eukaryotes

The similarities between Prokaryotes and Eukaryotes are following:


- Both have the same mechanism in protein biosynthesis.
- Both have the same genetic code.
The viruses are not considered cells they are parasites. They survive and multiply only in
the interior of the host-cells.

1.5. The biochemical Structure of the Cell

The chemical structure of the cell is the following:


- water.............................60-90%
- mineral substances..........4.3%
- sugar residues...............6.2%
- lipids.............................11.7%
- proteins.........................17.8%
The water represents the essential element of living organism. The water content varies
with the age, tissue, and species. An embryo contains approximately 80% water. In the
evolution process, water content diminishes, so that in adult it is 60%. Water content varies
upon the tissue. For example, the bone tissue has very small water content. Muscular and
connective tissues have more water. The water circulates and transports different substances
from one place to another. In the water take place the most biochemical reactions. The water
also represents the product of the biochemical reactions from the cell.
The mineral salts are three types:
- bioelements with structural role, formed by C, N, H, and O;
- most of metallic elements: Ca, Na, K, and Mg;
- most of nonmetallic elements: Cl, P, and S.
They have important functional roles in cell’s activity.
The organic substances are formed by 4 groups of substances: sugar residues, lipids,
proteins, and nucleic acids.
The sugar residues are the simplest elements. They have two major roles: plastic role and
energetic role.
The lipids have 3 important functions: plastic role, energetic role, and information
transmission.
The proteins have more roles: plastic role, proteins’ storing role, cell motility (actin,
tubulin, dineine), information transmission, protection role (antibodies, immunoglobulins).
The nucleic acids are organic elements directly implied in the coordination of all cell
activities.

1.6. The Morphologic Organization of Cell

The eukaryotic cells have two components:


- cellular cover;
- protoplasm that contains the nucleus and the cytoplasm.
The cytoplasm can be:
1) structured (morphoplasm). The structured cytoplasm builds the cellular organelles. The
cellular organelles are classified in: membranar organelles (endoplasmic reticulum,
Golgi complex, lysosomes, peroxisomes) and unmembranar organelles (ribosomes,
cellular center);
2) unstructured (the hyaloplasm). It contains the previous structure. There can also exist
temporary structures like the cellular inclusions.
CHAPTER II

THE CELL COVER

The cell cover is a complex morpho-functional structure, made up of three parts:


- glycocalyx (or the cell coat);
- plasma membrane;
- membrane cytoskeleton.
These elements define the cell extents and maintain the essential differences
between its content and the environment.
All biological membranes, including the plasma membrane and the internal
membranes of eukaryotic cells has a common overall structure: they are assemblies of
lipids and proteins held together by non-covalent interactions (Figure 2).
Absorbed glycoprotein

Glycolipid
Transmembrane
Glycocalix protein

Lipid
bilayer

CYTOPLASM
Figure 2. Schematic diagram of the cell cover, which is made up of three
components: cell coat (glycocalyx), plasma membrane and membranar
cytoskeleton.

2.1. The Plasma Membrane.

The plasma membrane is the most important constituent of the cell coat. Bowman
in 1840 and Remack in 1852 inferred the existence of the plasma membrane. They
assumed the presence of this structure at the boundary of the cell. Due to its thickness
and to the photonic microscope resolution power limits, the existence of the plasma
membrane was denied for a long period of time.
At the end of the nineteenth century and the beginning of the XXth century,
indirect data were brought about the existence of the plasma membrane.
Since 1950, after the discovery of the electron microscope, there have been
studied ultra-thin sections from animal tissues at a resolution under 50 nm.
The use of KMnO4 as a fixing agent and of epoxidic resins as embedding
materials permitted the study of plasma membrane and gave the proof of their
existence.
The data about the plasma membranes, obtained with the electron microscope, X-
ray diffraction, differential ultracentrifugation, biochemical analysis, freeze-fracture,
were synthesized in a membrane model, with dynamic features.
In 1976, Cifuentes assumed that the plasma membrane is a biochemical complex
with morpho-functional expression.

2.1.1. Morphologic Structure Of The Plasma Membrane.


The photonic microscope cannot offer data about the morphology of plasma
membrane due to its thickness, that is very small, inferior to the resolution power of
the microscope. Electron-microscopy studies showed that the plasma membrane is
composed by two electron-opaque, osmiophyllic, layers with a central osmiophobic,
electron-transparent layer between them.
The thickness of the plasma membrane varies among 7.5 and 10 nm; the
thickness of the two peripheral layers is about 2 nm and that of the central layer of 3.5
nm.
Danielli and Davson interpreted the electron-microscope images in 1935
concluding that the middle layer is composed by lipids, and the peripheral ones by
proteins.
In 1957, Robertson formulated “The Unit Membrane Theory”; according to this
theory, all cell membranes are built in the same way, by a lipid bilayer and proteins
adhering at the external and internal parts of the bilayer. This model ignores the
transmembrane proteins.
The freeze-fracture technique shows the granular aspect of cell membrane
because of the high number of intrinsic proteins (transmembrane proteins). The
Robertson model has two characteristic accepted today:
1. the lipid bilayer universality;
2. the plasma membrane chemical asymmetry.
This model does not include the transmembrane proteins and the plasma
membrane fluidity.

2.1.2. Molecular Structure of the Plasma Membrane.


Today is accepted that plasma membrane is composed by a lipid bilayer
comprised between 2 protein bilayers and crossed by transmembrane proteins.
Before the isolation of the plasma membrane in pure state, the knowledge about it
was based on indirect data.
1. In 1902, Overton observed that the substances solved in organic solvents
crossed easily the plasma membrane and concluded that cell membranes are formed
by a thin lipid layer.
2. In 1925, Göerter and Grendel extracted lipids from the red cells in the blood
and floated them on the surface of a aqueous solution The area of this single layer was
twice bigger than the surface of the original cells. They observed also that the
hemolysed red blood cells form spontaneously a lipid bilayer in an aqueous medium.
They concluded that the lipids are arranged in the membrane as a continuous bilayer.
3. Further evidence came from the measurement of the superficial tension, which
is about 0 dyne/cm at the cell surface and 5/10 dyne/cm at the oil-water interface. This
means that in the structure of membranes exist proteins that decrease the superficial
tension.
4. In 1935, Danielli and Davson concluded that the plasma membrane is formed
by a lipid bilayer with proteins attached on the inner and outer part of this bilayer.
5. In 1957, Robertson formulated “The Unit Membrane Theory”;
6. Other models were based on a structure formed by globular lipids and
proteins.
7. In 1975, Singer and Nicholson suggested “The Fluid Mosaic Model” that has
the following characteristics:
a. plasma membranes have lipids and proteins organized in systems like a
mosaic;
b. plasma membranes are quasi-fluid structures.
Proteins and lipids can move in the same layer or between the layers. The
movements suppose the existence of non-covalent bindings between the components.
Today it is known that the plasma membrane contains 52% proteins, 40% lipids
and 8% carbohydrates.

2.1.2.1. Lipids in the Membrane.


There are three kinds of lipids in biological membranes:
Š phopsholipids
Š glycolipids
Š cholesterol
In 1936, Hartley described the amphipatic character of lipids that means that all
lipids in a membrane have a hydrophilic part and a hydrophobic one.

LIPIDS HYDROPHILIC HEAD HYDROPHOBIC PART


Phospholipids Polar Group 2 fatty acid chains
- 1 saturated
- 1 unsaturated
Glycolipids Carbohydrate residue 1 fatty chain + 1
sphingosine chain
Cholesterol OH group from C3 The rest of the cholesterol
molecule

Phospholipids in the membrane.


Phospholipids in the membrane are divided into phosphoglycerids and
sphingolipids.
1. Phosphoglycerids rely on the glycerol molecule; an alcohol (that is choline,
ethanolamine, serine or inositol) with a phosphate residue represents the polar head
while the tails are represented by two fatty acid chains, one saturated and one
unsaturated.
In phosphoglycerids, two OH from glycerol are esterified with fatty acid chains,
and the polar group occupies the other OH. Usually, in the middle of glycerol (the
mild C) chain there is an unsaturated fatty acid, with one ore more double bounds, cis
type. The double bound confers a hunch to the fatty acid. This is important during the
molecular assembly and also for the fluidity of the plasma membrane. Opposite to the
polar group the esterification of the OH is done by a saturated fatty acid, usually with
12-24 atoms of C. (Figure 3).
Figure 3

The degree of saturation and the length of carbon chains are important for the
fluidity of the membrane. The polar head is formed by a phosphoric acid residue,
bound to an alcohol (that can be choline, serine or inositol).
The most important phosphoglycerids are: phosphatidyl-choline, phosphatidyl-
serine, phosphatidyl-inositol, phosphatidyl-ethanolamine.
2. Sphingolipids look like phospholipids, but contain sphingosine, an aminoalcohol
with a long unsaturated hydrocarbonated chain.
The hydrolipidic head is the polar group and an alcohol and a phosphate residue
form it. Two chains form the hydrophobic tails: one of them is always the same
(formed by a part of the sphingosine molecule) and the other can be different, being
linked with the rest of the molecule by an amidic bound. The most common
sphingolipid in the membrane is sphingomyeline, with the same polar group as
lecithin.

Membrane glycolipids.
The membrane glycolipids are asymmetric, being present in the external layer of
the membrane in animal cells. They have the polar group formed by a carbohydrate
residue, located in the external part of the plasma membrane. There are two classes of
glycolipids: cerebrosides and gangliosides.
1. The simplest glycolipids are the galactocerebrosides (neutral glycolipids) that
have coupled to the polar group a galactose residue; they form 40% of Schwann
cell’s membrane.
2. The gangliosides have a more complex structure. At the polar group they have
attached a sialic acid residue, which is negative charged. More than 30
gangliosides are known today, but their functions remains uncertain. They appear
to act as receptors on epithelial cells surface; they can also play a role in Ca++
sequestering at cell surface due to their negative charge. For example, the
ganglioside GM1 acts as a cell-surface receptor for the bacterial toxin that causes
the debilitating diarrhea from cholera; cholera toxin binds to and enters the cells
that have GM1 on their surface, including cells from the intestinal epithelia.
Although binding bacterial toxins cannot be the normal function of gangliosides
such observations suggest that the gangliosides may also serve as receptors for
natural signaling between cells.

Cholesterol in the membrane.


Cholesterol is the most important steroid compound in animal tissues. It can be
found in higher quantities in the membrane of mammalian cells and it is absent in
most of prokaryotes. Cholesterol is a large molecule, with 4 rings of carbon atoms and
one OH group situated on C3.
The cholesterol presence in the membrane of animal cells gives it stability and
mechanical rigidity. This feature is shown by the observation that the cells that are
unable to synthesize cholesterol are easily broken in culture mediums. When we add
cholesterol to the medium, the cells are not destroyed anymore. Cholesterol decreases
the membrane’s fluidity and the permeability of the cell.
In fact, cholesterol emphasizes the fence property of the lipid bilayer. In the
bilayer, cholesterol is oriented with OH group near the hydrophilic (polar) head of
phospholipids.
By decreasing the mobility of the first CH2 groups from phospholipid chains,
cholesterol induces a low alterability of membrane shape in this region, so decreasing
the permeability of the bilayer to small water-soluble molecules.
In higher concentrations, cholesterol in the membrane prevents the interaction
and crystallisation of phospholipid chains, so inhibiting the possible phase transitions
in the bilayer.

Membrane Lipids Features - Lipid Bilayer Features.


All the lipid bilayer features come from the common feature of membrane lipids:
the amphiphilic (or amphipatic).
1. Lipids form spontaneously micelles or closed vesicles, called lyposomes.
However, the most stable form is the lipid bilayer formed by an auto-assembly
process.
2. Fluidity is another feature; it is opposite to viscosity, and appears to be
essential for the normal cell growth and reproduction. There are different aspects for
fluidity, according to the movements allowed for lipid molecules:
• Movements of the whole lipid molecule:
„ lateral diffusion (or translation);
„ rotation its own axis - fast movements, frequently achieved;
„ cross-diffusion (flip-flop) - a slow and rare movement from one single-layer
to another (Figure 4).
• Movements inside the lipid molecule (intramolecular movements):
„ flexion of carbon atoms inside the methylene groups;
„ flexion of atoms in polar group.
Movements of lipids and proteins give fluidity to the plasma membrane.
Lateral diffusion

Figure 4. Different types of


Flip-flop movement possible for
(rarely occurs)
phospholipid molecules on a
lipid bilayer.
Flexion Rotation
3. Chemical asymmetry refers to the different ratios of different lipids into the
two layers. For example, carbohydrate residue is situated in the external part of the
membrane, glycolipids are in the exterior monolayer of lipid bilayer, and
phospholipids can prevail inside a monolayer, depending on the cell’s needs. For
example, in red blood cells membrane, almost all lipids that have choline on the polar
group are found in external half of the lipid bilayer (phophatidylcholine,
sphingomyelin) while most phospholipids that contain a primary terminal amino-
group (phophatidylethanolamine, phosphatidyl-serine) are found on the inner half of
the bilayer.
Because of the negative charge, phosphatidylserine is localized in the inner
monolayer; this causes a significant difference in the electric charge between the two
monolayers.
This chemical asymmetry of membrane lipids is generated in the endoplasmic
reticulum, where takes place lipid synthesis; the chemical asymmetry is essential for
membrane functionality.

2.1.2.2. Membrane proteins.


Proteins confer functionality to the membrane while the lipids confer the barrier
function. The percent of proteins in the membrane of different types of cells varies.
For example, myelin membrane that plays an electric isolator role in axons of neural
cells, contains around 25% proteins while the membranes involved in energy
transduction (e.g. the inner mitochondrial membrane) contain around 75% proteins.
Usually, protein ratio in a membrane is around 50%.
Because lipid molecules are smaller in comparison with proteins, usually in the
membrane there are more lipid molecules than proteins; there are around 50 lipid
molecules for 1 protein molecule in a membrane with 50% proteins.

Membrane Protein Functions.


1. Protein-enzymes - catalyses reactions associated with plasma membrane.
2. Receptor proteins - receive signals from the outer medium and send them into the
cell.
3. Transporting proteins - transport substances between the outer and the inner
medium.
4. Binding proteins of the cytoskeleton and of other cellular compounds, and
proteins, which bind cells together.

Membrane Protein Classification.


a. According to the molecular shape, there are fibrillar and globular proteins.
„ Fibrillar proteins have a spiral or filamentous shape and their functions are
performed by the outer-membrane part. Their inner part located in the membrane
needs a little place for anchoring. Fibrillar proteins contain a polypeptidic chain with
an α-helical structure. Usually, they are receptors and specific markers. An example
of fibrillar protein is glycoforin in red blood cells. Receptors for the immunoglobulins
on the B-lymphocytes surface are also fibrillar proteins.
„ Globular proteins have a more complex structure and are formed by
polypeptidic chains wrapped up. These proteins have a globular structure and need a
larger place for anchoring inside the bilayer. Some globular proteins form ionic
channels across the membrane. An example is the globular protein that form ionic
channels in the red blood cells for ion changes with the blood.

b. According to their position in the membrane, proteins are classified in:


„ transmembrane proteins or integral proteins (that are amphipatic, with
hydrophobic domains that interact with hydrophobic domains of the lipids comprised
at the interior of the bilayer, and hydrophilic domains). Integral proteins represent
70% of whole proteins; they are involved in membrane transport and can be removed
only by detergent action. The detergent displaces the lipids bound to the hydrophobic
side chains anchoring the membrane proteins. These proteins are then separated by
polyacrylamide gel electrophoresis, in the presence of SDS (sodium dodecyl sulphate)
detergent.
„ peripheral proteins that are associated to one of the single layers from the lipid
bilayer; they represent 30% of all proteins. They are receptors (when located in the
external monolayer). These proteins interact with the polar groups of membrane lipids
through electrostatic forces. They are easily extracted from the lipid bilayer by salt
solutions.
The association of peripheral proteins with the membrane can be done by:
• covalent bounds to a fatty acid chain;
• a prenyl group (usually located in the cytosol);
• an oligosaccharide to one of the membrane phospholipids;
• non-covalent bounds with other membrane proteins;
The way by which a membrane protein is associated to the lipid bilayer usually
reflects the protein function. By this way, only transmembrane proteins can function
on both sides of the lipid bilayer. For example: during the molecular transport, some
receptors are transmembrane proteins that bind signal molecules from the
extracellular space, so generating the intracellular signals on the other side of the
membrane. Some proteins involved in intracellular signaling are bound to the lipids of
the inner monolayer by multiple covalent bounds.

Membrane proteins Features.


1. Protein movements, similar to lipid movements, with small amplitude realize
fluidity.
2. Chemical asymmetry - some proteins are located only on the external
monolayer and others only in certain structures (for example marker enzymes, like
monoamine oxidase for the outer mitochondrial membrane).

2.1.3. Plasma membrane functions.


The plasma membrane is the border between the extracellular an intracellular
territories, connecting an amount of physical and chemical agents, which may
influence the cell activities. The membrane has two different functions:
1. Substance transport across the membrane, in both directions.
2. The information transfer.
These functions represent fundamental processes for the whole cell activity.
Information analyze entering the cell through the plasma membrane is the premise for
cell’s intervention in many processes.
Cell recognition allows the tissue genesis during the embryonic period. In
mature cells, cell recognition is involved in the defense mechanisms against either the
bacterial aggression or the heterogeneous grafting. Specialized cells that induce some
immunologic defense reactions, including phagocytosis by which the microbes are
engulfed and then destroyed by the cells recognize these aggressions. In the
heterogeneous grafting case, the host organisms recognize non-self structures and
then starts the grafting destroying reactions.
Contact inhibition means that a cell will stop the movements of the adjacent
cells. The neoplastic cells do not have this function, so they can migrate by the blood,
invading the normal cells located far from the initial center.
Cell adhesion maintains the cells joined in the same tissue. It is an important
function used in endocytosis of some substances, bacteria, viruses, the first
messenger, toxins and drugs.
Cell motility is realized by cytoskeleton contribution. For example, the Vibrio
cholerae penetrates in the organism by digestive way. Its pathogenic is the protein
toxin that is fixed on the GM1 receptor situated on the membrane of the enterocyte.
After the fixation of the ligand to the receptor, the ligand (the toxin) inhibits the
income of Na+ ions into cell. The Na+ bulk from the intestinal lumen increases the
osmotic pressure of the intestinal liquids. The water passes suddenly from the cells
and tissues in the intestinal lumen, being determinate by the increased osmotic
pressure. The water will be eliminated from the organism through diarrhea and
vomiting, thus leading to the dehydration of the organism. The modern treatment of
this disease includes GM1 receptor blocking substances that determines the inability
of the toxin to bind this receptor.

2.2. The Transport through the Membrane.

The transport is influenced by the biological membrane permeability that


determines a selective passage for different substances. The cell is able to maintain
different concentrations for substances on both sides of the membrane. The
permeability depends on the substance solubility, as well as on the dimensions of the
ions or molecules and their concentration at normal levels. In vegetal tissues, the
osmotic pressure in the intracellular medium is much higher than the extracellular
medium pressure. It means that the plasma membrane must have a rigid structure,
containing cellulose. The rigid structure is necessary for the protection of the vegetal
cells against the continuous variations of the pressure around it. In the case of the
superior cells, the internal pressure is almost the same as the external one, allowing
the movements and the flexibility of the membrane.
Several transport types across the membrane, which can coexist in different
ratios according to the cell type, are known. According to the dimensions of the
molecules that pass across the membrane, the substance transfer can be classified in:
A. Microtransfer Ö transport of small molecules across the membrane
It is done by:
I. Passive transport:
• simple diffusion:
„ through the lipid bilayer
„ through polypeptides (e.g. the ionophores)
• facilitated diffusion
• simple diffusion through channel proteins
II. Active transport:
• primary active transport (ionic pump mechanism)
• secondary active transport (molecular pump mechanism)
• group translocation (see figure 5).
B. Macrotransfer Ö transport of large molecules across the membrane
I. Endocytosis: two types of endocytosis are distinguished:
• Pinocytosis (cell drinking) involves the ingestion of fluid and/or solutes via
small vesicles. Pinocytosis can be of two types:
„ receptor dependent pinocytosis
„ receptor independent pinocytosis
• Phagocytosis (cell eating) involves the ingestion of large particles such as
microorganisms or cell debris via large vesicles (vacuoles).
II. Exocytosis
III. Transcytosis

Electrochemical
gradient

facilitated
diffusion ENERGY
simple
diffusion ACTIVE
PASSIVE TRANSPORT TRANSPORT

Figure 5. Schematic diagram of passive transport down an electrochemical


gradient and active tranport against an electrochemical gradient.

According to the direction followed by the transported molecules we can classify the
transport systems as follows:
Uniport system - a single molecule type is transported from one face of the
membrane to another.
Co-transport system - two molecule types are transported by:
- symport - both substances are transported in the same direction
- antiport - substances are transported in opposite directions (e.g. H+-K+
pump introduces 1 K+ in the cell moving out 1H+ at the same time).
There are two classes of membrane transport proteins that function in passive
transport.
1. Carrier proteins that have the characteristics of the membrane bound enzymes;
the carrier proteins bind the specific molecule to be transported and transfer it across
the membrane, in a process called facilitated diffusion.
2. Proteins that form transmembrane hydrophilic channels through which solutes
of appropriate size and charge can pass by relatively simple diffusion.

Simple Diffusion.
Simple Diffusion through the Lipid Bilayer.
This type of passive transport is realized according to the oil/water partition
coefficient. Overton observed that liposoluble substances passed easier through
membranes than non-liposoluble ones. There is a proportional dependence between
the permeability constant and this oil/water partition coefficient.
The permeability constant is expressed with the formula:
kxD P = permeability constant
P = K = oil/water partition coefficient
t D = diffusion coefficient
t = plasma membrane thickness
The oil/water partition coefficient (k) is measured by the oil/water partition
degree. We mix oil and water and we inject the substance whose k we want to know.
We shake the mixture and after the two phase's separations (oil/water), we measure
the substance concentration in oil and in water. When the coefficient k (that means
liposolubility) is high then the substance can pass easily through the plasma
membrane. Some exceptions of mentioned above are water, urea, and methyl alcohol.
This rule is important to explain the penetrating mechanism of some liposoluble drugs
through the membrane.
The diffusion coefficient (D) represents the substance quantity that passes from a
medium to another, in the time unit. To measure it, we have to know the surface
where changes are made and the substance concentration gradient in both mediums.
The permeability constant (P) represents the speed (cm/s) of the substance that
passes through the membrane.

Ionophores.
Ionophores are small hydrophilic molecules that dissolve in lipid bilayer and
increase the ionic permeability of the bilayer. Most of them are synthesised by
microorganisms and some are used as antibiotics. They have been widely used to
increase membrane permeability to specific ions in studies on synthetic bilayers, cell
organelles, and more recently on intact cells.
There are two classes of ionophores:
• mobile ion carriers
• channel formers
Valinomicin is an example of mobile ion carrier. It is a ring-shaped polymer that
increases the permeability of the membrane to K+. The ring has a hydrophobic
exterior that contacts the carbohydrate core of the lipid bilayer, and a polar interior
where a single K+ can fit. Valinomicin transports K+ down its electrochemical
gradient by picking up a K+ on one side of the membrane diffusing across the bilayer
and releasing K+ on the other side.
The ionophore A23187 is another example for a mobile ion carrier, but it
transports divalent cations such as Ca++ and Mg++. At low concentrations the
ionophore acts as an ion-exchange shuttle carrying two H+ out of the cell for every
cation it carries inside. This ionophore is widely used in cell biology experiments to
increase the concentration of free calcium in the cytosol. If the temperature of the
membrane is lowered below its freezing point, mobile carriers can not longer diffuse
across the lipid bilayer and the transport will stop. This temperature dependence
serves to identify an ionophore as a mobile ion carrier, since by contrast, channel
forming ionophores continue to transport normally the substances when the bilayer is
frozen.
Gramicidin A is an example of a channel forming ionophore. It is a linear peptide
of 15 aminoacids, all with hydrophobic side chains. Two such molecules are though
to come together in the bilayer to form a transmembrane channel that selectively
allows monovalent cations to flow down their electrochemical gradient. Gramicidin A
can transport about 2x107 cations per open channel in 1 second, that is a thousand
times more than it can be transported by a single mobile carrier at the same time.

The Facilitated Diffusion through Carrier Proteins.


The facilitated diffusion is an energy independent transport, realized in the
direction of the concentration gradient (from a higher to a lower concentration). It is
100,000 times faster than the simple diffusion although the transported molecules are
insoluble and larger than those involved in the simple diffusion. Trough this
mechanism the water, urea, alcohol and anions pass through the red blood cell
membrane, and glucose and aminoacids passes through the membrane of other cell
types. The facilitated diffusion depends on the existence in the plasma membrane of
some specialized transport proteins. A carrier protein specifically binds and transfers
molecules across the lipid bilayer. This process resembles an enzyme-substrate
reaction and the carrier involved behaves like specialized membrane-bound enzymes.
Each type of carrier protein has a specific binding site for the solute (substrate). When
the carrier is saturated (this happens when all such binding sites are occupied) the rate
of transport is maximal. This rate, referred to as Vmax is characteristic for each carrier.
In addition, each carrier protein has a characteristic binding constant for its solute,
KM, equal to the concentration of the solute when the transport rate is half of its
maximal value. The solute binding can be blocked specifically by competitive
inhibitors (which compete for the same binding site and may or may be not
transported by the carrier) or by non-competitive inhibitors (which bind elsewhere
and specifically alter the carrier’s structure).
The analogy with an enzyme-substrate reaction is limited, however, since the
transported solute is usually not covalent modified by the carrier protein. The
molecular details of the carrier proteins are unknown but it is highly unlikely that the
process involves the protein tumbling or shuttling back and forth across the lipid
bilayer. It is more likely that they are transmembrane proteins that undergo a
reversible conformational change in transferring the solute across the membrane.
The protein can exist in two different conformational states: in the state “pong”
the binding sites for the A solute are exposed on the outside of the bilayer; in the
“ping” state the same sites are exposed on the other side of the bilayer. Although this
process is completely reversible, if the concentration of A is higher on the outside of
the bilayer, more A will bind to the carrier protein in the “pong” conformation and
there will be a direct transport of A down its concentration gradient (figure 6).
Some transport proteins simply transport a solute from one side of the membrane
to another: they are called uniports. Other function as co-transport systems, where the
transfer of the solute depends on the simultaneous or sequential transfer of a second
solute, either in the same direction - called symport - or in the opposite direction -
called antiport (Figure 7).
solute

Pong Ping

Concentration
Lipid bilayer gradient

transport protein
meadiating facilitated
diffusion

Figure 6. Schematic drawing of a carrier protein that transports an uncharged


solute A

Transported molecule Co-transported ion

lipid
bilayer

SYMPORT ANTIPORT

UNIPORT COTRANSPORT

Figure 7. Schematic diagram of transport proteins functioning as uniport,


symport and antiport systems

The Facilitated Diffusion through Channel Proteins.


Channel proteins are used as carriers for hydrosoluble substances, while the
liposoluble substances pass through the lipid bilayer. The channels are found inside a
single protein, or are delimited by many intrinsic proteins. The inner part of the
channel is a hydrophilic one, their diameter is about 10A. These ionic channels are
well represented in the membrane of excitable cells. They are selective and some of
them allow the water passage, while the others allows only the ion passage (Na+, K+,
Ca++). The process depends on the diameter of the carried molecule.
Not surprisingly, transport through channel proteins can occur at a higher rate
than that of the transport mediated by carrier proteins. While some channels formed
by transport proteins are continuously open, other are opened transiently. The latter
channels are called “gated”. Some gated channels are open in response to an
extracellular ligand binding to a specific cell-surface receptor and are called ligand-
gated channels. Other gated channels are depended of the membrane potential, called
voltage gated channel, and others opens in response to changes in the intracellular
concentration of specific ions; for example, some K+ channels open when the
concentration of free Ca++ in the cytosol increases.
In many cases, gated channels have self-closing mechanism that rapidly closes
again the channels even if the stimulus that originally opened them is still operating.
For example, we consider the neuro-muscular junction where an impulse travels down
a nerve and stimulates a muscle to contract. This apparently simple response involves
the sequential opening and closing of at least four different sets of gated channels, all
within less than one second.

The Active Transport.


Bernstein started the study of active transport in 1912, on excitable cell
membranes. The active transport is realized against the concentration gradient and it
needs energy consumption and high specificity intrinsic proteins. The energy is
delivered by ATP hydrolysis. The active transport is coupled with the oxidative
phosphorylation, because the ATP synthesis is realized owing to this process. A
mechanic work is initiated after ATP hydrolysis and it lies at the origin of the primary
and secondary active transport.

The Primary Active Transport (Ion Pumping Mechanism).


We can classify the primary active transport as follows, according to the ionic
pump mechanism involved:
• the Na+-K+ ATP-ase
• the Ca++ ATP-ase
• the H+-K+ ATP-ase
• the proton pump

The Na+-K+ Pump.


The Na+-K+ pump is the most common pump, being encountered in all cells. One
third of animal total energy requirement is consumed to support this pump.
In the electrically active nerve cells that must continuously re-establish their
membrane potential following depolarization, this figure approaches 70% of total
energy requirements. A major advance in understanding the Na+-K+ pump functioning
came with the discovery in 1957 of an enzyme that hydrolyses ATP to ADP and Pi
requiring Na+ and K+ for their optimal activity. An important clue linking this Na+-
K+-ATP-ase with the Na+-K+ pump was the observation that a known inhibitor of the
pump, ouabain, also inhibited the ATP-ase.
The transport of Na+ and K+ is completely tight to ATP hydrolysis, so that one
can not occur without the other. Ion transport and ATP hydrolysis can occur only
when the Na+ and ATP are present inside the cell, and K+ is present outside the cell.
Ouabain is an inhibitor only when it is present outside the cell where it competes for
the K+ binding site. For each molecule of ATP hydrolyzed, 3 Na+ are pumped outside
the cell and 2 K+ are pumped inside the cell (100 ATP molecules can be hydrolyzed
by each ATP-ase molecule in every second) (Figure 8).
3 Na K and ouabain binding site

Sodium
concentration Potassium
gradient concentration
gradient

ATP ADP+Pi

2 K

Figure 8. Schematic diagram of the Na+-K+-ATPase actively pumping Na+


out and K+ in a cell against their concentration gradient

The Na+-K+-ATP-ase were purified and they consist of two subunits:


„ the α-subunit - is the catalytic subunit with a molecular weight (MW) of
100,000 Da.
„ the β-subunit - is the non-catalytic subunit, an associated glycoprotein with a
MW of 45,000 Da.
These subunits form a tetramer of 300,000 Da Mw to permit the function of the
ATP-ase (the tetramer consists of two α subunits plus two β subunits).
The complex has binding sites for Na+ ions and ATP on its cytoplasmic
surface and for K+ and ouabain on its external surface. The ATP-ase is reversibly
phosphorylated and dephosphorylated. The glycoprotein function is unknown.
The Na+ ions binding and the cytoplasmic ATP-ase face phosphorylation
induce the protein to undergo a conformational change that transfers the Na+ ions
across the membrane and releases them on the external part of the cell. Then, K+ ions
binding on the external face and the dephosphorylation return the protein
conformation to the original state. This conformational change transfers the K+ ions
across the membrane and releases them into the cytoplasm. These conformational
changes are similar to the “ping-pong” mechanism, except that here, the
phosphorylation and dephosphorylation processes induce these transitions (Figure 9).
Figure 9. A schematic model of the Na+-K+-ATPase. There take place
conformational changes similar to the ping-pong model.

Physiological roles of Na+-K+ pump:


1. The Na+ and K+ gradients maintained by the pump are responsible for
maintaining and generating the cell’s membrane potential. Two transport proteins are
crucial for generating and maintaining the membrane potential (which varies between
20 mV and 200 mV depending on the species and cell type).
a. Na+-K+-ATP-ase that actively pumps Na+ ions outside the cell and K+ ions
inside the cell, so that the concentration of K+ ions is higher inside than outside, while
the opposite is true for Na+ ions. In this way, this pump has a small direct contribution
to the membrane potential (20%).
b. K+ leak channel, that allows K+ ions to leak out of the cell down its steep
concentration gradient and make the membrane much more permeable to K+ ions than
to Na+ or anions. The internal part of the membrane becomes electrically negative
relative to the external part. In different cells, similar principles operate to generate
and maintain the membrane potential, although the membrane permeability to certain
ions is different. For example, the membrane of many cells is permeable to Cl-, which
therefore has an important contribution to the membrane potential.
2. The Na+-K+ pump also helps to regulate cell’s volume since it controls the
solute concentration inside the cell and thereby the osmotic forces that tend to make
the cell swell or shrink. The macromolecules confined inside the cell exert an osmotic
pressure on the membrane, furthermore, since these macromolecules are mostly
charged, they are necessarily accompanied by ions such as K+ which add the effect.
This pressure on the inner face of the membrane is counterbalanced by the osmotic
pressure due to the molecules in the extracellular fluid (Na+ and chloride ions). These
ions, however, tend to leak into the cell down their concentration gradient, upsetting
the balance and causing the cell to swell.
The Na+-K+-ATP-ase solves this problem in the following way: it directly
pumps out of the cell the Na+ that leak in, and at the same time it helps to prevent Cl-
ions from leaking by keeping negative the interior of the cell; in this way the effect of
chlorine ions concentration gradient is counteracted.
3. Na+-K+ pump is responsible for driving the active transport of sugars and
aminoacids.
4. Na+-K+ pump helps the cell proliferation.
5. Na+-K+ pump controls the neurotransmitters releasing from the brain.
6. The pump plays a role in thermogenesis.

The Ca++ ATP-ase.


This pump is found mainly in the excitable cell’s membrane. It is also found in
the smooth endoplamic reticulum membrane, as well as in the inner mitochondrial
membrane. The pump participates to the following activities:
• the maintaining of free cytoplasmic Ca++ concentration at lower levels (10-7)
comparing to the levels of the extracellular concentration (10-3).
• during the actin-myosin interaction, the stored Ca++ is released into the
cytoplasm
• inside the mitochondrion, Ca++ holds an important role in biochemical
reactions.
We know more about membrane-bound calcium pump in the sarcoplasmic
reticulum of muscle cells. The sarcoplasmic reticulum forms a network of fine
channels in muscle cells and serves as an intracellular store for calcium. The calcium
pump is responsible for pumping calcium ions from the cytosol into the sarcoplasmic
reticulum. When a nerve impulse depolarizes the muscle cell membrane, calcium ions
are relesead from the sarcoplasmic reticulum into cytosol, stimulating the muscle to
contract.
Like the Na+-K+ pump, the calcium pump is an ATP-ase that is
phosphorylated during its pumping cycle. Each molecule can hydrolyse up to 10 ATP
molecules per second on its cytoplasm surface and pumps 2 Ca++ ions into the
sarcoplasmic reticulum for every ATP hydrolysed.
Since this Ca++-ATP-ase is the only major protein in the membrane of the
sarcoplasmic reticulum (accounting for about 80% of its total proteins) it has been
relatively easy to purify. It is a single polypeptide chain containing 1000 aminoacid
residue.

The Hydrogen-Potassium ATP-ase.


It is found especially in the membranes of the cells in gastric epithelia mucous
membrane, being involved in the HCl synthesis. It realizes the transport of K+ ions
inside the cell and the H+ passage outside the cell, in the stomach.

The Proton Pump.


The pump exists in prokaryotes and also in eukaryotes. It represents the motor of
the cell metabolic process, in prokaryotes. Halobacterium Halobium membrane
contains proteins specialized in the photon absorption (green light ones). Thus it
disposes the hydrogen ions to the external medium, generating a pH difference, the
motor of the metabolic processes.
The pump also lies within the inner mitochondrial membrane, the residence of
cell respiration. This process presumes the proton pumping and electron transfer, both
phenomenons realizing ATP synthesis.

The Secondary Active Transport (Molecular Pumping).


It is realized during the sodium ions transport, requiring a transmembrane
transport, which presents on the exterior side two binding places: one of them binds
the sodium ions, the other binds the molecule to be transported (the aminoacids,
glucose). First, the Na+ and glucose (for example) are fixed on their binding places;
then, Na+ will be transported into the cell, according to their concentration gradient
involving the glucose transport into the cell. This transport model represents a
symport model. The glucose will be used for the cell energetic needs. Na+
concentrations on both sides of the membrane tend to equalize. In this moment acts
the Na+-K+ pump which will restore the concentration difference for Na+. So, Na+ will
be eliminated from the cell, K+ will enter inside, process powered by an ATP
molecule hydrolysis. As higher is the electrochemical gradient for Na+, a higher
quantity of glucose will enter inside the cell (figure 10).

Figure 10. Schematic drawing of glucose symport, showing how the active
transport of glucose is driven by a Na+ gradient that is generated and
maintained by the Na+-K+-ATPase

The same mechanism is used for the aminoacids transport, the carrier being
always the Na+. In this case, five-transport protein is used. Each protein type binds the
aminoacid with a similar chemical structure. Some bacteria, such Escherichia coli
uses the H+ pump to help glucose to enter into the cell.

Group Translocation.
It is found especially in bacteria. The mechanism involves the binding of a
molecule on the external face of a membrane protein, the molecule entering the cell
with a modified structure.
The protein that facilitates this transport is the phospho-transferase system.
For instance, the sugars will be fixed on the external face of the membrane protein.
The sugars will enter inside the cell, changing their chemical structure as a result of
the phosphorylation. The substrate for this process is the phospho-enolpyruvate,
which will be divided into P and pyruvate.
Many active sugars and aminoacid transport systems in bacteria involve a special
class of water-soluble proteins located in the space between the plasma membrane and
the cell wall - the periplasmic space. These proteins have specific binding sites for the
solute and are therefore called periplasmic binding proteins.
Each of these binding proteins that function in transport also seem to serve as
receptors in chemotaxis; chemotaxis is an important process that enables bacteria to
swim toward an increasing concentration of a specific nutrient (Figure 11).

Figure 11. Active transport of sugars into bacteria by group translocation

2.3. Information Transfer Across the Membrane - Chemical Signaling


Between Cells.

The evolution of multiple cell organisms depends on the ability of cells to


communicate between them. Communication between cells is required to regulate
their development and organization into tissues, to control their growth and division,
and to control diverse cell activities.

Cell-Cell Communication Types.


Cells are thought to communicate in three ways:
I. Indirect cell to cell communication (communication to a certain distance).
1. Nerve transmission - used to communicate rapidly and selectively some
signals.
2. The humoral way transmission - the biological active substances (hormones)
are secreted in the blood stream and reach different organism areas, bringing
information.
II. Direct cell to cell communication
1. Permanent - via “gap” junctions.
2. Transient - by the immune system cells communication, when it is made an
information change by transient cell contact.
III. The first two cell to cell communication types can be combined: e.g. the
nerve network interposed between the sensorial cells and the muscle cells (figure 12).
Figure 12. Schematic diagram showing three different ways in which cells are
thought to communicate with each other.

Endogenous Signaling Molecules (Extracellular Signaling Molecules).


Chemical signaling operates in three different ways:
1. Most cells in the body secrete one or more chemical signals that function as
local chemical mediators because they are fast uptake and destroyed that they act only
on cells in the immediate proximity. They realize two signaling ways:
- paracrine signaling - signals are transmitted locally, to the neighbor cells;
- autocrine signaling- signals are transmitted both to the neighbor cells and
to the emitter itself.
2. Specialized endocrine cells secrete hormones that travel through the blood
stream to influence target cells widely distributed in the body. They realize the
endocrine signaling way.
3. Nerve cells from specialized junctions (chemical synapses) with the target
cells and secrete very short-range chemical mediators called neurotransmitters. They
realize the neurocrine signaling way.
Endocrine and nerve cells are highly specialized for chemical signaling and work
together to coordinate the diverse activities of billions of cells in higher organisms.
Nerve cell transmit information much faster than endocrine cells because they do not
depend on diffusion or on blood flow to convey information over long distances;
instead, electrical impulses carry the signal rapidly along the nerve processes.
Only in nerve terminals, where a neurotransmitter is released, the electrical
impulses are converted into chemical signals. The neurotransmitter has to diffuse only
a microscopic distance to the target cells, a process that takes less than 1 ms.
Hormones are highly diluted in the bloodstream and therefore must be able to
act at very low concentrations (normally<10-8M), neurotransmitters are diluted much
less and can achieve high concentrations in the region of the target cell.
In other respects, however, the mechanisms of chemical signaling by
hormones and neurotransmitters are generally similar, and many of the signaling
molecules that are used by endocrine cells are also used by nerve cells (neurones).

Biologic receptors.
In 1878, Langley used for the first time the term receptor. In 1910, Erlich
sustained the existence of receptors.
A biologic receptor is a glycoprotein macromolecular structure that
complementary interacts with a ligand from the extracellular environment.

Receptors Classification.
Classification criteria:
- according to the position in the cell
- according to the transduction mechanism
- according to molecules that they are coupling (according to the ligand)
A. According to the position in the cell:
- membrane located receptors (receptors for polypeptide hormons)
- receptors in the cell (recptors for vitamin D, steroid hormons, retinoids,
and thiroid hormons )
- intracytoplasmatic receptors
- intranuclear receptors
Intracellular receptors can be coupled with inhibitor proteins that release the receptor
when the ligand is near.
B. Accoding to the transduction mechanism:
- ionic channel coupled receptors
- G protein coupled receptors (G protein receptors are 7-pass
transmembrane proteins; there are many types of G proteins - Gs,
stimulating, Gi, inhibitor, etc.
- enzyme coupled receptors - the receptors are enzymes that become active
when a ligand is bound or receptor activation leads to other enzymes
activation in cytoplasm (as proteinkinases)
C. According to the ligand, biologic receptors are of two types:
I. Receptors for endogenous substances:
1. Receptors for neurotransmitters located in the post-synaptic external
membrane (e.g. receptors for acetylcholine, histamine, noradrenaline).
2. Receptors for humoral transported substances:
• hormone receptors (receptors for insulin, gastrin, etc. )
• endogenous antigen receptors (on the surface of T lymphocytes), playing a
role in the immune response releasing.
• antibody receptors (receptors for IgE located in the surface of basophils and
mast cells)
• complement receptors (receptors that bind the serum C3 fraction of the
complement system and are specific for the phagocytic mononuclear cells).
• low density lipoprotein receptor - that induces receptor mediated endocytosis.
II. Receptors for exogenous substances:
1. For viruses and bacteria
2. For microbial toxins
3. For non-self exogenous antigens (receptors on B-lymphocyte surface)
4. For drugs
The receptor’s type and density varies with the cell type and the considered area.
The same receptor type can be present in one or more cell populations (e.g. insulin
receptors are present in hepatocytes, muscle cells and adipocytes).

Membrane receptor proteins properties


I. The specificity
The specificity that has two aspects:
1. The binding specificity means that a certain informational molecule with a
certain chemical structure is recognized by a certain cell receptor.
2. The response specificity means that a certain cell type when it receives
specific informational molecule responds in its own way, functions according to its
specialization and its specific location. The response can be cell secretion, contraction
or metabolite production.
II. The saturability
The saturability means that the receptor ability to bind signals is limited.
III. The plurality
The plurality means that for the same informational molecule there are many
receptor types and subtypes; cell respond depends on the type of activated receptor, or
the transduction mechanism used
That is why the same hormone can determine opposite reactions in different parts
of the body. For example, adrenaline has different effect on different tissues according
to its 4-receptor types: α1, α2, β1 and β2.
β1 receptors are found mainly in the heart and stimulates muscle fibber
contraction. β2 receptors are found in the lower airways (bronchia) where they control
bronchial muscle relaxation.
The noradrenaline is recognized by the β2 receptor, theoretically being useful
in asthma treatment; however, noradrenaline acts also on β1 receptor producing heart
activation that is an undesirable effect. For this reason, noradrenaline cannot been
used in asthma treatment.
The β1 and β2 receptors are structurally different and for that reason there
could be synthesized some substances that act mainly on β2 receptors (for example,
salbutamole as aerosols).

2.3.1. Information Transfer through Hormones.


There are two types of hormones:
1. Polypeptidic hormones are synthesized by compact endocrine glands or by the
diffuse endocrine system (that secretes and synthesizes hormone-like substances) e.g.
insulin, somatostatin, etc.
2. Steroid hormones are cholesterol derivatives (cortisol, sexual hormones).
To start a biologic response, polypeptidic hormones need a receptor structure
inside the cell and steroid hormones enter inside the cell and bind cytoplasmic
receptors.

Information Transfer through Polypeptidic Hormones.


The information transfer through polypeptidic hormones requires three
membrane-bound proteins that interact in the following sequence:
1. A protein receptor located in the outer layer of the membrane.
2. The translating protein represented by the Gs or Gi protein that take the
information from the receptor and transfer it to the third zone. G proteins are trimeric
proteins, formed by α, β and γ subunits. γ and β subunits are common to both Gs and
Gi. α subunit differs, in Gs is αs while in Gi is αi. In turn, there are many types of αi
type 1, 2, 3, etc
3. The amplifier that is the adenylyl-cyclase, located in the inner layer of the
membrane.

The Steps in Information Transfer through Polypeptidic Hormone.


I. The membrane stage:
The ligand binding to the receptor determines the receptor activation and a
ligand-receptor complex is formed. This ligand-receptor complex will diffuse through
the lipid bilayer, binding the Gs or Gi protein. This coupling will enable the α and β
subunit dissociation and the displacement of GDP by GTP from cytoplasm. After the
GTP molecule binding, the G protein conformation is altered and dissociates from the
activated receptor exposing a binding site for adenylyl-cyclase. GTP hydrolysis in
GDP and P will return the G protein to its original conformation, inducing the
activation of the cyclase to produce cAMP (Figure 13)
When the adenylyl-cyclase is activated, it begins the second stage. The activation
of cyclase is repeated until the dissociation of the ligand returns the receptor to its
original conformation. Each activated receptor protein activates many molecules of G
protein, thereby amplifying the response (Figure 14).
II. Intra-cytoplasmic stage:
The activated adenylyl-cyclase induces the cAMP formation from ATP, in the
presence of Mg++ ions. cAMP is a second messenger. Second messengers are:
1. cAMP is synthesised from ATP by the membrane-bound enzyme adenylyl-
cyclase, but it is also rapidly degraded in cells by one or more specific enzymes called
phosphodiesterases, which hydrolyse cAMP to 5’-AMP.
2. cGMP is synthesized from GTP by the membrane-bound enzyme - guanylyl-
cyclase.
3. Ca++ ions act to initiate neurotransmitter secretion, cause muscle contraction
and participate to phosphorylation reactions.
4. IP3 (inositol-triphosphate) is formed when act muscarinic substances.
The second messenger action is realized by activation of specific cell enzymes
called cAMP-dependent protein-kinases are formed by two subunits:
Š a catalytic subunit;
Š a regulatory subunit, capable to bind cAMP.
Figure 13. The intramembranar stage of the transmission through polypeptide
hormones

If cAMP is missing, the regulatory and catalytic subunits form an inactive


complex. The binding of cAMP to the regulatory subunit induces a conformational
change, causing this subunit to dissociate or no from the complex, and actives the
catalytic subunit.
Although the protein-kinase is shown as a dimmer for the sake of clarity, it is
actually though that is a tetramer consisting of two regulatory and two catalytic
subunits. Each regulatory subunit has two cAMP binding sites, and the release of the
catalytic subunits is a co-operative process requiring the binding of more than two
cAMP molecules to the tetramer.
Figure 14.Besides transducing an extracellular signal intoan intracellular one,
the coupling of cell surface receptors to adenylyl-cyclase activation largely
amplifies the initial signal.

The active catalytic subunits will catalyse the following type of reaction:

catalytic subunit of the protein-kinase


Protein + ATP Protein-P + ADP

cAMP-dependent protein-kinases are found in animal cells, where they


probably account for all of the effects of cAMP. While in many cases the substrates
for these protein-kinases are not known, clearly the differ in different cell types
explaining why cAMP vary according to the target cells.
There are many different protein-kinases in cells but only few of them are
regulated by cAMP. While some are regulated by Ca++ (PK-C) or by cGMP, the
regulation mechanism for most of these enzymes has not been yet identified. cAMP
also regulates the dephosphorylation of cell proteins; phosphorylated proteins are
rapidly dephosphorylated when cAMP levels fall.
Increased free Ca++ levels affect cells by binding calmodulin and altering its
conformation. The Ca++-calmodulin complexes in turn activate many different target
proteins, including Ca++-dependent protein-kinases (PK-C).
Thus, by using cAMP or Ca++ as second messengers, extracellular signals are
both highly amplified and become specific for each cell type.
Like cAMP, cGMP acts primarily by activating specific protein-kinases that, in
this case, are cGMP dependent.
Steroid Hormones Action Mechanism.
All steroid hormones are synthesized from cholesterol. Being relatively small
(300 Da MW) hydrophobic molecules, they cross the membrane by simple diffusion.
They are rendered soluble by reversible bindings with specific blood protein carriers
and circulate into the blood stream bound to these carriers.
When they arrive next to the target cells, they are released from their bindings
with the carrier and they cross the membrane by simple diffusion. Once inside the
target cell, each type of steroid hormones binds tightly but reversibly to a different
receptor protein that is found in the cytoplasm. The hormone binding causes the
protein receptor to undergo a conformational change that increases its ability to bind
DNA. Since the protein receptors are able to migrate through the nuclear pores, their
increased binding affinity for DNA causes the hormone-receptor complexes to
accumulate in cell nucleus. In the nucleus, the complexes bind on nuclear chromatin
and regulate the transcription of specific genes. Only steroid hormones directly
influence a small number of genes in any target cell.

2.4. The Glycocalyx.

Glycocalyx, also called the sweet cell cover (Bennett, 1963) is a glycoprotein
layer located over the membrane outer layer; its integrity is necessary for the normal
development of cell functions and activities.
The Morphological Structure.
In electron microscopy, glycocalyx looks like a fibrillar material of 50 nm thick,
made up of two layers:
• an internal layer or the surface cover, 20 nm thick, less electron-dense
• an external layer or the external lamina, 30 nm thick, denser than the first
Glycocalyx Molecular Structure.
The glycocalyx is a carbohydrate-rich peripheral zone at the surface of most
eukaryotic cells. The carbohydrate consists of the oligosaccharide side chains of
membrane-bound glycoproteins and glycolipids, although it often includes, in
addition, both glycoproteins and proteoglycans that have been secreted and then
absorbed on the cell surface. The proteoglycans (mucoproteins) consist of many
polysaccharide chains linked to a core protein.
Glycocalyx functions.
The most important functions of the glycocalyx are:
1. Protective role - it represents a resistant structure to mucolytic and proteolytic
enzymes, excepting the hyaluronidase and the neuraminidase.
2. Absorptive role - glycocalyx can absorb or fix cytophilic antibodies that
modify the phagocytosis. It has an important role in endocytosis, because in
glycocalyx are found specific receptor zones for different molecules: biological
molecules (hormones), drugs and toxins.
3. Glycocalyx permeability is emphasized by treatment with neuraminidase.
4. Cells adhesion - glycocalyx glycoproteins belonging to adjacent cells
participate to junctions' formation (e.g. desmosomes).
5. Role in cell recognition. Cells belonging to the same type, the same origin and
the same organism have the property to recognize one another. This function also
represents a defense mechanism (phagocytosis).
2.5. Cell membrane specialization.

The membrane specialization is complex morphological structures that confer


certain functions to the cell or the represent cell coupling zones. There can be:
I. Transient specialization:
1. Membrane and cytoplasm invaginations from endocytosis.
2. Membrane prolongation that appear in a certain functional state of the cell
(e.g. endocytosis, cell surface boiling movements during mitosis, pseudopodia
emission that realizes some movements).
II. Permanent specializations that confer a certain function to the cell or represent
cell-coupling zones. They are present:
1. At the apical pole - we can find the following differentiation:
• microvillia
• stereocilia
• cilia
2. At the base-lateral pole of the membrane - cell junctions

2.5.1. Microvillia.
Microvillia are cell expansions with a core of crossed-linked actin filaments
covered by membrane; they participate in absorption processes by:
1. Increasing the cell contact surface for substances that will enter the cell.
2. An increased number of receptors on the surface area unit.
Microvillia cover the exposed surfaces of many epithelial cells, especially where
cell functions require a maximum surface area for absorption, such as in the gut or
kidney. These finger-like extensions are about 1 μm length and 0.1 μm diameter.
Microvillia can be grouped in two ways:
1. Isolated microvillia, separated by a certain free space, having variable
dimensions and shape.
2. Grouped microvillia that concentrates a high number of receptors on the
surface unit (an enterocyte has 3000 microvillia per mm2).
In photon microscopy, microvillia look like perpendicular striations on the
membrane surface. Microvillia can be grouped in:
„ the striated plateau
„ the brush border
The striated plateau is formed by microvillia with the same shape and
dimensions; it is found at the apical pole of the enterocytes in the gut mucosa.
The brush border is formed by microvillia with unequal dimensions; it is
found at the apical pole.
In electron microscope, a three-layered external membrane, 9-10 nm thick,
similar in structure to common membranes, forms the structure of microvillia.
Inside the membrane, there are a lot of ATP-ases (carrier proteins). Under the
membrane there is an unstructured cytoplasm zone, called ectoplasm. The microvillia
core contains about 40-50 actin filaments that run in a parallel bundle along its length.
At the tip of the microvillia the actin filaments are embedded in an ill-defined cap of
amorphous material that contain myosin, while at their base they extend into a
perpendicular network, composed by many filaments, called terminal web (Figure
15).
Figure 15. Schematic
drawing showing the
polarity of actin filaments
in a microvillus (A) and in
a muscle sarcomere (B).

The actin filament bundle is tied to the inner face of the membrane of the
microvillia free pole by α-actinin. There are also fine lateral bindings between the
actin filament bundle and the entire length of microvillia membrane. Proteins called
fimbrin and villin form these bindings. The interaction of the actin filament bundle
with the lateral membrane of the microvillia is due to arms of myosin and calmodulin
(a Ca++-dependent protein). The microvillia membrane is covered by a rich
glycocalyx.

2.5.2. Stereocilia.
Stereocilia are cytoplasm extensions covered by the membrane. They are not
mobile but their structure is like the microvillia structure. Many of them agglutinate in
the same region and direct a secretion product. They are found in the epididymis and
in the deferent channel cells apical pole.

2.5.3. Cilia.
Cilia are cytoplasm extensions covered by a membrane; they have a high mobility
and are found in the superior airways mucous cells and in some parts of the oviduct.
The tail of the spermatozoon is a flagellum. Cilia are tiny hair-like appendages about
0.25 μm in diameter that contain a core formed by bundle of parallel microtubes.
They extend from the surface of many cell types and are found in most animal species
and some lower plants. They primary function is to move fluid over the surface of a
cell or to propel single cells through a fluid.
The cilia dimensions are under the resolution power of the photonic
microscope.
In electron microscope, cilia appear structured as follows:
1. Cilium stalk that is a free region formed by:
• an external membrane;
• an amorphous zone less electron-dense named ectoplasm;
• the axoneme, a complex structure composed entirely of microtubules and their
associated proteins.
The axoneme is formed by 10 microtubules pairs disposed as follows:
„ a central pair;
„ 9 peripheral pairs arranged in a ring, around the central pair.
The peripheral pairs are located from 50 to 50 nm. While central pair is formed
by complete wall microtubules, the outer doublets are composed by a complete wall
microtubule and an incomplete wall one; they are called A and B microtubule. They
fuse together to form a pair, A microtubule sharing its wall to the B one. A
microtubule is located closer to the central pair. Its wall is formed by 13 tubulin
protofilaments, each protofilament being formed by αβ tubulin dimmers. B
microtubule wall is incomplete, being formed by 10 protofilaments and it uses 3
protofilaments from A microtubule wall. In A microtubule wall, from 13 to 13 nm,
there are attached two fibrillar arms that project from each doublet and extend toward
an adjacent outer pair in the ring. These arms occur in pairs and are composed by a
protein called dynein. Dynein is an ATP-ase with a 300,000-400,000 MW and it
specifically interacts with tubulin. The interaction between tubulin and dynein plays
essential role in cilia movements, being stimulated by Mg++ and Ca++. By
electrophoresis there were found two types of dynein: dynein 1 and 2. In the cilium
stalk there is more dynein 1. A specific protein called nexin couples the adjacent
doublets in the outer ring; it is thought to be highly elastic and to form “straps” around
the entire axoneme, rather than hoops around a barrel. Projecting inward from each
doublet is a radial spoke, which ends in a globular zone, very near to the inner sheath.
The inner sheath is composed by slender protein arms that project outward from the
central pair of microtubules and curve around them. It apparently enables the central
pair to help regulate the movement of the axoneme. Each of the structures described -
dynein side arms, nexin links, radial spokes, the arms of the inner sheath - can be seen
as series of regular projections, each with its own periodicity. The radial spokes
maintain the geometric conformation of the moving cilia (figure 16).
2. The transient zone is the place where the central doublet microtubules stop in
an electron-dense structure, called basal-plaque. The outer doublet microtubules
continue and pass through this zone, forming the next structure.
3. The basal body (called also kinetosome) has a centriole-like structure. It has a
cylindrical shape, with the wall formed by 9 fused triplet microtubules. Each triplet of
microtubules contains the previous doublet and one more partial microtubule.
4. Actin microfilaments associations form the cilia root that form 70 nm thick
striations, structures that alternate with more clear zones. The cilia root has two
functions:
a. mechanical function for the kinetosome and other cilia structures anchorage;
b. contractile function due to actin microfilaments.
Figure 16. Cross section through the cilium stalk

Cilia movements.
Cilia movements are very difficult to study because its speed (600-1700 beats per
minute). There can be two types of movements:
• bending movements
• undulating movements
It is considered that cilia movements are the result of dynein-tubulin interactions,
guided by ATP, Ca++ and Mg++.

Cilia movements and its pathology.


Cilia immobility is due to:
• genetic impairment (nexin or dynein couldn’t be synthesised);
• acquired impairment (exposure to toxic mediums)
An example of genetic impairment is the Kartagener syndrome, characterised by
immobile cilia syndrome, and “situs inversus” - in which the normal asymmetry of the
body, as heat, liver or appendix positions, is reversed. It has therefore been suggested
that the unidirectional beating of the cilia during the early human development could
play a critical role in determining the normal left/right asymmetry of the body.
Cilia defects occurred in humans suppose also various hereditary forms of
male sterility due to the non-motile sperm. Depending on the form of this hereditary
disease, the sperm flagellum lack both dynein arms, only the outer or inner arms, the
radial spoke heads, or the inner sheath together with one or both of the central pairs of
microtubules. Exactly the same defects occur in the respiratory cilia of such
individuals, who commonly have long histories of airway diseases - with recurrent
bronchitis and chronic sinusitis - because their immobile respiratory cilia are unable to
clear the mucus from their lungs and sinuses.

2.5.4. Membrane Basal Pole Specialisation.


The epithelial cells basal pole is located on a continuous glycoproteic basal
lamina. This basal lamina can play an important role in the active transport and can
present some invaginations that part the cytoplasm, forming the basal labyrinth. The
number and the depth of basal labyrinth compartments depend on cell type and role in
the active transport. In choroid plexus, the basal labyrinth compartments are few and
superficial while in the distal contort tube in nephrocytes there are many deep
compartments belonging to the basal labyrinth. Inside the folds of the basal labyrinth
we can find mitochondria that delivers energy for the Na++ and K+ active transport.
For example, the saline gland epithelial cells in sea birds pull out NaCl (salt); that’s
why, sea birds can drink seawater. The epithelial cells of this gland have thousands of
invaginations, extended on the whole cell surface, near to the apical pole increasing a
lot the changing surface.

2.5.5. Cell junctions.

In multiple-cell organisms, cells become specialized in living and acting together


as a whole.
Specialized cells in multiple-cell organisms are generally organised in co-
operative assemblies called tissues; different tissues combine to form large functional
units called organs. Into the tissues, neighbouring cells are linked one to another by
specialized regions in their membrane called junctions.
Cell junctions are too small to be seen in photonic microscopy. They can be
distinguished, however, in freeze-fracture electron microscopy; we can observe that
the interacting membranes (and often the underlying cytoplasm and the intercellular
space) are highly specialized in these regions.
In a functional classification of cell junctions we can distinguish:
A. Anchoring junctions:
i. actin filaments attachment sites:
• cell-cell adherens junctions (e.g. adhesion belts);
• cell-matrix adherens junctions (e.g. focal contacts);
• septate junctions.
ii. intermediate filament attachment sites:
• cell-cell (desmosomes)
• cell-matrix (hemidesmosomes)
B. Occluding junctions (tight junctions)
C. Communicating junctions:
i. gap junctions
ii. chemical synapses
iii. plasmodesmata (plants only).

2.5.5.1. Anchoring Junctions are widely distributed in specific mechanical


stressed tissues like epithelium, skin or heart muscle. These junctions are connecting a
cell cytoskeleton to another or to extracellular matrix. They can base on actin
filaments or intermediate filaments interaction.
1. Adherens Junctions are connection sites for actin filaments. When they occur
in adjacent epithelial cells, they form the adhesion belt (zonula adherens), ancient
called belt desmosome. In epithelial adjacent cells the adhesion belt is located toward
the apical pole, just below the tight junction (Figure 17)
In adjacent cells, adhesion belts are apposed directly and the interacting plasma
membranes are held together by transmembrane linker molecules called cadherins. In
each cell, adjacent to the adhesion belt we can find a bundle of actin filaments that
runs parallel to the membrane. This actin bundle is linked to the membrane by
specific attachment proteins: α-, β-, and γ-catenin, vinculin, α-actinin, and
plakoglobin. Linked in this way, actin bundle forms an extensive transcellular
network of which contraction depends on myosin motor proteins. These junctions are
supposed to play a fundamental role in animal morphogenesis - the folding of
epithelial sheets into tubes and other related structures.
Figure 17. The structure of belt desmosomes.

If the cells make connection with the extracellular matrix, actin filaments ends at
the level of specialized regions in the membrane called focal contacts. The
transmembrane linker proteins that mediate the contact between actin filaments and
the extracellular matrix are some cell-surface matrix receptors called integrins. Their
extracellular domain binds protein compounds in the extracellular matrix while the
intracellular domain binds indirectly the actin filaments via a complex formed by
talin, α-actinin, and vinculin.
Septate junctions are found in invertebrate tissues.
2. Desmosomes and hemidesmosomes are connection sites for the intermediate
filaments. They act as rivets and they distribute tensile or shearing forces in specific
tissues like epithelium.
Desmosomes connect intermediate filaments in two adjacent cells. Inside the
cells they are anchoring sites for keratin filaments in epithelial cells and for desmin in
heart muscle cells. Three parts form desmosomes:
1. Extracellular elements represented by the linkers from cadherin family.
Intercellular space is kept intact (15-35 nm).
2. Cell elements:
• an internal plasma membrane thickening in both cells.
• cytoplasmic plate formed by a cytoplasmic condensation located under the
membrane.
• tonofilaments - keratin or desmin (intermediate filaments) - that enter the
cytoplasmic plate and return into cytoplasm. The cytoplasmic plate is separated from
the plasma membrane thickening by an ectoplasm zone, less electron-dense.
3. associated structures, common to both cells - actin filaments.
Hemidesmosomes or half-desmosomes seems to desmosomes except the fact
that they are joining the basal surface of epithelial cells to the underlying basal lamina
(a specialized structure of the extracellular matrix). Keratin filaments (tonofilaments)
are buried in the cytoplasmic plaque. The transmembrane linker proteins are integrins,
similar to focal contacts.
As we have seen cadherins in a plasma membrane anchor it to cadherins in the
membrane of adjacent cells; integrins in a plasma membrane anchor the cell to
extracellular molecules in the matrix.

Figure 18. Highly schematic drawing of a spot desmosome

2.5.5.2. Occluding junctions (tight junctions).


Tight junctions represent one of the major hallmarks of absorptive and secretor
epithelia; they are formed in all epithelia which mark a cavity (intestinal epithelium,
gland epithelium, nephrocytes, hepatocytes) and in the capillary endothelium in some
tissues (in brain capillaries) taking part to the hemato-encephalic barrier formation.
This barrier allows the glucose passage from blood to brain and the CO2 passage in
the opposite direction.
Tight junctions form an impermeable fence across cell sheets. Epithelial cell
sheets cover the surface of the body and line all its cavities. Despite extensive
biochemical differences, these cell sheets have at least one important common
function: they serve as highly selective permeability barriers, separating inner and
outer fluids that have very different chemical compositions.
Tight junctions play a crucial role in maintaining the selective-barrier function
of cell sheets. For example, the epithelial cells lining the small intestine must keep
most of the gut contents in the inner cavity (the lumen). The cells must pump selected
nutrients across the cell sheet into the extracellular fluid on the other side from which
they are absorbed into the blood. This transport depends on two different sets of
specialized membrane transport proteins: one is confined to the apical surface of the
epithelial cells (the surface facing the lumen) and pumps selected molecules inside.;
the other that is confined to the basal or lateral (so called baso-lateral surface) pumps
them out again on the other side. These pumps must not be allowed to diffuse from
the apical to the baso-lateral pole and transported molecules must be prevented from
leaking back into the lumen (Figure 19).

Figure 19. Schematic drawing of a small intestine epithelial cell showing


how tight junctions serve to confine different transport proteins to
different regions of the plasma membrane.

Tight junctions make possible the transport in two ways. First, they act as
diffusion barriers within the lipid bilayer in the plasma membrane. Thus, they prevent
the transport proteins in the apical membrane from diffusing into the baso-lateral
membrane and vice-versa. Secondly, they tie neighbouring cells together to create a
continuous sheet of cells between which even small molecules are unable to pass. In a
tight junction, the interacting plasma membranes are so closely apposed that there is
no intercellular space: if an electron-dense marker is added to one side of the cell
sheet, it will not pass beyond the tight junction. This junction can be disrupted either
by treatment with proteolytic enzymes or by agents that chelate Ca++ or Mg++, both
special proteins and divalent cations are required for maintaining their integrity. The
morphological structure of tight junctions shows a pentalamelar aspect, formed by
two peripheral electron-dense layers, two layers less electron-dense, situated under the
previous layer, and a central layer electron-dense. This structure is due to the tight
adhesion of the neighbour cell plasma membranes; the intercellular space is missing.
Because tight junctions can be very dynamic structures, they can assembly or
disassembly either partially or completely during many development and
physiological processes. These include the trans-epithelial migration of neutrophils
during inflammation, the migration of maturing spermatocytes across the
seminiferous tubule epithelium during spermatogenesis, changes in cell associations
during morphogenesis and perhaps even hormone regulation of the paracellular
pathway in ion transporting epithelia.

2.5.5.3. Communicating junctions.


Communicating junctions are of two types:
I. Gap junctions
II. Chemical synapses

I. Gap junctions are said to be communicating junctions because they allow small
water soluble molecules to pass directly from the cytoplasm of one cell to another; by
chemical synapses the cells communicate only indirectly, even though they are in
physical contact: the pre-synaptic cells secretes a chemical signal (called
neurotransmitter) that diffuses across the synaptic space and signals the post-synaptic
cell. Chemical synapses should not be confused with the less common electrical
synapses, at which electrical impulses pass directly from one nerve cell to another via
gap junctions.
The gap junction is the most common type cell junction, which is widely
distributed in tissues of all animals. This junction is build by proteins that extend out
from the plasma membrane to form structures called connexons, which are believed to
connect the two cells interiors by a continuous aqueous channel. Each of the two
interacting cells contributes enough protein to form one connexon, and these
connexon forms half the length of a channel (Figure 20).

Figure 20. Different conformations of the connexons.


The interacting plasma membrane are separated by a gap of 2 to 4 nm (thus the
term gap junction). Each gap junction can contain up to several hundred connexons.
The morphological structure of gap junctions shows a heptalamelar aspect formed by
two peripheral electronodense layers, two middle layers less electronodense two
internal layers electronodense and a space formed by the intercellular space (2-4 nm
thick) (Figure 21).
When fluorescent molecules of different sizes were injected it was found that
molecules smaller than 1000-5000 daltons could pass between cells through gap
junctions channel; larger molecules could not pass, suggesting a functional pore size
for connecting channels of about 1.5 nm. This pore size implies that coupled cells
share a variety of small molecules (such as inorganic ions, sugars, aminoacids,
nucleotides and vitamins) but do not share the macromolecules (proteins, nucleic
acids, and polysaccharides). Biochemical analyses of such preparations suggest that
the gap junctions are composed by one major protein of about 27,000 Da, called
connexine.

Figure 21. Schematic drawing of a gap junction model.

The Gap Junction Functions.


In some tissues, cell coupling via gap junctions serves an obvious function. In
principle, this type of coupling should allow metabolic cooperation between cells. It
is generally assumed that the electric and metabolic coupling detected between cells
in contact is mediated by gap junctions. For example, electrical coupling synchronises
the heart muscle cells contraction and the smooth muscle cells responsible for the
peristaltic movements of the intestine. Similarly, electrical synapses between nerve
cells permit action potentials to spread rapidly from cell to cell.
Cell coupling via gap junctions may be important in embryogenesis. In early
embryos, most cells are electrically coupled to each other. It is thought that metabolic
coupling may be an important mean of distributing nutrients in these embryos before
the blood circulatory system develops.
The permeability of gap junctions is rapidly (within seconds) and reversibly
decreased by experimental manipulations that decrease the intracellular pH or
increase the intracellular concentrations of free Ca++. That’s why gap junctions are
dynamic structures whose permeability can be controlled by the cells that form them.
If a cell dies or it is damaged, it is important that it rapidly will decouple from its
neighbours. This is thought to be achieved by a large increase in intracellular free
Ca++ - either by influx through a damaged plasma membrane or because the cell is no
longer able to pump Ca++ out of the cytosol efficiently.
CHAPTER III

THE CYTOSKELETON AND CELL MOTILITY

The earliest microscopic observations on living cells revealed that the cytoplasm
is a viscous fluid that can change from a more fluid state to one resembling to a
deformable solid. In the period among 1870 and 1885, it was widely believed
(because the appearance of the cells following fixation and staining) that the
cytoplasm contained a three dimensional network of protein fibers.
The term of cytoskeleton was introduced in 1929 by Koltzoff, who affirmed that
each cell has its own system of liquid compounds and rigid skeletons that gives its
shape. The cytoskeleton represents in fact the muscles and the bones of a cell.
The essential filaments in the cytoskeleton are extensively interconnected by a
three-dimensional network of fine threads, presumably composed by the some
proteins that can removed in detergent-treated cells. This network has been called the
microtrabecular network.
The cytoskeleton is a complex, three-dimensional network, formed by protein
filaments and microtubules, that cross in any direction inside the cell.
It has the following functions:
- it induces the cell’s shape
- it participates in cell motility
- it keeps the spatial internal organization of the cells (it maintain the
position of the organelles in a cell or it associates some metabolic enzymes)
Cytoskeleton is a very dynamic structure due to the continuous actin filaments
and microtubules polymerization and depolymerization. The actin filaments are in
equilibrium with actin monomers and the microtubules are in equilibrium with the
tubulin dimmers. The cytoskeleton contains also some accessory proteins that bind
together its different parts or bind these compounds with other cell structures. The
cytoskeleton can be seen after the treatment of a cell with fluorescent marked
antibodies oriented against the main cytoskeleton proteins. Actin filaments,
microtubules, intermediate filaments and their associated proteins are regulated by
unknown mechanisms in order to produce changes in cell shape and various cell
movements. In addition, the cytoskeleton seems to organize the cytoplasm by binding
various membrane-bounded organelles and soluble proteins. Microtubules emerging
from the cell center (also called MTOC – microtubule-organizing center) induce the
distribution of intermediate filaments and appear to be responsible to determine and
maintain the cell polarity. The organization of a cell’s cytoskeleton can be influenced
by that of its neighbors either through intercellular junctions or by the extracellular
matrix and it can be transmitted to its daughter cells when the cell divides.

3.1. The Actin Filaments.

In non-muscle cells there were described 5 different types of actin that differ in
molecular weight and aminoacid sequence. In non-muscle cells, actin form
microfilaments while in muscle cells it organizes in thin filaments. In non-muscle
cells, actin makes up 5-15% of the total cell protein mass.
Actin can exist in two forms:
Š the monomer form (the G-actin);
Š the polymerized form (the F-actin).
The globular actin is formed by a single polypeptide, with a molecular weight of
45,000 Da, a globular shape, a length of 6 nm and a width of 4 nm. The G-actin is
coupled with a protein called profilin and forms the profilactin complex and thus is
inhibited the polymerization of G-actin. The monomer form of actin has a binding site
for myosin, one for ATP and another one for Ca2+. In instead of Ca2+, Mg2+ is bound,
the profilin can detach from the actin and the latter can polymerize.
The fibrilar actin (the filamentous actin) is formed by the polymerization of G-
actin, in the presence of Ca2+ and energy brought by G-actin bounded ATP
hydrolysis. The polymerization of actin occurs in two steps:
1. At the beginning, in the phase called “lag-phase”, the polymerization
takes place at low-speed; the first 2-3 actin molecules are difficult to bind.
The lag-phase is thought to reflect the fact that the step in filament formation,
in which 2 or 3 actin molecules must come together in a specific geometric
conformation, is particularly difficult.
2. After this step, the ATP hydrolysis accelerates the actin
polymerization. The polymerization process stops when the cytoplasm
monomer concentration is at the “threshold level” (critical concentration).
The actin filament is formed by two strands of globular molecules twisted in a
helix with a length of 500 nm and a diameter of 6.5 nm. G-actin are stringed like
pearls in a necklace in both the F-actin chains.
Actin filaments are polar structures; their two ends differ. At one end prevail
the polymerization while at the other prevails the depolymerization. The polarity of
the actin filament, which is essential to its function in cell motility, is detectable in the
way it interacts with myosin. The actin filament polarity is important to realize the
actin-myosin interaction and to maintain the equilibrium between the monomer form
and the polymerized one. In non-muscle cells, actin filaments can be present
separately or in bundles; many kind of cellular extensions have a core of crossed
linked actin filaments. The actin bundles can be situated in the peripheral cytoplasm,
in the core cytoplasm and under the plasma membrane. Inside these bundles, the actin
filaments are parallel and are cross-linked by a protein called fimbrin. There are
various other actin-binding proteins in the actin bundle, for example villin.
The actin filament bundles forms some contractile rings:
a. The transient ring that appears at the cell division finish, dividing the
two daughters cells.
b. The actin filament belt from the latero-apical cell’s pole.
c. Inside the permanent cell specialization (microvillia and stereocilia).
d. Inside the cell’s temporary prolongation.
In non-muscle cells, the bundles of actin filaments have double functions:
1. Structural role
2. Dynamic role
The actin bundles in non-muscle cells are compared with the muscles. The actin
polymerization produces an increase in the cytoplasm viscosity. The actin
polymerization is influenced by actin binding drugs, cytochalasins (B, D and E) that
are metabolites excreted by molds; these drugs inhibits the addition of actin molecules
to actin filaments, leading to filament depolymerization and inhibit cell movements
based on actin-myosin interaction. The substance called phalloidin, a highly
poisonous alkaloid produced by the mushroom Amanita phalloides, inhibits the actin
depolymerization. By contrast with the cytochalasins, phalloidin stabilizes actin
filaments and inhibits their depolymerization. In muscle cells, the actin filaments form
the thin filament that interacts with myosin thick filaments in order to produce the
muscle contraction.

3.2. The Myosin Filaments.

Myosin filaments were observed for the first time in muscle cells, where they
form the “thick” filaments. In non-muscle cells myosin filaments are difficult to
evidentiate because they are transient structures.
Myosin can be extracted from almost every kind of cell in vertebrates and it is
always present there where actin bundles exist. The skeletal muscle myosin
polymerizes spontaneously in vitro and forms polymers that are much larger than
those formed by other forms of myosin. The myosin filaments are formed by the
addition of myosin molecules, associated by their tails. Myosin can be extracted from
the skeletal muscle by the treatment with concentrated salt solutions, in which myosin
is soluble. Muscle treated in this way loses only its thick filaments that are formed
only by myosin molecules. The latter have a molecular weight of about 500,000 Da
and are seen in electron micrographs as long rod-like molecules, each containing two
globular heads. 6 polypeptidic chains form the myosin molecule: two heavy chains of
200,000 Da each and two pairs of light chains of about 20,000 Da and 16,000 Da
each.
The heavy chain consist of two regions:
- a prolonged region, α-helical;
- a folded region, where they are separated.
This heavy chain segmentation is due to the myosin molecules configuration that
consists of a prolonged part named tail (with 2-coiled α-helices) and two heads which
emerge laterally from the rod-like tail. On each globular head are attached laterally
the two pairs of light chains. The tail is 134 nm length. Between the bicephalic head
and the tail is a flexible zone that allow the myosin head movement when it interacts
with the actin (figure 22).

Figure 22. The myosin molecule.

A protease called papain cuts the myosin subunit at the base of the head region
releasing an almost complete tail, called the myosin rod, and two separates myosin
heads. The myosin rod, as the intact myosin molecule, self-assembles at physiological
salt concentrations into large, ordered aggregates, which, however, lack side
extensions. The two separate heads produced by papain cleavage are each about
120,000 Da and are called myosin subfragment 1 (S1 fragments) (Figure 23).
The heads retain all the ATP-ase activity and the actin filament binding
properties of the intact myosin molecule and can be used to analyze the interaction
between actin and myosin. The two pairs of light chains form the S2 subfragment.
Myosin filaments are formed by the polymerization of myosin molecules
when the myosin tails are associated and the myosin heads project in the external part.
In muscle cells, thick filament have a bare central region where two oppositely S1
subfragments project to the outside (figure 24).

Figure 23. Limited


digestion with the
proteolytic enzyme papain
cleaves the myosin
molecule between its head
and tail, generating a
myosin rod and two S1
fragments

Figure 24. Schematic diagram of a myosin thick filament from skeletal muscle.

In non-muscle cells the myosin filaments are formed by a small number of


myosin molecules (10-20) polymerization while in muscle cells they are formed by
the polymerization of 500 myosin molecules (figure 25).

Figure 25. Phosphorylation of a light chain


of non-muscle myosin by Ca++ dependent
protein kinase causes the myosin molecules
to assemble into short bipolar aggregates.
3.2.1. The Actin-Myosin Interaction.
Accessory proteins to actin filaments:
1. Tropomyosin is a rigid, rod-shaped protein and lies in the long-pitched
grooves on either side of the actin filament, thereby stiffening the filament. It
corresponds to 6 globular actins.
2. The troponin complex is formed by three polypeptides:
- Troponin T has a binding site for the tropomyosin and is thought to be
responsible for positioning the complex.
- Troponin C binds up to 4 Ca2+ ions and is sensible to the increase in
intracellular Ca2+ concentrations. At the moment of Ca2+ binding it relieves the
inhibition of the actin-myosin interaction and it starts this interaction.
- Troponin I inhibits the interaction of actin and myosin, even in the presence of
2+
Ca (figure 26).

Figure 26. Schematic diagram of a muscle thin filament showing the position of
tropomyosin and troponin along the actin filament.

3.2.2. The Molecular Mechanism of Actin-Myosin Interaction.


a. When it intracellular Ca2+ concentrations increases, it starts the ATP
hydrolysis and ADP+P is bind to the myosin head.
b. The myosin head undergoes a change in shape accompanied by the
release of ADP+P, causing the myosin head to pull against the actin filament.
c. An ATP molecule binds to the myosin head, inducing the release of the
latter from the actin filament.
d. The bound ATP molecule is hydrolyzed and the myosin head returns to
its original conformation, after that binding the next actin monomer. Each
individual myosin head is therefore, thought to “walk” along an adjacent actin
filament. Each of the two heads on each myosin molecule is thought to cycle
independently of the other (figure 27).
Figure 27. (A) The interaction between a myosin molecule and an actin
filament.
(B) Schematic representation of the flexible hinge regions in the myosin
molecule.

3.3. The Microtubules

Microtubules are one of the two most important types of filaments of the
cytoskeleton and they participate to organize cilia, flagella, division spindle,
centrioles and the basal body, contributing to the cellular form maintenance and also
to the cellular motility; these functions are due to the tubulin-dynein system.
The microtubules morphology.
Microtubules consist by molecules of tubulin, a globular polypeptide with a
molecular weight of 50,000 Da. Tubulin is a dimmer of about 100,000 Da, each
dimmer being composed by two polypeptides, α-tubulin and β-tubulin, which have
closely related aminoacid sequences. It was described the third type of tubulin, γ-
tubulin, which is not polymerizing in the microtubules but which can play an
important role as nucleation center for the αβ dimmers. When tubulin molecules
assembles into microtubules, they first form protofilaments in which tubulin
polypeptides are aligned in rows, with β-tubulin of one dimmer joined with the α-
tubulin of the next dimmer. Usually 13 such protofilaments are arranged side-by-side
around a central core that appears to be empty on electron micrographs.
Even if a microtubule is usually composed by 13 protofilaments, there are
some variations in different species from 11 to 15 protofilaments. In some cases
protofilaments can associate in structures like doublets (in cilia or flagella) or triplets
(centriols).
The assembly of the tubulin molecules into microtubules resembles the assembly
of actin into filaments in several important features. It occurs spontaneously in vitro
and is normally accompanied by the hydrolysis of one molecule of bound nucleotide;
for tubulin polymerization, the nucleotide is rather GTP than ATP. The nucleotide
hydrolysis plays a crucial role in the kinetics of polymerization. But, in addition to the
exchangeable GTP that is hydrolysed to GDP during polymerization, each tubulin
dimmer contains one firmly bound molecule of GTP that does not participate to the
polymerization reaction and whose significance is unknown.
The resulting microtubule consequently contains one molecule of GTP and one
molecule of GDP for each tubulin dimmer.
The microtubules polymerize at high temperature (37°C) and depolymerize at
low temperature (4°C). Polymers of tubulin have a polarity due to the arrangement of
their asymmetrical subunits in a specific orientation in the polymer. The structural
polarity of the polymer is essential in cilia to induce the organized movements. It also
makes the two ends of the polymer different in a way that is important for the control
of polymer growth. It has recently been found that under certain unusual experimental
conditions, free tubulin molecules will add to the outside of the existing microtubules
to form curved protofilament sheets that in cross-sections resemble hooks. Depending
on the polarity of the microtubules, the hooks appear to be pointing either clockwise
or counterclockwise. Microtubules have been shown to extend with uniform and
identical polarity in nerve cell axons, in cilia, and from the centriole-containing region
in mitotic and interphase cells.
In microtubules, the two opposite ends grow and depolymerize at very different
rates. The growth asymmetry of the microtubules can be observed by allowing tubulin
molecules to polymerize for a short time on small fragments of ciliary axoneme and
then examining them in electron microscopy. In this way, one end of the microtubule
can be seen to elongate three times faster than the other end. The fast growing end
(defined as the (+)-end) has been shown to be the end that points away from the basal
body in a cilium and also the end to which tubulin molecules add in a growing cilium
in a cell.
The growth rate for microtubules differs according to the concentration of free
tubulin dimmers. They can assemble and disassemble rapidly in a cell, being always
in equilibrium with the microtubules. At equilibrium, tubulin molecules (as αβ
heterodimmers) are continuously adding to and are lost from both ends of the
microtubule. In the presence of GTP, however, there is a marked addition at one end
(+) and a marked loss at the other (-). Consequently, there is a continuous movement
of tubulin from one end of the microtubule to another.
The presence of (+) and (-) ends having different critical concentrations (the
critical concentration is defined as the concentration at which the rate of addition
balances the rate of dissociation) for polymerization also has important implications
for the spatial organization of filaments in cell.
Regarding the tubulin, here is evidence that the concentration of free molecules in
the cytoplasm is so low that an unattached microtubule will loose dimmers faster from
its depolymerizing (-) end than it would add the to it’s (+) end and would,
consequently disappear.
Many of the microtubule arrays in the cell are labile, and there are some whose
function depends on its lability. During interphase, microtubules radiate from the cell
center throughout the cytoplasm, but at the mitosis onset these cytoplasmic
microtubules disassemble as the microtubules in the mitotic spindle begin to form.
When mitosis finishes the process is reversed. The stability of different microtubular
structures varies considerably, the mitotic spindle being one of the most labile. This
lability can be seen in the extreme sensitivity of the spindle to various drugs that bind
to tubulin and prevent from polymerizing. From these, colchicine, an alkaloid
extracted from the meaow saffron, has the longest history, having been used
medicinally since ancient Egyptian as a treatment for gout. One molecule of
colchicine will bind tightly to one tubulin dimmer and there it prevents its
polymerization. The addition of colchicine (or of a related compound, colcemid) to a
dividing cell causes the disappearance of the mitotic spindle and blocks cells in
mitosis. For this reason, such compounds are known as antimitotic drugs.
Vinblastine and Vincristine, which also inhibit microtubule formation, have been
widely used as anticancer drugs; they are useful because the disruption of mitotic
spindle microtubules preferentially kills fast dividing cells.
The addition of a different type of drug, taxol, increases (rather than decreases)
the tubulin polymerization of tubulin: when added to the cells, it causes much of the
free tubulin to assemble into microtubules. Similarly, D2O (heavy water) causes a
shift toward tubulin polymerization and thus, for example, increases the number of
microtubules in a mitotic spindle. The additional stabilization of the microtubules in
the cell caused by either taxol or D2O is a deleterious to the function of microtubules
as their depolymerization: a cell with microtubules stabilized by either taxol or D2O is
unable to progress through its growth cycle and it stops at mitosis.

3.3.1. Microtubule associated proteins.


Microtubule associated proteins are specific molecules, bounded to α and β
tubulins, being responsible of stabilizing and cross-linking of the microtubules. The
accessory proteins can be classified into two major classes according to their
sequence:
- First group includes MAP1A and MAP1B that have a specific
aminoacid sequence that stabilizes tubulin polymer by diminishing the
electrostatic repulsion between the both negative-charged tubulin subunits.
They are big molecules, found especially in axons and dendrites of neurons
but even in other non-neuronal cells.
- Second group includes MAP2, MAP4 and Tau protein. MAP2 is
found only in dendrites of the neurons, where it forms fibrillar bridges
between microtubules and couple the microtubules to the intermediate
filaments. MAP4 is a non-neuronal protein that cooperates with microtubules
during the mitosis and in interphase. The Tau proteins appear to bind several
tubulin molecules simultaneously, there by enhancing tubulin polymerization
and the cross-linking by an 18-nm length domain. Tau proteins have a
molecular weight of 55,000 to 62,000 Da. Microtubule stabilization by Tau
protein gives a high resistance to the axons in neurons.
MAPs function.
MAPs association to the lateral parts of the microtubules affects their dynamic
during assembling and disassembling of the tubulin subunits. MAPs play also a role
to the extremities of the microtubules by controlling their length. Phosphorylation-
dephosphorylation process control MAPs, a phosphorilated MAP being incapable to
bind microtubules. The specific kinases involved in these processes are the mitogen-
activated protein-kinase and cdc-kinase that plays major roles in protein control
during cell cycle. As a conclusion, MAPs, are also targets for different signaling
ways.

3.3.2. The Cellular Center. The Centrioles.


The cellular center is present during the interphase. It is found in the cytoplasm of
the cells that divide. Usually, it is situated near the nucleus. It was considered, till
present days, that the cellular center misses in the neurons, cells that have lost the
division capacity (after neuronal death, their empty place will be occupied by the glial
cells). Recently, it was demonstrated that the center also exists in neurons, having the
microtubules organizing function (they are called neurotubules).
At the photon microscope, cellular center appears to be a sphere, more basophilic
than the cytoplasm, being called centrosome. Within the centrosome, there are one or
two basophilic and refracting corpuscles, called centrioles. A dense material that
participates to form the centrosphere (which can be observed in interphase) surrounds
the centrosome. During the mitosis, from the centrosphere arise some radial structures
that form the aster.
The centrioles are the only visible elements at the electron microscope. The radial
structures belonging to the aster’s structure represent the arising microtubules of the
division spindle. Centrioles are non-membrane structures that play an important role
in cell’s mobility. The centriols are a like cylinders of 300 to 700 nm length and 150
to 200 nm diameter. The nine-fold array of the triplets in the centriole is the same as
that in the basal body of the cilia. The microtubules of each triplet have equal sizes
and are noted with A, B, and C. The A microtubules are the innermost while the C
microtubules are externally located. Packing together 13 tubulin protofilaments makes
up the microtubule wall. The electron microscope shows that the A microtubule is
joint to the C microtubule belonging to the neighboring doublet through a fibrillar
bridge, that ensures the cylindrical structure of the centriole.
The centrioles have two extremities:
- the proximal extremity, that looks to the cellular center.;
- the distal extremity, that looks to the cell periphery.
In the middle of the distal extremity we observe an electron-opaque walled
structure, that represents the origin of 9 linear structures, with a radial geometry.
These structures join to the central structure at the A microtubules of peripheral
triplets (spoke-like).

3.3.2.1. Centrioles Associated Structures.


The centrioles associated structures are:
1. Diplosomes are couples of centrioles associated one perpendicular to
another and they lie in every cell during the interphase. The centrioles,
likewise the cell, have a centriolar cycle. The cells have a diplosome during
the G1 and the S phases of cell cycle. Each centriole will form a procentriole,
the origin of a mature centriole, at the end of S phase. During cell evolution,
the procentrioles will grow and will reach the size of a mature centriole. Each
centriole together with it’s procentriole migrates during the M phase of the
cell cycle to the cell’s opposite poles. So, after cytodieresis, each cell has one
diplosome.
2. Pericentriolar sattelites are globular proteins, bounded to the
centriol’s wall through a microtubule fragment.
3. Microtubules are attached to the centrioles through the pericentriolar
satellites and they have not direct contact with the centrioles.

3.3.2.2. The Centriole Roles


1. The centriole is the kinetic center of the cell, being the real cause of
the chromosomal movements during the mitosis, cilia and flagella movements
(the central bodies derives from the centrioles).
2. The centrioles are microtubule organizing centers. A denser
cytoplasm containing proteins and ARN molecules is found around the
centrioles. This zone is responsible for the microtubule polymerization. The
microtubule (-) extremity is embedded in the pericentriolar cytoplasm, while
(+) extremity, where the fast polymerization takes place, is free in the
cytoplasm. Centrioles determine the overall polarity of the cell. Fibroblast and
macrophage have always the centriole situated in front of the nucleus, in the
direction of their movement. Thus, centrioles appear to have a role in guiding
cell movements.

3.3.2.3. The Microtubule Roles.


1. Structural role – microtubules may be the overall organizers of the
cytoskeleton. All eukariotic cells have a distinct spatial geometry that can be
recognized by the position of their organelles and by features of their external
surfaces. While all compounds of the cytoskeleton reflect this geometry, microtubules
often seem to play and unique part in determining it. It is a common observation that
microtubules are aligned with the long axis of the cell and in many cases their
presence is essential to maintain the asymmetry cell’s shape.
a. Microtubules clearly influence the distribution of intermediate
filaments in cultured cells. For example, in cultured fibroblasts, intermediate
filaments spread out in a radial pattern quite similar to that of cytoplasm
microtubules extending from a region near the cell nucleus toward the
periphery. If the cells are treated with colchicine, the microtubule network
will depolymerize rapidly, and over the next several hours the intermediate
filament network will gradually collapse into a dense filamentous cap lying
adjacent to the nucleus. If the drug is removed, the microtubules will rapidly
repolymerize and the intermediate filaments slowly return to their normal
distribution.
b. There is some evidence that microtubules also influence the
distribution of actin filaments in the cell. The clearest example is the
contractile ring whose action completes the process of the cell division. In this
case it is vital that the activity of the actin-based contractile ring will be
coordinated with those of the microtubule-based spindle. In fact, if the
position of the forming mitotic spindle is displaced mechanically, the position
of the contractile ring subsequently forms will correspondingly change.
c. The usual microtubule function is to organize the other compounds of
the cytoplasm. The role is evident in plant cells. In mammalians, spiral rings
of microtubules become wrapped about the axoneme of a developing sperm
tail and then disappear, leaving a spiral ring of mitochondria that supplies the
axoneme with ATP.
2. Dynamic role – microtubule in cytoplasm determines cell’s polarity and
coordinates various parts of the cytoskeleton that are responsible for complex
movements in the cell (movement of cilia and flagella, chromosome movements
during mitosis).
In cultured cells, various type of local surface activity that are actin based,
such as membrane ruffling, microspike formation and phagocytosis, often appear to
occur in regions of the cells close to microtubules end; these movements can be
influenced by colchicine treatment. In the presence of colchicine, the normally
coherent cultured cell movements become disorganized; membrane ruffling now
occurs around the entire periphery of the cell, which wanders “like a ship without a
rudder” rather than moving in straight line.

3.4. The Intermediate Filaments.

Intermediate filaments (IF) forms the third compound of cell cytoskeleton. Their
name derives from their diameter (about 10 nm) that is intermediate between that of
actin microfilaments and that of microtubules. IF are found in almost all the human
cells, especially in epithelial cells and neurons. They are organized in a network that
crosses the cytoplasm and that insures the intracellular organization and the
disposition of the cells in tissue. In the following table we can see the classification
and the tissue distribution of the IF.
Intermediate filaments Tissue type
TYPE I
Acid keratins Epithelia
TYPE II
Basic keratins Epithelia
TYPE III
Desmin Muscles
Acid fibrilar glial protein Glial cells and astrocytes
Vimentin Mezenchimal cells
Peripherin Neurons
TYPE IV
NF-L Mature neurons in central and
NF-M peripheral nervous system
NF-H
Internexin
Nestin
TYPE V
A laminin In all cells
B laminin
C laminin
NF = neurofilaments
L,M,H = low, medium, high refers to the molecular weights
IF Type I and II:
Keratins are formed by complementary acid and basic intermediate filaments
that form hetero-dimmers. They are frequently found in epithelial cells, associated
with the desmosomes or to hemidesmosomes, and they form flexible networks; in this
way they play a supportive role.

IF type III contains 4 proteins: vimentin, desmin, acid glial fibrillar protein, and
peripherin.
Vimentin appears in endothelial cells, in some of the epithelial cells and in
mezenchimal cells as fibroblasts, forming fibers that end in the nucleus membrane.
They can maintain the localization of the nucleus and of other organelles.
Desmin is found especially in muscle cells, bundling myofibrils. In striated
muscle fibers, desmin filaments are circling the Z disks and connect it to the neighbor
Z disk or to the membrane aligning the disks.
Acid glial fibrilar protein forms the IF in glial cells in central nervous system
and in astrocytes.
Peripherin is less known and is found in neurons in peripheral nervous system.

IF of IV type are called neurofilaments and they fill the axons core in neurons,
where they are usually associated to the microtubules. Another protein called nestin,
found in neurons and miocytes, and is included in this class because of genic
similarity.
Neurofilaments function is to support radial growth of the axons in neurons.

IF of type V are found exclusively in the nucleus, playing a supportive role for
nuclear membrane. These IFs are composed by laminins A, B and C. Nuclear
laminins form the nuclear lamina, a filamentous network situated on the ineer part of
the nuclear membrane. Laminins play an important role in nucleus assembly.

The assembly of IF is probably irreversible. There is no evidence for a pool of


unpolymerized intermediate filament proteins in a cell, or for a dynamic equilibrium
between their soluble and polymerized shapes, as there is for actin and tubulin.

3.5. The microtrabecular network.

The microtrabecular network is made up of fibril structures, crossed in a


cytoplasm network with dense meshes whose content is represented by ions, H2O. It
represents the microscopic support of the bio-structured matter. Many associated
proteins adhere to this support. The microtrabecular lattice’s roles are to join together
the cytoskeleton elements and also to facilitate the interaction between cytoskeleton
and other structures, therefore participating to cell motility. Some of the associated
proteins are: α-actinin, vimentin, nexin, desmin, fimbrin.

3.6. The Cytoskeleton Roles.

Cytoskeleton has two major roles:


1. Structural role – the maintaining of the shape of the cell, with the
compartmentalization of the cell and also with cell shape change as against to the
cellular needs: pseudopodia emission, realized through the interaction between
actin, myosin and cell membrane or during endocytosis.
2. Dynamic role, including the following aspects:
a. The cellular motility and the movements are realized through the
interaction between cytoskeleton elements and the cell membrane.
b. The organelle’s migration in the cell is conditioned by the interaction
between them and the cytoskeleton elements. For example, membrane-
bounded organelles such as mitochondria and lysosomes often move
individually in a highly characteristic manner, known as saltatory movement.
In this way they travel in rapid bursts along straight but usually invisible
tracks, stopping briefly now and then before setting off again, often back
along the same track but sometimes in a different direction.
c. The ciclase is the phenomenon representing the molecular movement
into the cytoplasm.

3.7. The Cellular Movements.

Cellular movements are classified according to the proteic system involved in the
movement:
1. Mobility due to muscle contraction, realized through the actin-myosin
system activation.
2. Cellular mobility that modifies the relation between the cell and the
environment realized by:
a. Ameboidal movement realized by the actin-myosin system: import of
substances during the phagocytosis.
b. Flagellary movement based on the interaction between dynein and
tubulin: the spermatozoon movement.
c. Ciliary movement.
3. Cell mobility that do not modify the relation between the cell and the
environment, realized by:
- exocytosis;
- endocytosis;
- boiling movements, chromosomal movements during the cell
division.
CHAPTER IV

MACROMOLECULES AND PARTICLES TRANSPORT ACROSS THE


MEMBRANE: EXOCYTOSIS AND ENDOCYTOSIS

Macrotransfer system is a special type of transport across the membrane being


realised via vesicles. While transport proteins mediate the passage of many small
polar molecules across cell membranes, they cannot transport macromolecules as
proteins, polynucleotides or polysaccharides.
Most cells are able to eject and take in specific macromolecules across their
plasma membranes; some even are very different from those that mediate small solute
and ion transport, because they involve sequential membrane bounded-vesicles
forming and fusion.
There are three types of vesicle transport, depending on transport direction: into
the cell, out of the cell or passage through the cell.

A. The first type is called endocytosis; there are two types of endocytosis,
distinguished according to vesicle size:
I. Phagocytosis (“cell eating”), that involves the large particle ingestion such
as micro-organisms or cell debris, via large vesicles (often called vacuoles).
II. Pinocytosis (“cell drinking”), that involves fluid and/or solutes ingestion
via small vesicles.
B. The second transport type is called exocytosis.
C. The third transport type is called transcytosis. By transcytosis macromolecules are
transported through the capillary endothelium cells. Transcytosis can be done by:
„ independent vesicles that pass through endothelial cell, from the luminal
face to the interstitial one.
„ through a channel formed by fused vesicles.
Transcytosis realises the changes between plasma and interstitial fluid.
Macromolecules transport depends on particles dimension, their electric charge
and their chemical properties.

A. Endocytosis is the process where cells ingest macromolecules and liquid or solid
particles from extracellular medium. It is a kind of nutrition for cells or a defence
modality against external aggressors (intercellular space is cleaned by specialised
cells);
I. Pinocytosis (“cell drinking”) is a process that involves the ingestion of
liquid particles. Virtually all eukaryotic cells are continuously ingesting bits of their
plasma membrane as small endocytic (pinocytic) vesicles. The extracellular fluid (and
every substance that can dissolve in it) becomes trapped in the vesicles and is ingested
as well. There are two types of pinocytosis:
Fluid phase endocytosis (receptor independent pinocytosis) is visualised with the
aid of a tracer, such as an enzyme peroxidase, introduced into the extracellular fluid.
A liquid molecule binds on the external face of plasma membrane produces
interactions between plasma membrane and membrane cytoskeleton; plasma
membrane invaginates and the liquid drops are introduced in the cytoplasm, covered
by a plasma membrane fragment. The structure formed is called pinosome and has a
diameter of 50-250 nm. Once formed, the pinosome can evolve in three ways:
a. It binds a primary lysosome and forms a secondary lysosome where the
ingested molecule is disrupted in simple elements, because of lysosomal
enzymes; by this process, from greases there are obtained fatty acids, from
polypeptides result simple peptides). Simple substances pass through
secondary lysosome membrane, enter the cytoplasm and are used to synthesise
new cell structures or are used in cell metabolism.
b. The pinosome remain in cytoplasm as stock substances.
c. The pinosome content is released to the exterior by exocytosis.
The vesicle substance concentration is the same as in the extracellular fluid
concentration (it is a non-selective type of endocytosis). Cell surface area and volume
remain unchanged during this process because the membrane is added to cell surface
by exocytosis as fast as it is removed by endocytosis.
Receptor-mediated endocytosis (adsorptive endocytosis) is called also
concentrative or selective pinocytosis. It takes place in some plasma membrane
depressions that concentrate a large number of receptors for different molecules.
These depressions are coated on the inner face by specific molecules as clathrin and
adaptin that plays an important role in receptors recognition. Receptors are
recognized by these proteins and are concentrated at plasma membrane depressions
level. The depressions act as molecular filters, being permissive only for specific
molecules.
After ligand recognition and binding by the receptor, the depression (also called
caveolae) goes deep inside and then closes. As a result, it appears a clathrin-coated
vesicle. Clathrin coat has a specific structure, being an assembly of three-legged
protein complexes (or triskelion). Three clathrin molecules and three small
polypeptides compose each triskelion. Triskelions are arranged as a basket-like
network of hexagons and pentagons at coated-vesicles surface. The second major
protein of coated vesicles is adaptin. Adaptin plays an important role in clathrin-coat
binding to the membrane and in receptor concentration in caveolae. Adaptin is
recognizing a signal sequence formed by 4 aminoacid residues from the cytoplasmic
receptor tail.
Immediately after forming, the vesicle loses its clathrin coat; clathrin elements
are directed by cytoskeleton components to the inner face of the plasma membrane,
where they will coat another caveolae.
Once formed, the vesicle can follow three ways:
1. It stops in early endosomes. Early endosomes represent a membranous cavities
network with an acid interior (pH=5). This is a dynamic compartment that appears
and disappears according to cell needs. They act as a sorting compartment on the
endocytic way. Because of acid medium, receptors that are bringing ligand
molecules are changing their conformation, delivering the ligand. Endocyted
molecules (ligands) will group in a specific region of the endosome. Here are
formed some vesicles by budding processes. These vesicles contain endocyted
molecules. At the same time, receptors are accumulating in another region of the
endosome. Another budding processes take place in this region including the
receptors in vesicles; these receptor-containing vesicles will be sent back to the
membrane to fit in new caveolae – receptor-recycling way.
2. The second way is similar with the first but receptors reach the baso-lateral pole
of the cell instead the apical pole, serving for transcytosis.
3. The third way seems also the first one before the vesicles enter the early
endosome. Receptors are included in vesicles together with the ligand molecules.
In both Ist and IIIrd ways, vesicles reach the late endosome. From here, part of vesicles
is directed toward lysosomes or to Golgi complex. In lysosomes these structures will
be digested while in Golgi complex they will be used.
Early and late endosomes differs by forming time: 1 minute for early endosome
and 5-15 minutes for late endosomes. Both types interior is very acid, with a pH
around 6, maintained by a proton pump located in their membrane.
An important example is cholesterol uptake by animal cells; this process is
realised by receptor mediated pinocytosis, and it is important in order to prevent
cholesterol accumulation in the blood, with aterosclerotic plaque formation. Most of
the cholesterol is transported in the blood compacted (bounded) in low-density
lipoproteins (LDLs). These LDLs are large spherical particles (22 nm diameter) that
contain a cholesterol core of about 1500 molecules, surrounded by a lipid bilayer
containing a single protein species. When a cell needs cholesterol, it expresses LDL-
receptors on its surface.
Most receptors for LDL associates spontaneously to coated pits but there are also
receptors that bind the coated pits only after LDL-binding. All particles that bind to
LDL receptors are internalised very fast. The coated vesicles lose their coat and fuse
with early endosomes, than with late endosomes, and than with lysosomes. Thus,
within 10-15 minutes of binding to cell-surface receptors, the LDL is delivered to
lysosomes where the cholesterol esters are hydrolysed to free cholesterol and
therefore become available for new membrane synthesis in the cell. If too much free
cholesterol accumulates in the cell, the latter stops cholesterol and LDL-receptor
synthesis (Figure 28).
Concluding, LDL-receptors are following the Ist way. They dissociate by LDL in
endosomes and they are recycled while LDL passes into lysosomes.
A more complex recycle undergoes transferin receptors after transferin
endocytosis. Transferin is a plasmatic carrier-protein for Fe. It can be found as
apotransferin if it lacks Fe. Surface cell receptors bind transferin and deliver Fe to
early endosomes. Low pH in early endosomes induces transferin to deliver Fe and
apotransferin. Apotransferin remains bounded to its receptor, being recirculated to the
apical pole and then delivered at neutral pH in order to bind another Fe molecules. By
this way transferin delivers Fe to the cell, without entering the lysososmes. Transferin
receptors follow the same Ist recycling way.
The second recycling way is followed by EGF-receptors (EGF – epidermal
growth factor, a small protein that induces epidermal cell growth). EGF-receptors
accumulate in the coated pits only after EGF binding. Most of the receptors are not
recycled, being degraded into the lysosomes, together with EGF. Concluding, EGF
binding to EGF surface receptors induces a decrease in receptor number so a negative
receptor feedback.

II. Phagocytosis (“cell eating”) is the process that involves solid particle
ingestion. On membrane surface there are receptors that recognise, bind the ligand
and start the ingestion and the bacteria digestion of bacteria, parasites,
microorganisms, senescent and damaged cells and cell debris. Metchinikov described
it in 1905 in protozoa, where it represents a special type of nutrition. In metazoan,
phagocytosis is a type of feeding and of defence against the infection by
microorganisms.
According to the size of ingested material, the phagocytes are:
„ mobile microphages (polymorphonuclear leukocytes).
„ mobile macrophages (big circulant monocytes).
„ fixed macrophages (hystiocytes from lax connective tissue, adventitial cells in
vessels walls, some cell in the lung, Kupffer cells, in liver).

Figure 28. Receptor mediated pinocytosis for LDL (up) and the relation late
endosome - Golgi complex.

Phagocytosis is an energy-consummating phenomenon. The scavenging


senescent and damaged cell function is more important as quantity: in each of us,
macrophage phagocyte more than 1011 senescent red blood cells every day.
Phagocytosis steps are:
1. When a bacterium enters an organism, the first step is the opsonisation. The
surface bacteria non-self antigens are released in the digestive system and act
on the immune system, starting the specific antibody synthesis (called
opsonines) and their binding on bacteria surface (opsonisation process). Thus,
the bacterium can be recognised by macrophage surface.
2. The chemotactism: circulating macrophages came to the infection centre
when they are found by a chemical signal that can be lymphokines (secreted
by T memory lymphocytes), immunoglobulins(secreted by mature
plasmocytes – derived from B-lymphocytes). When they receive the chemical
signal, the macrophages extend psudopodia, pass through the vessel’s wall
and flow over the infection core.
3. The adhesion: on macrophage surface there are specific receptors (Fc
receptors for opsonised bacteria, C3b receptors for complement) and non-
specialised receptors (for senescent and damaged cells).
4. Engulfing: phagocytes extend some pseudopodia that engulf the particle and
fuse to form a phagocyte vacuole.
5. The digestive step and the killing of phagocyted cells are realised after the
fusing of the phagosome with a primary lysosome and like that resultsthe
secondary lysosome.
The Endocytosis Mechanism.
In endocytosis, molecules bind to the outer face of the plasma membrane
immobilises membrane proteins, determining an increased permeability for Na+ ions.
Na+ enters the cell and induces a depolarisation wave that determines Ca++ release
from endoplasmic reticulum, where it is stored. Ca++causes the retraction of
microfilaments inserted on the inner face of the membrane. During their contraction,
microfilaments induce the invagination of the plasma membrane followed by vesicle
closure (a zippering mechanism). Fragments of plasma membrane are lost during
endocytosis, because plasma membrane fragments coat the pinosome and the
phagosome.

B. Exocytosis is the vesicle transport phenomenon produced by joining the cytoplasm


vesicles with the plasma membrane; the material inside the vesicles is eliminated in
the extracellular medium. These processes appear on synapses level when the vesicles
contain chemical mediators and fuse with the neurone plasma membrane (or
presynaptic membrane). Chemical mediators are eliminated in the synaptic space and
bind to specific receptors from post-synaptic membrane producing its depolarisation.
In this way, nervous influx is transmitted at synapses level. Plasma membrane
intervenes in the excretion process and eliminates in the extracellular medium the
cell-elaborated products, like hormones, enzymes, lipoproteins, glycocalyx
components, and useless final products of cell metabolism. Normally, exocytosis is a
discontinuous phenomenon, but it can be continuous under certain conditions.
1. Discontinuous exocytosis is a controlled phenomenon and it is realised by a
discharge induced by certain stimuli. For example, secretion granules
containing insulin, produced by β-pancreatic cells are delivered into
bloodstream when the glucose level increases.
2. The continuous exocytosis is an uncontrolled phenomenon, specific to sick
cells. Plasmocytes can continuously secrete immunoglubulins, being the real
cause of some secreting tumors.
Exocytosis mechanism.
Secretion vesicles migrate from the arising place to cell periphery, thus
contacting the inner plasma membrane face. The ATP, cAMP, and Ca++ produce
microfilament contraction, determining vesicle opening. The content is eliminated in
the extracellular space. In exocytosis process, the plasma membrane remake the loses
produced during exocytosis.
CHAPTER V

THE CELL’S NUCLEUS

The cells are compartmented membrane structures. A eukaryotic cell has two
compartments:
- the first one is the nucleus which hosts the genetic memory of the cell and the
process of DNA replication and RNA synthesis by DNA transcription;
- the second one is the cytoplasm that contains the translation and transport
mechanism of RNA and the machinery that helps the cell biosynthesis.
The prokaryotes have no individual nucleus, even they have in their structure
genetic material condensed in nuclear equivalents.
The eukaryotes have in interphase an individual nucleus that is the control center
of all the cellular activities.
Eukaryotic DNA is tightly complexed with specialized proteins-histones.
The nucleus of all the cells, except nervous cells, has two functional states:
- the metabolic state (interphasic nucleus);
- the mitotic state.

5.1. The Nuclei’s Shape

The nuclei’s shape corresponds generally to the cell’s shape:


- a spherical nucleus at the spherical cell;
- an ovoid nucleus at the prismatic (columnar) cell;
- a stick-like nucleus at the spindle-like cell;
- a flattened nucleus at endothelial cell;
- a polylobate nucleus at polymorphonuclear neutrophil;
- an uncompleted segmented at basophil.

5.2. The Nuclei’s Position

Usually, the nucleus occupies inside the cell a central position that can be
modified by numerous factors:
- the accumulation of reserve substances (for example, in fat cells the nucleus
is pushed at cell periphery-eccentric nucleus);
- the accumulation of secretion granules (for example, in the cells with serous
exocrine secretion-parotid gland, exocrine pancreas, etc. – the nucleus
occupies a medio-basal position).
In the cells with mucous secretion (for example, the goblet cells from the
intestinal epithelium) the nucleus occupies a basal position.

5.3. The Nuclei’s Dimensions

The nucleus size and volume vary with the cellular type. It also depends on the
phase of the cellular cycle and its functional activity, age, etc.
The usually dimensions of the nuclei are between 3 and 25 μm. In the granular
cells from cerebellums cortex nucleus diameter doesn’t exceed 2-3 μm;
spermatozoon’s nucleus has 4 μm and that of the ovule 20-25 μm. Very big nuclei
are also found in the basophilic megakaryocytes from haematogene bone marrow.
Nucleus’ size and volume depend on cell dimensions; there is a nucleoplasmatic ratio
(NPR) which is constant for each cellular type.

Nv 1 1
NPR = = ÷
Cv - Nv 3 20
Where:
Nv – nucleus’ volume
Cv – cell’s volume
Cv-Nv – cytoplasm’ volume
When cell’s volume grows more than nucleus’ volume, the nucleoplasmatic ratio
will be remarked by a cellular division or by a growing in nucleus’ volume. In
pathological conditions, for example in neoplasic cells, NPR can be complete
modified.

5.4. The Nuclei’s Number

Usually, the cells have a single nucleus they are mononucleated cells. There are
some exceptions: adult red cells that have lost the nucleus in order to adapted to the
function of gas transport (oxygen and carbon dioxide).
They are non-nucleated cells. In some cellular types with more intense metabolic
activity there are two nuclei double-nucleated cells (for example, hepatocytes and
condrocytes). There also are cells with more nuclei multi-nucleated cells (e.g., rough
muscular fiber that contains hundreds of nuclei).
According to their forming process, multinucleated cells are classified in:
- Plasmodium cells that are formed from a mononuclear cell where take place
successive nuclear divisions (cariokinesis) without cytoplasm division
(cytodierhesis). An example is the rough muscular fibers’ osteoclasts.
- Sincitium cells are formed by the fusion of more mononucleated cells with the
disappearance of intercellular plasma membrane (e.g., the trophoblastic
sincitium on the surface of placenta villosities).
Multinucleated cells also appear in pathological cases: in tuberculosis appear
giant Langhans cells.

5.5. The Nucleus’ Molecular Structure

To study the chemical composition of nucleus is necessary its isolation by


differential ultracentrifugation. Using biochemical analysis it was established that the
nucleus contains:
1. DNA ................................................................14.00%
2. RNA ................................................................12.00%
3. Basic proteins or histones ................................22.50%
4. Acid proteins or unhistones .............................51.50%
5. Lipids, nucleotides, sugars residues, ions ......in small amounts
The DNA enters in the chromatic structures and is the stable element that stays
only in the nucleus.
The RNA enters inside the nucleolus and is a transitory element.
The basic proteins are rich in aminoacids such as: lysine, histidine and especially
arginine. The histones form complexes with DNA named histone-DNA complexes.
They have roles in DNA arrangement, mitosis starting and the activation of some
genes.
The most of the acid proteins are enzymatic proteins with roles in the
autoreplication and transcription process (DNA and RNA polymerizes,
transcriptases).

5.6. The Nucleus Morphological Structure

The nucleus is formed by four elements:


A. Nuclear cover (nuclear envelope)
B. Chromatin
C. Nucleolus
D. Nuclear juice (the carioplasm, nucleoplasm)

5.6.1. The Nuclear Cover


The nuclear cover is a complex lipo-protein structure that separates the nuclear
content from the cytoplasm in the interphasic period and that controls the exchanges
between nucleus and cytoplasm. It is formed by:
1) External membrane (outer nuclear membrane)
2) Internal membrane (inner nuclear membrane)
3) Perinuclear cistern (inter-membrane space)
4) Nuclear pores

The external nuclear membrane is a lipoprotein, three-layered structure, thick of


5÷7.5 nm. Their three-layered organization is less evident than that of plasma
membrane. On the external nuclear membrane continues with the rough endoplasmic
reticulum membrane.
The internal nuclear membrane is a lipoprotein, three-layered structure, thick of
5÷7.5 nm. On the internal face of internal nuclear membrane there is a fibrin-granular
structure, thick of 15÷60 nm, named nuclear cover.
The perinuclear cistern continues with the endoplasmic reticulum lumen and has
a thickness of 20 ÷40 nm.
The nuclear pores: at these regions the outer membrane is connected to the inner
nuclear membrane (Figure 29).

The nuclear Envelope’s Molecular Structure


By differentiated ultracentrifugation and biochemical analyses, there were
established the components of the nuclear envelope. The inner and outer nuclear
membranes have a percentage of lipids less than 30%; from these, 60-80% are
phospholipids (lecithin, phosphatidil-coline, phosphatidil ethanol-amine), 10% are
greases (cholesterol, glycerides). The protein’s percentage is about 70%, some
proteins are enzyme proteins (adenylat cyclase, acid phosphatase) and other are
receptor proteins (steroid hormones’ receptors). In the perinuclear space there are
some enzymes (peroxidases), Ca2+ ions or some metabolites.

5.6.1.1. The Nuclear Lamina


The nuclear envelope has an electron-dense layer lying on the nucleoplasmic side
of its inner membrane. This fibrous lamina (or the nuclear lamina) varies in thickness
in different cells, in some it cannot be detected at all by microscopic techniques.
There are biochemical evidences that a nuclear lamina is present in almost all-
eukaryotic cells, and it is thought to play a crucial part in organizing both the nuclear
envelope and the underlying chromatin.
Figure 29

A fibrous network that can be isolated as a membrane-free sheet containing


specialized proteins that form pores in the nuclear membrane composes the nuclear
lamina. In vertebrates, the lamina is formed by the spontaneous assembly of three
major polypeptides that are thought to bind to specific proteins embedded in the lipid
bilayer of the inner membrane. Other components associated with the lamina are to
specific sites on chromatin and thereby guide the interactions of chromatin with the
nuclear envelope.
The lamina polypeptides are probably instruments in the dissolution and
reformation of the nuclear envelope that occur during mitosis. In prometaphase, most
of these proteins are released from the nuclear membrane and become diffusely
distributed in the cytoplasm. This reversible disassembly is believed to be controlled
by a transient phosphorylation of the three lamina proteins, that in turn causes the
breakdown of the nuclear envelope observed during mitosis (Figure 30).

Figure 30

5.6.1.2. The Nuclear pores


The nuclear envelopes of all eukaryotes, from yeast to man, be perform by
nuclear pores, each one being surrounded by a large disk-like structure known as the
nuclear pore complex (~ 80 nm inner diameter). On both surfaces of the envelope
each complex is defined by:
- eight large protein granules arranged in an octagonal pattern that form the
annulus;
- eight cones that are disposed radial from the pore wall to the lumen and form
a structure named diaphragm;
- a central particle, with a granular or stick-like shape and a diameter of 15-25
nm;
- eight bundles of nucleoplasmic filaments that are inserted on the internal
annular granules and are directed toward the nucleus interior.
The pore numbers vary in large ranges (11-60 pores/μm2), function of the cellular
type. For example, the human lymphocyte has 3 pores/μm2, the amphibian oocyte has
50 pores/μm2, the rat hepatocyte has 30 pores/μm2.
The nuclear pores are dynamic elements, appearing and disappearing function of
the communication necessities (Figure 31).
Fig 31. Structure of the nuclear pore complex

5.6.1.3. The Nuclear Envelope Functions


1) The metabolic function is due to the nuclear envelope molecular structure like
that of the endoplasmic reticulum membrane. Due to these similarities, the
nuclear cover participates at metabolic processes: the fatty acid chains
elongation and de-saturation, phospholipids and cholesterol synthesis, lipids
and proteins’ glycosilation, cellular detoxification. The ribosomes from the
outer faces participate at the protein biosynthesis. The synthesized proteins
resemble to that synthesized by the rough endoplasmic reticulum’s ribosomes.
2) The permeability function
a) The transport through nuclear membranes:
The nuclear envelope is a lipoprotein complex that allows the passage of
molecules with molecular weight less than 5,000 Da in both senses.
There pass aminoacids, nucleotides, and metabolites. Through these
nuclear membranes pass also macromolecules by vesicle transport.
b) The transport through nuclear pores:
A cell synthesizing DNA needs to import about 106 histone molecules
from the cytoplasm every three minutes in order to pack the newly
produced DNA into nucleosomes; this means that there pass about 100
histone molecules per minute per pore. The nuclear export of new
ribosomal sub-units is particularly problematic. Since the diameter of
these particles is about 15 nm, they are much too large to pass through
the 9 nm channel. They are specifically transported through the pores by
an active transport system. Messenger RNA molecules, complexed with
special proteins to form ribonucleoprotein particles are selectively
exported in this way. The passage is possible due to the pore proteins’
phosphorylation that magnifies the pore diameter. The enzymes’ passage
is due to the changing of enzyme conformation, when they become
fibrillar structures.
3) The information transfer function: is realized in double sense: from nucleus
to cytoplasm and from cytoplasm to nucleus. For example, the viruses enter
into the host cell. The viruses are formed by cytoplasm and a proteinic
4) The cellular organelles’ genesis. By the burgeoning of nuclear envelope
external membranes there are formed new structures of endoplasmic
reticulum or Golgi complex.

5.7. The Chromatin – The Chromosomes in Interphase

The exam in light microscopy of the nuclei treated with basic staining shows the
existence inside the nuclei of an intense basophilic substance named chromatin. The
spaces less stained between the chromatin masses are named inter-chromatinic spaces.
The chromatin and the chromosomes are two aspects of the same genetic material
(DNA). In interphase the genetic material is represented by chromatin and during the
cellular division it is organized in the shape of chromosomes.

The Chromatin’s Aspect in Light Microscopy


The chromatin is found:
1) Dispersed into the nucleoplasm in the following shapes:
a) Granules of different sizes and shapes named chromocentres.
b) Very fine granules uniformly dispersed inside the nucleus.
c) A fibrillar structure that forms a network.
2) Condensed at nucleus’ periphery, on the internal face of the nuclear cover
where it forms the chromatic membrane.

The Euchromatin and Heterochromatin


Chromatin’s aspect and disposing are identical into the nuclei of same cell type
and represent a criterion to identify the cells. The fine granular chromatin uniformly
dispersed inside the nucleus is the euchromatin. It is a little bit stained with a basic
dye and it shows an intense metabolic activity in the cell.
From functional point of view, there are the following types of euchromatin:
1) Active euchromatin represents the chromatin fractions where takes place a
continuous transcription of the genetic material.
2) Permissive euchromatin is the part of the chromatin that is able to become
transcriptional in the presence of chemical modulator signals.
The condensed chromatin in the shape of big granules is named heterochromatin
or untranscriptional chromatin and is stained very intensively with basic staining.
From functional point of view there are two shapes of heterochromatin:
1) Constitutive heterochromatin is constant condensed in interphase; it is
inactive genetically; this means that it does not contain structural genes and
the transcription process does not take place here. It contains repetitive DNA
with short sequences of nucleotides repeated many times; it represents 15% of
the whole quantity of DNA.
2) Facultative heterochromatin is a condensed chromatin that contains
repressed (inactive) structural genes. They activate under the influence of
depressor factor and they transform in euchromatin.
The facultative heterochromatin is classified function of the type of chromosomes
formed in:
1) Autosomal facultative heterochromatin that corresponds to the condensed
parts of the 22 pairs of somatic or autosomal chromosomes.
2) Gonosomal facultative heterochromatin is a chromocentre known as sex
chromatin.
a) The Barr corpuscle lies inside the nuclei of female somatic cells and
results from the interphasic condensation of the two X sexual
chromosomes. Barr corpuscle has the shape of crescent or disk with a
diameter of 1 μm. It is found at nucleolus periphery (nervous cell) or on
the internal face of the nuclear cover (epidermis, epithelium of the
buccal mucous). In the medical practice it is often used the Barr test that
establishes the nuclear sex of the cell. This test allows the diagnosis of
some congenital genetic diseases with number or structure anomalies of
the X chromosome. The positive test at female (a single Barr corpuscle
into a cell) reflects a normal karyotype (44 + XX). There are cases
when female somatic cells’ nuclei contain 2 or 3 Barr corpuscle; this
means that they have 47 or 48 chromosomes, 3 or 4 of them being
gonosomes. This test has a practical importance because X chromosome
contains sexual and unsexual genes that in such diseases give troubles
in social adapting. When Barr corpuscle is absent, the Turner syndrome
appears (44 + XO). Mental retard, gonad dysgenic, somatic and visceral
malformation characterize it.
b) In 1970, Pearson used the fluorochrome technique to identify the
chromosomes and showed inside the male cells’ nuclei an intense
fluorescent corpuscle named F corpuscle. It represents the condensation
of Y chromosome. The presence in such a cell of two Y chromosomes
(XYY – this means two F corpuscles), indicates that these persons are
predisposed at an antisocial comportment with criminal tendencies.

The Chromatinic Fiber


At the electron microscope the chromatin appears in the shape of fibrils with a
diameter of 20 Å name chromatinic fibrils; these fibrils represent the structure units of
the interphasic chromatin and of the chromosomes in mitosis (the condensed form).

The Biochemical Structure of Chromatinic Fiber


By differential ultracentrifugation and biochemical analysis, there were obtained
the following components of the chromatinic fiber:
- DNA macromolecules ..............30%
- RNA macromolecules ................5%
- Basic proteins ...........................40%
- Acid proteins ............................25%
- Other substances in small amounts: lipids, ions (Ca, Mg), etc.
The DNA histone complex represents the basal structure of the chromatinic fiber,
the RNA synthesized at this level is a transient element that passes into the cytoplasm
where proteins’ biosynthesis takes place.
The instructions about the types of protein and the quantity of each that a cell can
synthesize come from nucleic acids. Cells have two chemically similar information-
carrying molecules: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Both
DNA and RNA in their primary structures are linear polymers composed of
monomers called nucleotides (monomer representing the single chemical unit).
Cellular RNAs range in length from less than one hundred to many thousands of
units, but the number of units in a cellular DNA molecule can exceed a hundred
million. Both DNA and RNA consist of only four different nucleotides, three of then
being similar. A nucleotide has three parts:
- a phosphate group
- a pentose (a five carbon sugars molecule), which be:
• RNA ribose
• DNA deoxyribose
- an organic base
The difference in the nucleotides of DNA and RNA is that one of the four nucleic
acid bases differs between the two polymers. The common bases are: adenine and
guanine (known as purine bases), abbreviated A and G, and cytosine (a pyrimidine
base) abbreviated with C. The fourth base (also a pyrimidine one) is different:
thymine (found only in DNA), and uracil (found only in RNA) abbreviated with T
and U, respectively.
A purine has a pair of fused rings, and a pyrimidine has only one ring. Both
purines and pyrimidines are heterocyclic, that means the rings are built of more than
one kind of atom, nitrogen in addition to carbon.
Cells and extracellular fluids contain small concentrations of building blocks
called nucleosides, combinations of a base and a sugar without a phosphate. Are also
four ribonucleosides and deoxyribonucleosides. Nucleotides, which have one, two, or
three attached phosphate groups, are also referred to as nucleoside phosphates. The
nucleoside phosphates that are used as building blocks are esterified at the 5’
hydroxyl. Nucleoside monophosphates have a single esterified phosphate,
diphosphates contain a pyrophosphate group and triphosphates have a third
phosphate. A supply of nucleoside triphosphates is necessary for the synthesis of
nucleic acids. When nucleotides polymerize to form nucleic acids, the hydroxyl group
attached to the 3’ carbon of a sugar of one nucleotide forms an ester bond to the
phosphate of another nucleotide, eliminating a molecule of water.
The links between the nucleotides are called phosphodiester bonds. Like a
polypeptide, a nucleic acid strand has a chemical orientation: the 3’ end has a free
hydroxyl group at the 3’ carbon of a sugar; the 5’ end has a free hydroxyl or
phosphate group at the 5’ carbon of a sugar. This directionality, plus the fact that (as
we shall see) synthesis proceeds 5’ to 3’, has given rise to the convention that
polynucleotide sequences are written and read in the 5’ - 3’ direction (from left to
right); for example, the sequence AUG is assumed to be (5’)AUG(3’). (Although,
strictly speaking, the letters A, G, C, T, and U stand for bases, they are also often used
in diagrams to represent the whole nucleotides containing these bases.) The
orientation of a nucleic acid strand is an extremely important property of the
molecule.

5.7.1. The DNA


The DNA macromolecules are informational molecules formed by the
polymerization of some monomers named nucleotides or deoxyribonucleotides. Such
a nucleotide is formed by 3 chemical structures:
- a monosaccharide (pentose) represented by a 2-deoxyribofuranose
- a nitrogenous base
- a phosphoric monoacid radical - PO4H
There are 4 nitrogenous bases that enter the chemical structure of DNA
macromolecules. They are classified in:
a) Purinic bases - adenine (A)
- guanine (G)
b) Pirimidinic bases - tymine (T)
- cytosine (C)
By polymerization of the nucleotide it is formed a polydeoxyribonucleotidic
chain that represents the primary or monocatenar structure of the DNA. In this chain,
the nitrogenous bases are bounded through a deoxyribofuranose and monoacid
phosphoric radical. Usually, DNA macromolecule is formed by two complementary
polynucleotidic chains, representing the secondary or bicatenar structure of DNA; the
DNA macromolecule is formed by two complementary chains. They have the aspect
of a flexible ladder twisted in its long axis. The connection between the two
complementary chains is made by hydrogen bridges between guanine and cytosine.
In the double helicoidal structure of DNA, the distance between two succesive
nucleotides is of 3.4 Å. Both chains form a complete spiral at 34 Å intervals. The
diameter of the helix is of 20 Å. DNA molecule is the biggest biological molecule. Its
mass is of 12 - 16 x 106 Da.

5.7.2. The RNA

Similar to DNA, RNA is formed by three components:


1) A monosaccharide (pentose) = ribose
2) A nitrogenous base
3) A monoacid phosphoric radical
The nitrogenous bases are grouped in:
a) Purinic bases – adenine (A), and guanine (G)
b) Pirimidinic bases – uracyl (U), and cytosine (C)
Instead of tymine (T) from DNA molecule there is uracyl (U), a nitrogenous base
with a structure similar to that of tymine.
In the protein synthesis participate 3 types of RNA:
- messenger RNA ..............................1 – 2 %
- soluble or transfer RNA ................10 – 15 %
- ribosomal RNA .............................80 – 90 %

Messenger RNA
It takes the genetic information from DNA and transits it into the cytoplasm, to
the organelles implied in protein synthesis. It is used as mould for aminoacids
assembling in a polypeptide chain. mRNA is very quickly synthesized and destroyed.
It has a single chain, complementary to one of the DNA chains where it was
synthesized. mRNA molecules are responsible for ribosomes association in
polyribosomes functional elements in protein synthesis. The fact that the smallest
proteins have a polypeptide chain formed by 100 aminoacids means that the minimal
length of mRNA chain is of 100 x 3 ribonucleotides (a codon is formed by 3
nucleotides). The molecular weight of mRNA is of 5 x 105 ÷ 40 x 105 Da.

Transfer RNA (Soluble RNA)


This type of RNA forms transitory complexes with the aminoacids and it places
the role of specific adapter for aminoacids at the level of a certain codon on mRNA,
joined by ribosomes. From the combination of tRNA with the aminoacids result
tRNA-aminoacyl complexes that arrive at the place where proteins are synthesized
(polyribosomes and mRNA). After the inclusion of the specific aminoacid into the
polypeptide chain, tRNA is delivered, being able to take and transport the same
aminoacid to the place where the protein is synthesized. Inside the nucleus there are
synthesized at least 20 types of tRNA for the 20 aminoacids that are in the cells.
tRNA molecule has 4 arms, each of them having regions with complementary
nitrogenous bases called stem (strains) and regions without complementary bases
named curls. The stems have a double helix structure that gives to tRNA molecule a
high stability.
In the structure of tRNA molecule there are 3 characteristic regions (curls)
necessary for the recognition of molecules or cellular structures with which they have
contacts:
- the region or the curl for the recognition of aminoacid;
- the curl of the anticodon: 3 nitrogenous bases complementary to a certain
codon from the chain of mRNA;
- the curl for the recognition of the ribosome.
- In comparison with the other two types of RNA, tRNA has the smallest
molecular weight, 25,000 Da.

Ribosomal RNA
It is synthesized inside the nucleus and forms two subunits:
- the small subunit (40S)leaves the nucleus together with the molecules of
mRNA; it contains rRNA 18S and 34 ribosomal proteins.
- The large subunit (60S) will combine in the cytoplasm with the small subunit
in the moment of protein biosynthesis and both the subunits will form the
ribosome; the large subunit contains rRNA 28S, rRNA 5, 8S, rRNA 5S and
45 ribosomal proteins.
5.7.3. The Basic Proteins
Generally, basic proteins have a small molecule and a basic character because of
their content in aminoacids (arginine, histidine, and lysine).
They are classified in:
1) Protamines lie especially in the nucleoproteins from fish’s spermatic cells.
They are also found in small amounts in the acids from mammalian cells.
2) Histones are basic proteins tighly bound with the DNA molecule. By
biochemical researches it was found that eukaryotic cells’ nuclei contain 5
types of histones:
- H1 very rich in lysine
- H2A, H2B moderate rich in lysine
- H3, H4 rich in arginine
These kinds of histones, especially the last 4 are found in equal amounts and have
a small molecular weight (10 ÷ 15,000 Da). The molecular weight of H1 is double. In
almost all cellular types the DNA/histone ratio is equal to the unit.

5.7.4. The Acid Proteins


The acid proteins or unhistones form a very heterogeneous group whose number
and chemical structure vary function of the cellular type; they are found in large
amounts in liver and in small amounts in thymus.
Some of them have enzymatic activity: RNA polymerase, proteinkinase,
transferase, etc. They do not have a role in the formation of chromatinic fibers. In
some tissue there are small amounts of unhistone peptides that enter the structure of
chromatinic fiber.

5.7.5. The Nucleosomes


Using special techniques it was established that the chromatinic fibers have a
pearl-collar aspects, being formed by repetitive globular units. These units are formed
by double octamers of histones (H2A, H2B, H3, and H4). They are surrounded by 1.75
whirls of DNA. This fragment of DNA contains 140 ÷ 160 nitrogenous bases. This
assembly is named nucleosome. The histone octamers core is formed by histones rich
in arginine (H3, and H4). It has the shape of a disk with diameter of 11nmn and the
height of 5.7 nm.
In the chromatinic fibers, a DNA fragment covered by H1 histone binds two
successive octamers. Histone biosynthesis is achieved in the same time with that of
DNA (the S phase of the cellular cycle). The unhistone protein does not participate at
the obtaining of the chromatinic fibers. They form nuclear enzymes: kinases,
polymerizes, transferazes that catalyze the replication and DNA transcription reaction.

5.8. The Interchromatinic Spaces

The nuclear zones between the chromatinic bundles are called interchromatinic
spaces. The ultrastructural autoradiography and cytochemical studies showed that
these spaces are occupied by heterodispersed nuclear RNA. It can have the following
aspects:
1) Perichromatic fibrils are represented by fibrillar elements with a diameter of
30-50 Å, disposed at chromatin’s periphery. From chemical point of view
RNA molecules form them.
2) Perichromatinic granules are found in all the animal cell types and appear in
the shape of granules with a diameter of 250-450 Å. They are formed by
particles of ribonucleoproteins. It is supposed that they are evacuated in the
cytoplasm through the nuclear pores in the shape of mRNA with the small
subunits of the ribosomes atatched.
3) The interchromatic granules are groups of particles with a diameter of 200
Å, formed by mRNA molecules covered with proteins.
4) The spiral bodies are spherical aggregates of 0.3-0.5 μm formed by spiral
fibrils of ribonucleoproteins whose role is unknown.

5.9. The Nucleolus


The nucleoli are corpuscular nuclear organelles responsible for the biogenesis of
the ribosomal ribonucleic acids. Nucleolus has no membrane to keep it together, but it
seems to be constructed by the specific binding of unfinished ribosome precursors to
each other by unknown means. They are found in all eukaryotic cells, only in
interphase. Nucleoli’s dimensions vary with the cellular type. Usually their diameter
is of 1-2 μm. In the cells with intense protein synthesis their dimensions are larger.
Generally, nucleoli’s shape is round oval; when the cell is older, it has an irregular
contour. The nucleoli can occupy very different position: central, paracentral or near
the nuclear cover, especially when rRNA is synthesized.

5.9.1. The Nucleolus’ Structure at the Light Microscope


The exam of living cells at the contrast phase microscope shows that the
nucleolus is a very refringent corpuscle. It has an irregular contour and a heterogenous
content. With the usual staining with hematoxylin-eosin, the nucleolus appears as a
basophilic corpuscle. It is very visible in some neurons with euchromatic nucleus.
Feulgen staining is negative in the middle of the corpuscle that contains RNA and
positive at the exterior. It stains the chromatin formed by DNA in violet red. The
silver impregnation shows two components in the nucleolus’ structure:
- nucleolema or perinucleolar chromatin at the periphery;
- pars amorpha or nucleolar body.

5.9.2. The Nucleolus’ Structure at the Electron Microscope


there are 4 components in the structure of nucleolus:
- Pars fibrosa (fibrillar component) contains fibrils with a diameter of 50 Å
disposed as a network (RNA).
- Pars granulosa (granular component) contains granules with a diameter of
200 Å, similar to those of ribosomes (RNA).

-
Nucleolus ultrastructure
- Pars cromosoma (chromosomal component) contains fibrils with a diameter
of 100 Å disposed both at nucleolus’ periphery (perinucleolar chromatin) and
in its interior (intranucleolar chromatin-DNA). This pars cromosoma contains
DNA from the nucleolar organiser region of a chromosome.
- Pars amorpha (unstructured component) is homogenous, less
electronodense.it is disposed among the first 3 components and is formed by
proteins.

5.9.3. The Nucleoli’s Classification


According to their structure, they are classified in:
1) Reticular nucleoli represent the commonest shape where the fibrillar and
granular components are disposed in a dense matrix, like a network.
2) Compact nucleoli contain very agglomerated granular and fibrillar elements.
They are found only in the nuclei of some cells, for example in lymphocytes.
3) Annular nucleoli are unusual structures where fibrillar and granular
elements are peripherally disposed.

5.9.4. The Biochemical Structure of Nucleoli


The main chemical components of the nucleolus are:
1) Nucleolar RNA is found in different types according to the sedimentation
coefficient (in Svedberg units): RNA 45S, RNA 41S, RNA 32S, RNA 20S,
etc. They correspond to different maturation stages of rRNA. The nucleolus
also temporary stores mRNA and tRNA found in their way to the cytoplasm
(7%).
2) Nucleolar DNA (3%).
3) Nucleolar proteins represent 90% and they can be:
a) Basic proteins or histones from the chromatinic fibers
b) Acid proteins or unhistones are represented by the enzymes that catalyse
the biosynthesis and maturation of the RNA reactions [the transcriptase
(RNA polymerase III-DNA dependent), ribonucleases, metilases, etc.].
Besides these major chemical components, in the nucleolus there are also found
in small amounts: Mg2+, Ca2+, Zn2+, etc.

5.9.5. The Nucleolus’ Roles


1) Role in rRNA synthesis
Inside the nucleolus there are DNA fragments used as a mould only for rRNA
synthesis (ribosomal RNA). The rRNA precursor is a 45S RNA that appears after
rRNA transcription catalyzed by transcriptase. In some biochemical reactions
catalyzed by endonucleases, convertases, methylases, 45S RNA is transformed in 41S
RNA. This is divided in 32S RNA and 20S RNA.
By methylation, 32S RNA is transformed in its mature shapes, 28S RNA, 5,8S
RNA that together with 5S RNA (molecule transcripted by an extranucleolar DNA
fragment) and with proteins form the large ribosomal subunit 40S. Both subunits
leave the nucleus and assemble in the cytoplasm on the mRNA molecule.
2) Role in mitosis’ preparation
Nucleoli’s presence in interphase is absolutely necessary for the development of a
normal mitosis. Nucleoli’s alteration with a UV microfascicle blocks the cells in G2
phase that precedes the mitosis.
3) Role in mRNA and tRNA transfer in the cytoplasm
The nucleolus is an intermediate station for mRNA and tRNA in their way to the
cytoplasm.

5.9.6. The Nucleoli’s Biogenesis


In a cellular cycle, the nucleoli are not permanent corpuscle: they are present in
interphase and disappear in mitosis. Their appearance in telophase is commanded by
some autosomes: 13, 14, 15, 21, and 22 that are called nucleolar organizers.
The nucleolar organizers are responsible for the forming of the chromosomal
component of the nucleolus (rRNA); the other 3 components: fibrillar, granular and
unstructured are synthesized in the G1 phase of the cycle when rDNA genes are
reactivated in view of the transcription.

5.10. The Genome

The DNA molecule is linear polymer containing more billions nucleotides


arranged in irregular sequences. The genetic code (the “words”) is written by three
nucleotides, usually called the codon and each DNA molecule is packed and stored in
the chromosome. For example, Escherichia coli genome contains around 4.7 x 106
base pairs (nucleotides), and the human genome contains 3 x 109 base pairs found in
24 chromosomes (24 DNA molecules). Because each human cell result from parental
cells, the human cells contain 46 chromosomes, that means 6 x 109 base pairs.
The mainly genome function is to produce RNA molecules. Thus, some DNA
nucleotide sequences are copied in complementary RNA sequences. Each DNA
region, that produces a functional RNA molecule, forms a gene. Usually, the genes
from eukaryote chromosomes would contain maximum 2 millions of base pairs.
CHAPTER VI

CELL SYNTHETIC PROCESSES AND CELL SECRETION

One of the most important phenomena that happen in the interphase is the synthesis and
cell secretion, the central place being occupied by the protein synthesis. The process takes
place in cytoplasm. The organelles involved in this function are the ribosomes, endoplasmic
reticulum, and Golgi complex.

6.1. The Ribosomes.

Ribosomes are non-membrane organelles found in the cytoplasm of all eukaryotic cells.
They play an important role in the protein biosynthesis, by placing the aminoacids according
to some fragments from messenger RNA.
The ribosomes lies generally in cell cytoplasm, free or attached to the rough endoplasmic
reticulum membrane or to outer nuclear membrane.
The number of ribosomes in a cell depends on cell type; in the neurons or secretor gland
cells, their number is higher than in other cell types. Their number is variable, according to
the functional state of the cell. In the active mammary gland secreting cells, the number of
ribosomes is higher than in repose period.

6.1.1. Morphologic Structure.


The ribosomes cannot de detected in photon microscopy. Their presence was detected by
electron microscopy, using detection methods for ribonucleic acids. They were found in
tigroid corpuscles (or Nissl corpuscles) in neurons and in Berg corpuscles in hepatocytes,
corpuscles formed by agglomerations of RER (rough endoplasmic reticulum). G.E.Palade
realized in 1956 a structural model by electron microscopy.
The ribosomes are ovoid structures, little elliptic on their large axis, with the large
diameter of 20-25 nm and the small one of 15-17 nm. It was used the negative staining
method. By differential ultracentrifugation method we can separate ribosomes from the other
cell components that are in a suspension. We take a thin copper wire and over it we put a fine
layer of weigh metal (uranyl-acetate). The layer is impermeable for electrons. On a
photographic plate, the ribosomes appear as white spots (the electrons cannot pass through the
ribosomes and do not impress the plaque).
Ribosomes are composed by two subunits:
) the small subunit Ö in eukaryotes has a 40S sedimentation coefficient (30S in
prokaryotes)
) the large subunit Ö in eukaryotes has a 60S sedimentation coefficient (50 S in
prokaryotes)
A ditch that can be crossed by the electrons and appears dark separates the subunits.
More sophisticated researches are the immune-electron-microscope studies, neutron's
diffraction, these researches giving rise to a three-dimensional image of each ribosomal
subunit.
The small subunit consists of a prominence called head (1/3 of a subunit), a platform and
a base or body (2/3 of the subunit). Between the head and the base there is a strangulation.
The large subunit has three prominences: the central one named central protuberance and
the others are the stem and the crest. Between the central protuberance and the crest there is a
depression called valley and a channel that cross all the subunit and where is located the
polypeptidic chain. The large subunit is tied from the small one only at the protein synthesis
start, in the presence of Mg++. At the end of the synthesis the subunits are separated (figure
32).

Figure 32. Ribosome


subunits and their
conformation.

6.1.2. The Molecular Structure.


By differential centrifugation there were separated the two subunits and a biochemical
analysis was done. In the chemical structure of the ribosome we distinguish: RNA ribosomal
molecules, basic proteins, acid proteins, ions (Mg++, K++, Ca++).
The small ribosomal subunit, R40S, consists of a single molecule of 18S rRNA
formed by 2000 nucleotides (the molecular weight is about 600,000 Da) and a group of 34
different proteins (these compounds raise the molecular weight of the small subunit at
900,000 Da).
The large subunit, R60S, consists of 3 molecules of rRNA (one of 28S formed by
5000 nucleotides and the other with 5.8S coefficient formed by 120 nucleotides) and 45 types
of different proteins; there is a total molecular weight of 1,600,000 Da. Some of the
associated proteins play an important role in the three-dimensional organization of the
ribosomal subunits.

6.1.3. Polyribosomes.
They are the functional structures of ribosomes that are tied at the rRNA. The number of
ribosomes involved in the protein synthesis varies according to protein dimensions that must
be synthesized. The mRNA molecule includes the molecule-organizing plan. When the
mRNA is long, the protein is complex. According to mRNA length, the number of ribosomes
organized in polyribosomes varies a lot. The mRNA molecules that code for globine synthesis
have a length of 2600 nm.
The number of ribosomes attached to mRNA is about 4-6. The number of ribosomes that
codes for collagen molecule is around 100.
The polysomes can be free in the cytoplasm, attached to the mRNA where they are
involved in protein synthesis mechanisms, or attached to the RER (rough endoplasmic
reticulum) membrane, involved in the export of synthesized proteins. In the mammary
secretor cells, there are many ribosomes attached to the RER membrane.
The ribosomes are tied to the RER membrane by two glycoproteins, called
glycophorines. There are two types of glycophorines, type I (MW = 65,000 Da) and II (MW =
63,000 Da).

6.1.4. Protein Synthesis.


The proteogenesis or the protein synthesis represents the bulk of reactions that takes place
into cytoplasm and by which, using aminoacids as raw material, proteins are synthesized.
Proteins are complex substances that individualize the living world and that represent the
material support of their specificity. Polypeptidic chains made from different aminoacids
linked by polypeptidic links form proteins.
The fundamental elements that decide protein structure and function are aminoacid
number, type and sequence.
Proteins represent 50% from the total weight of dehydrated cells, having the following
functions:
1. Structural role (they enter in the structure)
2. Catalytic role (proteins enters the enzyme structure)
3. Energetic role proteins delivers energy in cell metabolic processes
4. Sending information (chemical messengers)
5. Motility (actin, myosin, dynein, tubulin)
6. Stocking role
7. Defending role (antibody production).

6.1.5. Protein Synthesis Mechanism.


In order to induce protein synthesis there are some conditions:
1. Inside the cell there have to exist the bricks to build the proteins and these bricks are the
20 types of aminoacids.
2. Protein synthesis needs a lot of energy that is delivered by two macroergic molecules:
ATP, GTP, each of them being involved in certain steps of the process.
3. Protein synthesis needs some building plans. mRNA is formed by the transcription of
monocatenar DNA fragments. MRNA has the protein building plans in the codons, each
of them specifying a certain type of aminoacid. tRNA bring the aminoacids in the
polypeptidic chain according to mRNA codons.
4. Protein synthesis needs a key to understand the information from the building plans. The
key that decodes the information is represented by the ribosomes that move along the
mRNA molecule, reading the information from codons, permitting the aminoacyl-tRNA
complex insertion in the correct position.
Protein biosynthesis is realized by the following steps:
I. The aminoacyl-tRNA complex synthesis.
II. The polypeptidic chain initiation.
III. The polypeptidic chain extension.
IV. The polypeptidic chain terminalization.
V. The protein maturation
Stage I. The correct protein synthesis depends on the correct activation of the tRNA
molecule. A protein enzyme that is called aminoacyl-tRNA-synthase realizes this thing. The
complex is realized in two steps:
1. The aminoacyl-adenylate complex activates the aminoacid. This reaction takes
place between an aminoacid and a molecule of ATP. The reaction is catalyzed by
aminoacil-tRNA-synthase,K+, Mg++. An aminoacyl-adenylate complex is formed.

ENZYME
AA + ATP AA-AMP + P-P
++ +
Mg , K

2. The reaction between one molecule of aminoacyl-adenylate and one molecule


of tRNA gives the final complex:
ENZYME
AA-AMP + tRNA AA-tRNA + AMP

The reaction is catalyzed by aminoacyl-tRNA-synthase and there is obtained aminoacyl-


tRNA. The enzyme participates to both reactions because it has two binding ends: one for a
certain aminoacid and the other for a certain mRNA molecule. In a cell there are 20 AA
(aminoacids) and 20 tRNA, tied by the enzyme (there are 20 different types of aminoacyl-
tRNA syntase). This stage utilizes energy delivered by ATP hydrolysis.

Stage II. The stage consists in some reactions that have like purpose the first aminoacid
insertion into the polypeptidic chain and the protein biosynthesis release. It is formed an
initiation complex, mRNA-ribosome 80S-aminoacyl-tRNA. In these reactions participate
some proteins, individualized by saline extraction from the ribosomes, called initiation
factors. The initiation factors are: IF1, IF2, IF3, IF4A,B,C,D, IF5. The IF1, IF2, IF3 factors plays an
important role in polypeptidic chain initiation. IF1 participates to the binding process between
R60S and the mRNA-aminoacyl-tRNA-R40S complex. IF2 participates to the binding process
between R60S and the mRNA-R40S and aminoacyl-tRNA complex. IF3 binds R40S to
mRNA. This stage has 3 steps and uses as energy source the GTP.
1. In the nucleus are formed mRNA molecules, with their codons. Here are
formed ribosomal particles with two subunits. This step takes place in the nucleus and
R40S binds to mRNA, at a distance of 10 nucleotides from the 5' extremity, there where it
is an AUG codon (the initiation codon). The binding is realised with the intervention of
IF3.
2. The second step is the insertion of the first AA-tRNA initiation complex,
which has always the same aminoacid type named methionine. The initiation complex that
brings the first aminoacid in the polypeptidic chain is formil-methionil-tRNA. It
recognises by the azotate bases of the anticodon the azotate bases of the initiation codon
AUG. The IF2 factor participates to complex initiation and brings a GTP molecule to
supply energy. When the complex is ready, IF3 is released in the cytoplasm.
3. Finally, the large ribosomal subunit joins the complex. The IF1 factor is
essential for this process, bringing another GTP molecule for hydrolysis. Now, ribosomes
presents two sites: P-site (from peptidyl) - occupied by formil-metionil-tRNA and the
neighbour place A (from aminoacyl) - it is free and placed where it is the following
codon of mRNA molecule.
Figure 33. Initiation step in protein biosynthesis.
Stage III. Now takes place some cycle-repeated reactions, for a time. The number of
cycles depends on the number of codons on mRNA that codifies the polypeptidic chain. This
stage has also 3 steps:
1. The insertion - it consists in occupying the A place with the second AA-tRNA
complex, according to the second codon configuration. New complexes aminoacyl-tRNA
are formed (the aminoacid can be lysine) by the intervention of two free cytosolic proteins
named elongation factors (EF-Tu = transfer unstable and EF-Ts = transfer stable). They
participate to the insertion of the second aminoacyl-tRNA complex in the A place,
bringing the second aminoacid in the polypeptidic chain. The reaction needs energy
delivered by a GTP molecule hydrolysis; both places, P and A, are occupied by an
aminoacyl-tRNA complex.
2. The elongation. In this process participates an enzyme called peptidyl-
transferase that cuts the bind between methionine and the tRNA in the P place and makes
the first peptide bound between the first and the second aminoacid. The tRNA in the place
P become free and it will be delivered into cytoplasm. Now, A place is occupied by a
dipeptide (a chain of two aminoacids). EF-Tu, EF-Ts and GDP+P are also delivered from
the complex into cytoplasm.
3. The translocation. First, the tRNA molecule is moved off from the P place into
cytoplasm and the P place is free. The tRNA will be able to bind another aminoacid that
will be always methionine (because of the accepting specificity). Then, the ribosome
moves off along the mRNA molecule toward the 3' end. The translocation needs energy,
delivered by a third GTP molecule hydrolysis. The P place is occupied by a dipeptidyl and
the A place is free. After this, the reaction repeats cyclically (figure 34).

Figure 34. The elongation and terminalization steps in protein biosynthesis


Stage IV. The terminalisation and delivery of the polypeptidic chain.
It begins when the A place is located over a stop codon (terminalisation codon). There are
3 stop codons: UAA, UAG and UGA. These three codons have 3 azotate bases that are not
complementary with the azotate bases on mRNA so they do not allow aminoacyl-tRNA
addition. Instead of tRNA, the anticodons binds two releasing factors RF1 and RF2. The
peptidyl-transferase participates to the polypeptidic chain delivery cutting the links between
the last aminoacid in chain and the last tRNA by hydrolysis. The ribosome is moved off from
the mRNA and the two subunits separate. The polypeptidic chain can be delivered directly in
the cytoplasm or in the rough endoplasmic reticulum and perinuclear cistern forming the
export proteins. The polypeptidic chain undergoes maturation processes. Generally, the first
aminoacid (methionine) if moved off by breaking the peptidic bound.

6.2. The Endoplasmic Reticulum.

Introduction.
A single continuous sheet enclosing a single closed sac forms the endoplasmic reticulum.
It is highly convoluted and covered by a lipoprotein membrane.
In 1879, G. Garnier studying the gland cells, observes in their cytoplasm the presence
of an intense basophilic organelle, that he named ergastoplasm.
In 1945, Porter observes the presence of a canalicle network, delimited by a
membrane that he named endoplasmis reticulum. Electron microscope complex studies
performed by Porter (1953) and G.E.Palade (1954) show the ultrastructure and the functions
of this organelle.
The endoplasmic reticulum is present in cell types, being largely represented in
secretor cells. It has two functionally distinct regions that differ by structure and function;
they have the same membrane with a thickness of 5-6 nm. These regions are named rough
endoplasmic reticulum and smooth endoplasmic reticulum. The rough endoplasmic reticulum
is studded with ribosomes on the cytoplasmic side of the membrane; the smooth endoplasmic
reticulum lacks of ribosomes. These two regions differ considerably in shape. Rough
endoplasmic reticulum (RER) is organised in stacks or flattened sacs, called cisterns; smooth
endoplasmic reticulum (SER) consists of a meshwork of fine tubules (figure 35).

6.2.1. The Rough Endoplasmic Reticulum (RER).


The characteristic elements of the RER are fine membranes that form oriented stacks or
flattened sacs called cisterns, each having a luminal space. The outer side of the membrane is
studded with ribosomes, isolated or in active polyribosomal groups.

Figure 35. A schematic view of the


endoplasmic reticulum.
RER is visualised in photon microscope as intense basophilic corpuscles because the
presence of ribosomes. The large ribosomal subunit has a communication channel that makes
the connection with the endoplasmic reticulum lumen, where are located the polypeptidic
chains synthesised in ribosomes tied to the mRNA. It has a different disposition, depending
on cell's type. In gland cells of the exocrine pancreas, in parotid, it occupies the basal pole. In
hepatocytes, the sacs are concentrically disposed, forming the Berg bodies. The RER is
grouped in small areas in nerve cells - e.g. motoneurons in the spine marrow - forming the
Nissl bodies. The ergastoplasmic sacs occupy almost all the hyaloplasm in plasmocytes; they
have a high density and they are disposed concentrically around the nucleus. In the stem
embryonary cells, RER is found in small amounts. With the progress of the differentiation
process, the number of ergastoplasmic sac increases and the morphology become more
complex. The lumen diameter is about 50-60 nm; some canalicles can be dilated, looking like
a vesicle or a sac with a 200 nm diameter. Their content can be homogenous and few dense to
electron waves or heterogenous, with rough aspect, intense osmiophilic, depending on the
functional state. The RER membranes are continued with the nuclear extern ones and the
lumen with the perinuclear cistern.

6.2.1.1. The Smooth Endoplasmic Reticulum.


An interconnected canalicle labyrinth that communicates with the RER sacs forms it.
They have no ribosomes. It is found in all cells, but especially in:
- cells that synthesize steroid hormones: the adrenal gland, interstitial cells from
testis and ovary;
- cells that synthesize a large glycogen quantity: hepatocytes and myocytes;
- cells that synthesize pigments: melanocytes.
In the rough muscle cell, the smooth endoplasmic reticulum is named sarcoplasmic
reticulum and it plays the role of Ca++ and ATP receiver, participating to the excitation-
contraction coupling process.

6.2.1.2. Chemical Structure of the Endoplasmic Reticulum.


There have been used the differential ultracentrifugation techniques to know the chemical
structure of this organelle. In this time of the procedure, cells are torn and the endoplasmic
reticulum form vesicles, called microsomes. They are treated with ribonucleases to move off
the ribonucleoproteins and are chemical analyzed.
The chemical structure of the ER is composed by:
- 60% proteins – like structure proteins, protein-enzymes or export proteins;
- 40% lipids – like phospholipids and cholesterol.
The known enzymes are: NADH cytochrome b5, the ATPase, glucoso-6-phosphatase (the
endoplasmic reticulum marker).

6.2.1.3. Endoplasmic Reticulum Biogenesis.


The origin for ER is not well known. It seems that it comes from the outer nuclear
membrane burgeoning, or de novo, from the synthesis and assembly of the membrane
molecules. G.E. Palade described the transforming possibility of an endoplasmic reticulum
shape to another by ribosome joining or separating, by using the electronomicroscopic and
hystoradiographic studies.

6.2.1.4. Endoplasmic Reticulum Functions.


There are two types of functions:
I. RER specific functions.
a. Protein synthesis – due to the attached ribosomes – there are two types of
proteins:
- export proteins;
- membrane structure proteins.
Because of this function RER is well developed in exocrine pancreas, in hepatocytes (it
secretes albumin), in plasmocytes (it secretes immunoglobulins) and in neurons (membrane
protein biosynthesis). Proteins are synthesized in ribosomes and enter inside the lumen
through the channel from the large subunit, in the lumen, they aquire the secondary and
tertiary syructure, close to the final one, they reach the Golgi complex in 25-40 minutes and
here they suffer a maturation process.
b. The polypeptidic chain glycosylation takes place when it passes the elongation step.
Proteins synthesized in RER differ from that synthesized in cytosol because the polypeptidic
chains from RER are glycosylated.
c. The modifications of the lateral aminoacid chains are realized by the bisulphidic
bindings.
II. SER specific functions.
a. Lipid synthesis is well developed in gonads and intestinal mucus cells. In SER are
synthesized 3 glycerids that can be evidentiated like grease drops (kilomicrons).
b. The organism detoxification means some toxic drugs metabolism and their nocive
effect neutralization. Detoxification reactions are oxidation, hydrolysis, reducing or
conjugation reactions. After these reactions, the substance is transformed, being more soluble
and able to be eliminated through the membrane and after that sends by the kidney. These
reactions are very important in biliary salts, steroids, hemoglobin and drugs metabolism.
c. SER in hepatocytes modifies the hepatic glycogen in the presence of phosphorylase
and from glycogen is released glucoso-1-phosphate that is changed in glucoso-6-phosphate.
On this glucoso-6-phosphate acts glucoso-6-phosphatase, the marker enzyme of ER
membrane. In the presence of glucoso-6-phosphatase, glucose is released and sent into the
bloodstream.
II. Common functions to both endoplasmic reticulum types.
a. ER is an intracytoplasmic circulatory system and it induces a functional
compartimentation of the cytoplasm;
b. ER plays a mechanical role and supports cytoplasm;
c. ER synthesizes the phospholipids; this process take place in the endoplasmic reticulum
membrane, the active center of synthesis being through the cytosol that contains the
precursors (acetyl-coenzyme A).
d. ER is the cell membrane factory and it synthesizes structural proteins and lipids. When
these elements are synthesized the ER membrane grows and there are formed vesicles by the
burgeoning of the ER membrane. These vesicles contain portions of ER lumen and
membrane. The vesicles come at the plasma membrane or at other organelles and bring with
them the products made inside the endoplasmic reticulum. The exception is represented by
mitochondria that receive the phospholipids carried by transport proteins. Mitochondrial
proteins come inside as precursor proteins and are transformed by mitochondrion function
according to the necessities. Most proteins are synthesized by free polyribosomes and not in
ER.

6.3. The Golgi Complex.

The Golgi complex is a membranous cellular organelle formed by a heterogenous group


of membrane limited compartments, represented by a bunch of smooth cisterns. Camillo
Golgi discovered it in the cerebellum Purkinje cells in 1898.
Golgi complex ultrastructure

A. J. Dalton and M. D. Felix showed the real existence of the Golgi complex using the
electron microscope. They showed that the organelle is formed by a fine lamellar structure
and named it “Golgi complex”. It is a cellular organelle, present in all cellular types,
excepting the adult red cells and is well represented in the glandular and nervous cells.

6.3.1. The Morphologic Structure at the Photonic Microscope


Using some special techniques with OsO4, chromium, silver salts, we put in evidence the
morphologic polymorphism of this organelle; it can have various aspects: vacuoles,
anastomosed trabecules. It’s shape is variable, depending of the cellular type, but even in the
same cell it is modified, depending on its functional state. Dalton and Felix observed the
canallicular and microvesicular aspect of Golgi complex, on cells “in vivo” state.
The Golgi complex position differs according to the type and activity of the cell. In the
nervous cells, it is disposed perinuclear. In the exocrine secretion cells, Golgi complex is
situated supranuclear, between the nucleus and the apical pole, close to the active zone were is
realised the secretion products’ releasing. In the endocrine cells, it is situated between the
nucleus and the basal pole. This structure is in a continuous transformation and movement.
The structure depends on the functional state of the same cellular type. For example, in
hepatocytes, thyroid follicular cells the Golgi complex moves from a pole to another,
depending on the metabolic activity.

6.3.2. The Morphology at the Electron Microscope


Electron-microscope studies showed that this organelle is formed by two components,
both limited by a lipoprotein membrane, 6-8 nm thick.
Its structure contains:
1. Plate sacs’ groups or cisterns - they present dilatations at the extremities. More
cisterns form a dictyosome. The space between the sacs is about 6-20 nm. Each
dictyosome has two faces:
„ a forming one or an immature one - the “cis” face refereed to the nucleus or
the endoplasmic reticulum:
„ maturation one or the “trans” one - where are formed the secretions vesicles
it is reversed to the cellular membrane.
2. Microvesicles that come at the “cis” face.
3. Macrovesicles that detach from the “trans” face.

6.3.3. The Golgi Complex Functions


Cellular secretion functions:
1. The secretor granules’ formation: The compounds synthesized in E. R. are
enclosed in microvesicles and sent to the dictyosome. Here, the microvesicles bind to the
dictyosomes and their content migrates through the dilated zones of the sacs where the
Golgi complex enzymes act on the E. R. products and form the prosecretor granules. When
all the prosecretor granules are in the dilated dictyosome zone, the dictyosome interrupt in
different places and form vesicles with prosecretor granules. Inside the vesicles, the
prosecretory granules form secretor granules and the vesicles are directed through the
secretor cell’s pole. There are situations where the secretion product concentration is made
inside the vesicles where have place maturation, concentration and glycosylation reactions
(specific to the pancreas).
2. The terminal glycosylation of proteins: the secretion products synthesized and E.
R. are glycosylated at their end, in the presence of the glycosyl-transpherase and α-
manoxidase.
3. The cerebrosides and gangliosides glycosylation have place in brain and kidney
and take place in the presence of specific glycosyltranspherase.
4. The sulfatation of the endoplasmic reticulum products in the presence of
sulphotranspherases; the Golgi complex has essential role in mucopolysaccharides
secretion.
5. The secretion product concentration is realized in the Golgi sacs’ dilated zones
or in the prosecretor vesicles. The secretion products interact with some
proteinpolysaccharides complexes with opposite electrical charge and reduce the osmotic
activity. The water is eliminated and the products are concentrated.
6. The secretion products maturation (the proinsulin forms the insulin). The
products synthesized in E. R. come at the Golgi complex enclosed in vesicles. Here they
have place biochemical reactions that form mature reaction products (sulfatation,
condensation, maturation, glycosylation, segregation and packing in Golgi sacs’ portions,
when are formed macrovesicles that are accumulated at the secretor cells’ pole.

Lysosomes’ biogenesis
The lysosomal enzymes are maturated and transformed in Golgi sacs, recognized by a
receptor from the Golgi complex membrane due to their manose-6-phosphate residue. Thus,
they are segregated, packed in chlatrin coated vesicles and they come in cytoplasm and form
primary lysosomes.

Membrane traffic directing and membrane recycling


The membrane traffic supposes that from E. R. go microvesicles in Golgi complex where
form macrovesicles that are eliminated to the secretor pole. In the exocytosis process, the
plasma membrane increase its surface. In the endocytosis process, it decreases the membrane
surface and forms endocytotic vesicles that stop at the Golgi complex where are transformed
and used again. This is an energodependent process that passes only in the Golgi sacs. Thus,
Golgi complex can receive the information through hormones and modify the cell’s
metabolism (Figure 36).
Figure 36. Membrane traffic directing
and membrane releasing function of
Golgi complex.
CHAPTER VII

THE MITOCHONDRION

Mitochondria are organelles present in all plant and animal cells and it occupies a
substantial fraction of the cytoplasm of virtually all eukaryotic cells. Although they
are large enough to be seen in the light microscope and were first identified in the
XIX-th century, but the real process in elucidating their function had to wait until
1948, when a procedure was developed for isolating intact mitochondria.
Mitochondria are usually depicted as stiff, elongated cylinders with a diameter of
0,5-1μm. The size of mitochondria is different upon the kind of the cell: in
hepatocytes and nephrocytes they have a length of 3 μm and a diameter of 0,5 – 1 μm;
in muscular cells their length may be until 8 μm and in cells from exocrine pancreas
10 – 12 μm. The number of mitochondria varies with the energetic requires of the cell
– for example in liver, each cell contains 1.000 to 2.000 mitochondria and a fifth of
the total cell’s volume is occupied by this organelles; in fat cells, lymphocytes, etc.,
their number is reduced.
The observation of the living cells reveals a mobility and plasticity of the
mitochondria – rapid changes of shape and that belie their static image in electron
micrographs. As they move about in the cytoplasm, mitochondria often appear to be
associated with the microtubules of the cytoskeleton. This association may determine
the unique orientation and distribution of mitochondria in different types of cells: the
mitochondria of some cells form long moving filaments or chains, while in other
types of cells they are fixed in position near a site of unusually high ATP
consumption.
In the nephrocytes from distal contort tubes they occupy the basal labyrinth, the
delivered energy being used for the active transport of substances; in ciliary cells’
mitochondria are found near the basal corpuscle (the kinetic center of the cilium).

7.1. The Structural Organization of Mitochondria.

Any mitochondrion consists of two concentric specialized membranes that play a


crucial part in its activities. Each membrane has a thickness of 6 – 8 nm and contains
a lipid bilayer and a unique collection of proteins, and together they enclose and
define two separate mitochondrial compartments: the internal – matrix space – and a
narrower intermembrane space.

Figure 37. A schematic


view of the mitochondrion
ultrastructure.
The inner membrane forms a series of infoldings, known as cristae, in the matrix
space, that increase its total surface area. The number of cristae is threefold greater in
the mitochondrion of a cardiac muscle cell than in the mitochondrion of a liver cell,
presumably reflecting the greater demand for ATP in heart tissue. An amorphous
material, less electronodense occupies the intermembrane space. The matrix space is
occupied by a fine granular material that contains sometimes granules with a diameter
of 3–5 nm (intramitochondrial granules that represents the places where bivalent
cations are fixed, especially Ca2+ and Mg2+). It is more electronodense than the
intermembrane space (fig.38).
From 10 to 10 nm the inner membrane presents the tripartite units (F1 particles)
formed by:
- a spherical head with the diameter of 9 nm that profane in the matrix space;
- a cylindrical stalk with the length of 5 nm and the diameter 3.5 nm;
- a quadrilateral base with the side of 14 nm and thickness 4.5 – 5 nm (Figure
38).

Figure 38. General


organisation of
mitochondria.

7.2. The Molecular Structure of Mitochondria.

1) The outer membrane contains many copies of a transport protein that forms
large aqueous channels through the lipid bilayer; it is permeable to all molecules of
10.000 Da or less, including small proteins. Such molecules can enter the
intermembranespace, but most of them can not pass the impermeable inner
membrane. This means that, while the intermembrane space is chemical equivalent to
the cytosol with respect to the small molecules it contains, the composition of the
matrix space is much more highly specialized. The outer membrane contains 60%
proteins and 40% lipids (the highest percentage is occupied by cholesterol and
phospholipids). It contains two enzymes such as: monoamine-oxidase (marker
enzyme), NADH2-c-reductase.
2) The inner membrane contains a higher fraction of proteins (80% of the total
membrane weight) and 20% lipids. Cardiolipin, the lipid concentrated in the inner
membrane, is believed to reduce the permeability of the phospholipid bilayer to
protons.
Freeze-fracture studies on the inner membrane indicate that it contains many
protein-rich intramembrane particles that are laterally mobile in the membrane plane.
Some of these particles are permeases that allow otherwise impermeable molecules,
such as ADP and other phosphorylated compounds, to pass from the cytosol to the
matrix. The marker enzyme for the inner membrane is cytochrom-C-oxidase.
It contains 3 major types of proteins:
- the enzymes of the respiratory chain;
- an enzyme complex called ATP synthetase that makes ATP in the matrix;
- the Krebs cycle enzymes (succinate-dehidrogenase-marker enzyme) and
specific transport proteins that regulate the passage of metabolites inside and outside
the matrix.
Since an electrochemical gradient that drives the ATP synthetase is established
across this membrane by the respiratory chain, it is important that the membrane be
impermeable to most small ions.
3) The intermembrane space contains several enzymes like adenilakinase that
maintains the equilibrium ATP/ADP/AMP; it catalyses the reaction: ATP+ AMP=
2ADP. Other enzymes are nucleoside mono and diphosphokinase, etc.
4) The matrix contains a highly concentrated mixture of hundreds of different
enzymes, including those required for the oxidation of pyruvate and fatty acids and
for the citric acid acid cycle. It also contains several identical copies of the
mitochondria DNA genome, special mitochondria’s ribosomes, tRNAs, and various
enzymes that are required for the expression of the mitochondria’s genes (Figure 38a).

Figure 38a

7.3. The Mitochondrial Respiratory Chain.


The components of the mitochondrial electron transport chain are:
1) NADH-CoQ reductase – 85.000 Da and prosthetic groups FMN, FeS.
2) succinate-CoQ reductase – 87.000 Da and prosthetic groups FAD, FeS.
3) CoQH2-cytochrome c reductase – 280.000 Da and prosthetic groups hem b,
hem c1, FeS.
4) cytochrome c oxidase – 200.000 Da and prosthetic groups hem a, hem a3, Cu.
5) cytochrome c – 13.000 Da and prosthetic groups hem c.
Except for the succinate-CoQ reductase complex each multiprotein complex is
a site for proton transport across the inner mitochondrial membrane during electron
movement. For example, the NADH-CoQ reductase complex pumps four protons per
pair of electrons transported (or, equivalently, four protons per NADH molecule
reduced). The cytochrome c oxidase complex pumps two protons for every electron
pair transported (or, equivalently, for every two molecules of cytochrome c oxidized).
The CoQH2 – cytochrome c reductase complex pumps two protons per electron. In
this process (that is known as the Q cycle), UBIQUINONE (CoQ) plays a key role.
Current evidence suggests that 10 protons are pumped across the inner mitochondrial
membrane as one electron pair is transferred from NADH to O2. The NADH-CoQ
reductase complex, four by the Q cycle in the CoQH2 – cytochrome c reductase
complex and two by the cytochrome c oxidase complex, pumps four protons.
Ubiquinone (CoQ) is a carrier of hydrogen atoms (protons plus electrons). It can
accept either one or two electrons. Cytochromes, Cu2+ ions and FeS proteins are some
electron carriers: they accept and release a single electron at a time.
Cytochromes – electron carriers – are generally membrane proteins that contain a
hem prosthetic group similar to that in hemoglobin. The Fe in the center of the hem is
the electron transporter. The different cytochromes in the chain – a, a3, b562, b566, c
and c1 – have slightly different heme structures and oxide ligands of the Fe atom.
Therefore, each cytochrome has a different reduction potential, or tendency to accept
an electron – an important characteristic dictating unidirectional electron flow along
the chain.
When NADH is oxidized to NAD+, two electrons and one proton are released.
Electrons are passed from NADH or FADH2 to O2 along a chain of electron carriers,
most of which are prosthetic groups (such as flavine, hem, iron-sulfur clusters and
copper) bound to protein particles on the inner membrane.
Ubiquinone, also termed coenzyme Q(CoQ), is the only electron carrier that is
not a protein-bound prosthetic group.
The NADH-CoQ reductase complex carries electrons from NADH to ubiquinone.
During this part of the electron transport chain, NADH is oxidized to NAD+ and the
two released electrons move through a series of carriers until CoQ is ultimately
reduced to CoQH2.
The succinate-CoQ reductase complex transfers electrons from succinate
(released during its oxidation to fumarate) to FADH, and than to CoQ.
Coenzyme Q is the “collection point” for electrons formed by the oxidation of
NADH, FADH2 and succinate.
CoQH2-cytochrome c reductase, transfers electrons from reduced CoQH2 to the
water-soluble protein cytochrome c to obtain its reduced form.
The cytochrome c oxidase complex transfers electrons from reduced cytochrome
c to O2, the ultimate electron acceptor, to yield H2O.
Each of these four electron transport complexes is laterally mobile in the
mitochondrial membrane plane. The complexes are present in nonequal amounts: for
each NADH dehydrogenase complex, there are about three CoQH2 – cytochrome c
reductase and seven cytochrome c oxidase complexes.
Furthermore, there do not appear to be stable contacts between any two
complexes: electron transport from one complex to another occurs only by diffusion
of electron shuttles.
The NADH-CoQ reductase, CoQH2 – cytochrome c reductase, and cytochrome c
oxidase complexes are sites for pumping protons across the inner mitochondrial
membrane (Figure 39).
7.4. The Mitochondrial ATP Synthetase.

This enzyme is a large protein complex through which proton flows back down
by the electrochemical gradient into the matrix. Like a turbine, this protein complex
converts one form of energy to another, synthesizing ATP from ADP and Pi in the
mitochondrial matrix in a reaction that is coupled to the inward flow of protons.
ATP synthetase is a large complex, with a molecular weight of about a million,
composed of at least nine different polypeptide chains. The entire complex is also
known as the F0F1ATP-ase. Five of its polypeptide chains make up the spherical head
of the complex, known as the F1ATP-ase. ATP synthetase serves as a reversible-
coupling device that interconverts electrochemical-proton-gradient and chemical bond
energies. Its direction of operation depends on the net free-energy change for the
coupled processes of proton translocation across the membrane and the synthesis of
ATP from ADP and Pi. In fact, three parts form ATP synthetase:
1) A hydrophobic proteic complex named the base, situated inside the lipidic
bilayer, that has the H+ translocation system, from the matriceal side to the
cytoplasmic one.
2) A spherical complex, with 9 nm diameter, named the head. This one extends
through the mitochondrial matrix. The complex is named also F1 subunit and has
catalytic role in the reaction of ATP synthesis from ADP+Pi.
3) A proteic stalk that binds the other two parts and contains a protein that gives
olygomycine sensitivity and another protein that binds the F1 portion to the
membrane. When the H+ are transported from the matriceal face to the cytoplasmic
one, it results a pH difference and an electrochemical potential accumulation that
gives the motrice force and determines the H+ back entering the matrix. The internal
mitochondrial membrane is not permeable for H+. These ions enter through the
protonic tunnel from the ATP-synthetase. When the H+ ions pass through this tunnel
and enter inside the matrix, at the F1 level is realized the ATP synthesis from ADP+P.
When is synthesized more ATP than the cell needs, the synthesis stops. When the cell
needs again ATP, the F1 subunit begins again the ATP synthesis from ADP+P.
CHAPTER VIII

THE ORGANELLES OF CELLULAR DIGESTION

Cellular digestion is the phenomena that lead to the digestion of the molecules
penetrated inside the cytoplasm through the endocytosis of some old organelles, some
cell fragments or other structures.
In the digestion phenomena are implied two kinds of cellular organelles:
- lysosomes
- peroxisomes

8.1. The Lysosomes.

The word lysosome was introduced by Champy in 1913 and it names some
intense osmiophillic granules. Christian de Duve discovered them in 1957 by
biochemical researches. He ultracentrifugated mitochondria's suspension and he
isolated a subfraction rich in acid phosphatase, an enzyme that is found in great
amount into this “ lytic particles”. In the same period Novikoff, morphogically
identified these organelles on electronomicrographs.
The lysosomes have a spherical or oval shape, with a diameter of 0,25- 8 μm and
they are found into the cytoplasm of all eukaryote cells. They have a rich content of
hydrolazic enzymes.

8.1.1 The Ultrastructural Organization


The lysosomes are formed by:
1) Lysosomal membrane has a structure similar to other endomembranes
(lipoproteic, trilayered, with a thickness of 6-8 nm) but it is doublet on its interior face
by a glycoproteic layer that gives to the lysosome enzymatic latency. The lysosomal
membrane can be broke by:
- physical agents (UV radiation or very low temperatures);
- chemical agents (K vitamin, washing agents).
2) Lysosomal matrix or the core can have different aspects: into some of them it
appears homogenous or fine granular; into other the matrix has a very heterogeneous
aspect because of the different structures that they contain (membrane fragments,
bacteria, etc.) (Figure 40).

Figure 40. The


lysosomes in electron
microscopy

The lysosomes contain approximately 36 kinds of acid hydrolazic enzymes. They


are represented by proteases, peptidases, nucleases (DNAses and ribonucleases),
phosphatases, lipases, esterases, glucosidase, etc.
The acid phosphatase is the marker enzyme for the lysosomes. Not all the
lysosomes have a complete enzymatic equipment. There are differences in the number
and concentration of the enzymes from their structure. The delivery of the lysosomal
in the cytoplasm is achieved by the alteration of the morpho-functional structure of
the membrane by physical, chemical or metabolic agents. In pathological cases, the
breaking of the membrane and the delivering of the enzymes into the cytoplasm give
cell’s death. The lysosomes are also named “the suicide bags of the cell”. In small
amounts, the lysosomes also contain structural proteins and lipids.

8.1.3. The Classifying of the Lysosomes.


Upon their structure and content, the lysosomes are classified in:
I. Primary lysosomes or protolysosomes are organelles that did not step in the
cellular digestion yet. They are small and present large differences of their content
function of the species, cellular type, functional state, etc. For example, the primary
lysosomes of the neutrophil polymorphonuclears (mobile microphages) contain a
great amount of neuraminidases that destroy bacteria membrane; the lysosomes of the
macrophages have a content rich in acid phosphatases.
Their content rich in hydrolazic enzymes determine the role of the primary
lysosomes. From the place where they are formed (Golgi complex) they migrate
toward endocytosis vesicles or membrane rests, fusing with them.
II. The secondary lysosomes result from the fusion of primary lysosomes with
vacuoles with different materials. They are big structures. According to the nature of
their content, the secondary lysosomes are classified in:
1) Heterolysosomes are secondary lysosomes that result from the fusion of
primary lysosomes with fagosomes or pinocytosis vacuoles that contain materials
from the extracellular medium. From the fusion of these two structures result a single
vacuole – the secondary lysosome – whose content is represented by the endocytosis
material and the lysosomal enzymes. The hydrolytic enzymes will digest that
material, process named heterophagy. In this way the substances are transformed in
small and simple molecules of glucids, lipids and proteins. These molecules pass
through the lysosomal membrane and are used into the cell to build structural
elements, secretion granules or hyaloplasm.
2) Autolysosomes or cytolysosomes are vacuoles where the proper structures of
the cell that finished their activity and are worn-out (rests of mitochondria,
cytomembranes, cell parts) are digested. The membranes of the smooth endoplasmic
reticulum form the membrane of the autophagic, vacuole. The substances resulted
from this digestion process pass through the membrane of the secondary lysosome
and will be used in the building of new structures. The autophagy is a general
phenomenon where the cell destroys its proper old organelle without the fewer of
chemical components.
3) The crinolysosomes result from the fusion of exo- or endocrine secretion
granules with a primary lysosome. It is a special kind of autophagy named
crinophagy. The substances resulted from crinophagy will be used in a new secretory
cycle.
4) The multivezicular bodies are vacuoles where the digestion process is finished
and which do not contain anymore-undigested substances. In electron microscopy the
multivezicular bodies appear as big vacuoles (0,2-0,5μm diameters) that contain
microvesicles. It is supposed that as the digested substances pass in the cytoplasm, the
secondary lysosome diminishes its volume and its membrane is invaginated as in
pinocytosis and form microvesicles that will be enzymatic digested (Figure 42).
5) The residual bodies or telolysosomes are heterolysosomes or autolysosomes
where the digestion was finished but which contain substances that could not be
digested by lysosomal enzymes. The residual bodies can contain:
- myelin figures – that represent degradation products of the phospholipids;
- lipofuscin granules – brown pigments;
- biliary pigments – hemosiderine granules;
- injected foreign substances – ferritine.
The undigested materials from the residual bodies are delivered outside the cell
through exocytosis. In different kinds, for example in neurones, the residual bodies
are accumulated, forming the lipofuscin granules.

Figure 41. Different


lysosome types, in
evolution.

8.1.4. The Lysosomes Biogenesis.


Lysosomal enzymes’ synthesis is achieved at the level of the ribosomes attached
on the membranes of the endoplasmic reticulum. From this place they are transferred
into the functional units of the Golgi complex where the maturation process and the
packing up into membranes take place.
The way of the substances that will become lysosomal enzymes is known as
“Golgi associated endoplasmic reticulum where from lysosomes arise”(GERL).

8.1.5. The Lysosomotrop Treatment.


Christian de Duve presented the lysosomotrop treatment in 1979 at the first
symposium in the Institute of Normal and Pathological Cellular Biology – Bucharest.
It is based on the phenomenon of accumulation into the lysosomes of some specific
medicinal substances, introduced into the cell by endocytosis.
This kind of endocytosis has as a first stage the binding of the specific drug to the
target cell by a carrier that can be endocytated. The simple specific drug cannot be
endocytated by the cell.
The drug-carrier complex is endocytated because the membrane of the target cell
has receptors for the carrier. The endocytosis vesicle that contains the drug-carrier
complex fuse with the primary lysosome, forming a heterolysosome. The lysosomal
enzymes digest the carrier and the drug delivered from the complex will pass through
the lysosomal membrane into the cytoplasm and will act on ill organelles.
At the same symposium, Christian de Duve exposed a method for the
lysosomotrop treatment in some neoplasia. There are associated DNA and
adriamicine (an antimitotic). These complexes arrive into the lysosomes where DNA
(the carrier) is digested by the enzymatic content and the adriamicine pass into the
cytoplasm where it acts as antimitotic. This method gave good results on animals and
the use of this complex avoided the toxic effects of adriamicine.
In some congenital genetic diseases named tezaurismoses, characterised by the
lack of lysosomal enzymes, these are introduced into the blood flow in a lipid cover,
complex named liposome. These are endocytated by the target cells, completing the
enzymatic equipment of the lysosomes.
The lack of an enzyme may determine the accumulation of undigested
polysaccharides or lipids that occupy the cell and compromise its function. The most
frequent affected organs are the brain, the liver and the spleen. Some examples of
such congenital diseases with enzymatic lack are the following:
- the Niemann-Pick disease characterized by a sphingomielinase absence;
- the Gaucher disease characterized by a glycocerebrosidase lack;
- the Pompe disease characterized by the absence of glycogenolytic enzymes.

8.2. The Peroxisomes

The peroxisomes (microbodies) are constant cellular organelles characterized by


a rich content in peroxidases that catalyze the forming reaction (the oxidase) and the
decomposing reaction (the catalase) of the oxygenated water (hydrogen peroxide) that
is a toxic element for the cell.
They were discovered with the electron microscope in the contort renal tubes by
Rhodin in 1954. The subsequent studies of Christian de Duve showed the
peroxisomes’chemical structure rich in enzymes. K.R.Porter and J.B.Caulneld in 1958
and H.H.Mollenhauer in 1966 showed the peroximes’presence in the vegetal cells.
Function of their dimensions, the peroxisomes are classified in:
- proper peroxisomes with a diameter of 0,5-1,5μm;
- microperoxisomes with a diameter of 0,2-0,4μm.
The peroxisomes’ number varies function of the cellular type and functional state.
For example, in hepatocytes, the number of peroxisomes may be greater than the
number of lysosomes (Figure 42).

Figure 42. Electron-microscopy


diagram of the peroxisomes.

8.2.1. The Peroxisomes’ Ultrastructure.


The proper peroxisomes are formed by constant structural elements as the
membrane and peroxisomal matrix and inconstant elements represented by the
nucleoid and the border plate.
1) The membrane that delimits at the periphery the peroxisomes’ content
has a structure similar to other cytomembranes: it is a trilayered structure formed
by proteins and lipids assembled in a mosaic model. In some cell types the
peroxisomes’ membrane continues with the membrane of the smooth endoplasmic
reticulum.
2) The matrix is a homogenous, fine granular, more electronodense than
lysosome’s matrix. In some cellular kinds the matrix can contain ramified
microfilaments with a diameter of 4-4.5 nm.
3) The nucleoid is found in all vertebrates except birds and human. It is
an electronodense structure, formed by microtubes with a diameter of 4,5 nm,
honey comb-like disposed.
4) The border plate is linear and occupies peroxisomes’ periphery. The
internal face of the limiting membrane through a narrow electronolight space
separates it. It exists into all peroxisomes in mammalian. The border plate has a
thickness larger than peroxisome’s membrane (8.5 nm) and is very electronodense
(Figure 43).

Figure 43. Peroxisomes


ultrastructure with nucleoid and
border plate.

8.2.2. The Microperoxisomes’ Ultrastructure.


The word microperoxisome was first introduced by Novikoff to define some
particules into the enterocytes of Guinea pig and rat. These particles have an intense
peroxidasic activity.
1) Microperoxisomes’ membrane has the same characters as other
cytomembranes. It continues with the membranes of the endoplasmic reticulum.
2) The matrix is homogeneous or heterogeneous and doesn’the contain
nucleoid or border plate.
The microperoxisomes are found in almost all cellular types. Their number is
greater into fibroblast, endothelium, eosinophyl granulocytes and into the cells that
synthesize steroid hormones.
The peroxisomes and microperoxisomes differ one from another in some
morphological characters like size, the absence or presence of the nucleoid or border
plate, etc. These two kinds of peroxisomes have a common character – the presence of
peroxidasic enzymes.

8.2.3. The Chemical Structure of Peroxisomes.


The use of differential ultracentrifugation and biochemical analysis showed that
the peroxisomes are formed by: glucids, lipids, structure proteins and enzyme
proteins. The catalase is the marker enzyme for peroxisomes and it represents 30% of
the total enzymatic equipment. Are also found the uricase or uratoxidase, d-aminoaci-
doxidase, the enzymes of the glixylate cycle (isocytrate-lyase and malat-synthetase).
8.2.4. The Peroxisome’s Biogenesis.
The peroxisomes may be born from expansions of the smooth endoplasmic
reticulum, subsequent separated or from Golgi complex.
In the embryonic period they are formed through cytodifferentiation from the
rough endoplasmic reticulum (the peroxisomes with nucleoid from rat hepatocytes).
8.2.5. The Peroxisomes’ Functions
1) The respiratory function is achieved by some oxidases that use molecular
oxygen for the direct oxidation of some substratum producing hydrogen peroxide
(H2O2). It is also used the catalase that decomposes the hydrogen peroxide.
2) The glucose catabolism adjustment function is achieved by the peroxisomal
enzyme α-hydroxiacid-oxidase that catalyses the glycocolat oxidation in glyoxilate
and the lactate oxidation in pyruvate. α-hydroxiacid-oxidase and D-aminoacidoxidase
or aminotransferase has a role in glycogenesis (glucose synthesis from unglucydic
substances). These enzymes and especially the last one make the irreversible transfer
of the amino groups from leucine, phenylalanine or other aminoacids. There are
formed the α-ketoacids that represent the substratum for glyconeogenesis.
3) The uric acid decomposing reaction is achieved by the uratoxidase (uricase)
enzyme that is found only in nucleoid, and it catalyses the degradation reaction of the
uric acid (catabolism product of purines) in the reaction:
Uric acid = Alantoine + CO2 + H2O
The human beings excrete uric acid as a final product of the purinic metabolism
because they do not have uratoxidase and are predisposed at gout.
4) The thermogenesis and the producing of a small amount of energy function are
achieved by the peroxisomal oxidations.
5) The lipid metabolism function is achieved by the β-oxidation of the fatty acids,
similar to those that takes place in mitochondria. The peroxisomal β-oxidation
produces acthyl-CoA necessary for cholesterol and biliar acids’ synthesis in liver
cells.

8.2.6. Pathology Implications.


Similar to lysosomes, the peroxisomes are implied in 2 congenital diseases:
1) The acathalasemy is characterized by cathalase absence from peroxisomes.
2) The brain-liver-kidney syndrome (Zellweger) is a rare congenital disease
where the numbers of peroxisomes from hepatocytes and proximal contort tube is
very reduced or they lack, although the activity of the liver catalase is normal. The
mitochondria from brain and liver have ultrastructural and functional defects. The
smooth endoplasmic reticulum is found in a reduced proportion and in the hepatocytes
the glycogen is found in excess.
CHAPTER IX

THE CELL CYCLE

All beings have a life cycle where take place growing and differentiation
processes, having as final aim the life conserving.
The cells that represent structural and functional units of the organism have their
own cycle called cell cycle.
Cell cycle means the assembly of biochemical, morphological and functional
changes that take place in a cell from its appearance by mother-cell division to its own
division into daughter cells.
Cell cycle has two steps: the interphase and the cell division (Figure 44).
As it is known, the cell cycle can be divided into four stages: G1 (extracellular
environment control, cellular growth before DNA replication), S (DNA synthesis), G2
(the cells verifies if the DNA replication is completed and prepares for division)
(Figure 44 a).
In fact, all the signals that regulates the cell cycle converge to the nucleus-placed
control horologe which is based on a group of interacting proteins capable to trigger
or stop cell division, thus co-ordinating the mechanisms leading to cell division as the
"cell executive manager " (R.Weinberg-1996).
The control horologe of the cell cycle uses a large number of proteic molecules in
order to insure this succession of stages: e.g. cyclins (first D, then E, A and B1),
cyclin-dependent kinases (CDK) playing a major role in controlling the shift from one
stage of cell cycle to the next .
There are several molecular systems that control the activity of different CDK:
the one for synthesis, control and cyclins regulating subunits destruction (R.
King,J.Petors, M.Kirschner-1994), post-translational changes of the kinase subunit
alone by highly specific kinases and phosphatases (T.Coleman,W.D.-1994) as well as
binding/ unbinding to a large variety of inhibitory proteins capable of inhibiting most
of CDK types; among these types there are: p21 (active during all stages of cell cycle;
its expression it is adjusted by p53 protein that is encoded by a tumour suppresser
gene; p53 controls cell 's health. DNA integrity and the proper succession of cell cycle
stages; in the cells do not posses p53, p21 is very reduced and is not able to associate
with CDK),p27 (also called IcK, kip- 1 and Pic- 2 -K.Polyak, M.Lee, A.Koff,
J.Roberts et al.- 1994) and p57; other inhibitory proteins are specific to CDK- 4 and
CDK- 6 e.g. p16, p15, p18 and p19/p20 (also named InK 4D and InK 6B- H.Hirai,
M.Roussel, J.Cato et al.- 1995) .
These inhibitors regulate the cell cycle by inhibiting CDK4- cyclin D1, CDK2-
cyclin E and CDH 2- cyclin A complexes. The importance of these molecules in cell
cycle control and carcinogenesis is proved by the fact that, directly or indirectly, they
are the targets for mutations as it happens in a large number of human malignant
tumors and tumoral cell lines.
Figure 44. The cell cycle.

In the last years, studies linking oncogenesis and the cell cycle found proves
indicating that a misfunction of the cell cycle control horologe induces both
uncontrolled cellular proliferation and genetic instability, which are characteristics of
the tumor cells (T, Hunter and J. Pines - 1994).
The end of stage G1 is crucial for the normal progress of a cell cycle since this is
the moment when the cell decides whether the cell cycle continues or not. Also, the
misfunction of the control horologe of the cell cycle during stage G1 is one of the
most frequent molecular anomalies seen in malignant tumors. When this phase is
concluded and the cell enters the stage S, a “molecular switch” must be turned from
“off” to “on” (Figure 44b).
The way this switch functions is: as the D and E cyclins concentration increases,
they associates with CDK and, as a result they become active. Thus activated, the
cyclin-kinases complexes initiate Rb protein phosphorylation, the main inhibitor of
cell cycle control horologe, and it remains hyperphosphorylated until mithosis is
initiated when it is dephosphorylated. During the first phases of stage G1, Rb protein
is associated to the transcription factors from E2F family, thus inhibiting the
transcription of the genes implicated in DNA synthesis. After phosphorylation, the Rb
protein releases E2F factors, allowing the cell to enter the stage S. If Rb protein is not
phosphorylated at the end of stage G1, the cell cycle is blocked by maintaining the
"switch" in the position "off ".

9.1. The Interphase.


The interphase occupies the largest part of the cell cycle (approximately 22 hours
in mammalian). It lasts from the end of a division to the beginning of the next division
(the period between two successive divisions).
The name interphase was given because it was thought that in this period the
cell is inactive. Accordingly, in this period, there are not registered morphological
changes; the nucleus is in the metabolic phase, the most evident structures being the
chromatin and the nucleoli. The chromatin that contains DNA is stained in green and
the nucleoli that contain RNA are stained in red (by methyl-pyronine green method).
Not even in the cytoplasm, changes were observed.
By biochemical point of view, the interphase is the period characterised by an
intense metabolic activity: DNA autoreplication and transcription, protein
biosynthesis, all of these reactions preparing the cell division.
The interphase has three successive steps, known as G1, S, and G2.
G1 phase (pre-synthetic stage) is the period that follows to one division and lasts
approximately 5-10 hours. In this stage, the cytoplasm mass grows because of the
intense processes of cell biosynthesis. These are determined by DNA transcription in
RNA that transmits the genetic information from nucleus to cytoplasm where
synthetic reactions take place.
S phase (DNA synthesis) has a constant time of 6-8 hours. The passing of a cell
from G1 to S phase depends on the accumulation into the cytoplasm of a protein that
at a certain concentration starts DNA autoreplication. In this period, DNA amount is
doubled; it is assured the qualitative and the quantitative maintaining of DNA. The
new formed molecules have the same structure and genetic characteristics like
mother-molecules. At the same time with DNA autoreplication the histones are
synthesised RNA and protein synthesis are continued.
G2 phase (post-synthetic stage) is short (approximately 4 hours) and begins at the
moment when DNA autoreplication stops. In this period, RNA and protein synthesis
continue. At the end of G2 phase there are all the materials for the next division. All
cytoplasmic structures and ultrastructures are multiplied so they can be equally shared
between the two daughter-cells. The passage from G2 phase to that of multiplying (M)
is conditioned by the phosphorylation of H1 basic protein that condenses the
chromatin fibres in chromosomes. The action of different physical (UV rays) or
chemical (colchicine) factors may stop the cell in a certain stage of the cell cycle.
Some authors introduce the rest stage (or G0) in the interphase.

9.2. Cell Division.


All cells, excepting the red blood cells and neurones are able to multiply; it
means that they form two daughter cells that in almost all cases have the same
morphological and functional characters as the mother cell.
The first observation on cell division were obtained in 1841 by Remack on blood
bird cells. The accumulation of observations on vegetal and animal cell multiplying,
determined Virchow in 1850 to affirm that omnis cellula e cellula. Flemming, in
1882, and Van Beneden, in 1850 observed the presence in animal cells in division of
some intense basophilic corpuscles. They were named chromosomes in 1888 by
Waldayer.
Cell division is achieved by:
1. Mitosis, indirect division or cariokinesis that have two aspects:
a. somatic mitosis or equational mitosis.
b. reductional mitosis or reductional mitosis that takes place in germ cells in their
maturation process.
During the indirect division, morphologic changes appear, involving:
„ all nuclear elements (cariokinesis)
„ all cytoplasmic elements (cytokinesis)
2. Amitosis or direct division.

9.2.1. The Somatic or Equational Mitosis.


During this type of cell division are multiplied most of the cells in pluricellular
organisms (all the somatic cells). It is characterised by:
• the spiralling of the chromosomes and their separation in two halves so that
each daughter cell will have the same number of chromosomes as the mother cell;
• the appearance in the cytoplasm of the division spindle (microtubes that are
formed by tubulin dimmers polymerisation) by which the chromosomes are linked to
the centrioles, structures that direct their movement.
The somatic division has 4 successive stages and lasts in 1-1.5 hours.
The prophase is the longest stage, lasting 25-35 minutes and is characterised
by the following morphological changes:
• in the nucleus appear small chromatin granules that will form the filamentous
or stick-like chromosomes; they approach the nuclear cover, leaving a wide centro-
nuclear space. The nucleoli become less visible and the nuclear membrane is
fragmented.
• in the cytoplasm, each centriole of the diplosome forms a procentriole; each
centriole together with its procentriole moves away one from another and between
them appear the microtubes of the division spindle.
The prometaphase is an intermediate stage between prophase and metaphase
where cell membrane and nucleoli disappear as structures. The chromosomes become
more evident through the light spiralling of the chromatides; the centromeres, of
which position is constant, become visible. They occupy nucleus place in which they
will move because of its fluidity.
The metaphase together with the prophase lasts 25-35 minutes and is
characterised by chromosome organisation in the equatorial plan of the cell, forming
the equatorial plaque. Each chromosome has near its centromere a more dense
granule called kinetochore. The kinetochore fixes the microtubes of the division
spindle. The chromosomes have U or V shape; the centromeres are located inside,
forming the equatorial plaque and the chromatids are directed toward cell periphery.
The centrioles are directed toward the opposite poles of the cell. At the metaphase
end, on each chromosome appears a longitudinal cleavage determining the appearance
of a double number of chromosomes (92 chromosomes); each of them contains a
genetic equipment, identical to that of the interphasic chromosomes (chromatin
fibers).
The anaphase lasts 5-8 minutes and is characterised by the simultaneous
migration of the son-chromosomes toward the opposite cell poles with centrioles and
microtubes of the division spindle intervention. In the second phase of the anaphase,
in the cytoplasm zone that separates the two chromosome groups we find the
microtubes of the division spindle called interzonal microtubes.
During metaphase and anaphase, on cell surface we can observe boiling
movements that are a consequence of the intense physico-chemical changes that take
place in cell mitosis.
The telophase lasts approximately 20 minutes and begins with the two groups of
chromosomes arrival at the opposite cell poles. They form a compact, hyperchromatic
mass (despiralling). The nuclear cover appears and it separates chromatin by
cytoplasm. The nucleoli appear from nucleolar organisers located in chromosomes. In
the equatorial segment of the cell appear a strangulation that progresses to form two
daughter cells. During cytodieresis, the organelles are equally shared in the two
daughter cells. The surface movements diminish and then disappear. With these
phenomena the telophase is considered finished. It marks the end of a cell cycle and
the beginning of another cycle (figure 45).

Figure 45. Phases of


the mitosis.

The mitosis can be influenced or stopped by the action of some antimitotic


factors. They can be represented by chemical substances (adriamicine, colchicine) or
physical factors (X-rays). Somatic mitosis gives birth to two cells that keep the
number of chromosomes of the mother cell (they are diploid cells - 46 chromosomes).
Depending of the degree of morpho-functional similarity between the mother
cell and the two daughter cells, somatic mitosis can be classified as follows:
1. Homotypic where there are no differences between the mother cell and the two
daughter cells; this form is characteristic for the very young not-differentiated cells.
2. Heterotypical where the two daughter cells resemble, but are different from
the mother cell.
3. Homo-heteroplastic or asymmetrical: the two daughter cells are different one
from another; one of them is identical with the mother cell while the other differs.
4. Differential mitosis or rejuvenation mitosis: cell that divides form cells less
differentiated than itself. It is observed during blast transforming of the lymphocytes.
9.2.2. Reductional Mitosis or Meiosis.
The meiosis is a special kind of mitosis that interests only the germinal cells
during their evolution toward the mature forms, capable of fecundation. The primitive
germinal cells (spermatogonia and ovogonia) in their evolution toward mature forms
(spermatogenesis and ovogenesis) suffer more divisions that give morphological and
functional changes. The result of this transforming is double:
1. It reduces the chromosome number in the mature forms at a half in comparison
with the number of chromosomes from primitive germ cells (from 46 they arrive to 23
chromosomes). Reductional division or meiosis achieves the halving of the
chromosome number in the gamete maturation stage. It is necessary for the zygote or
fecundated egg to keep the characteristics of the species; it must contain the same
number of chromosomes like the mother and father (meiosis absence makes the
number of chromosomes double from one generation to another).
2. It modifies gamete shape regarding the fecundation; the male germinal cells
are first spherical and big. It loses a part of the cytoplasm and becomes a mobile cell
through the differentiation of the flagellum. The female germinal cell grows a lot
through the growth of the cytoplasmic mass. It synthesises nutritive material
necessary for the embryo in the first developing stages.
The first maturation division or meiosis interests the first degree ovocytes and
spermatocytes (diploid cell - 46 chromosomes); it is quickly followed by a second
maturation division that is an equal division. From it result the second degree
ovocytes and spermatocytes (haploid cells - 23 chromosomes) that are then
differentiated in gametes - spermatozoon and ovule.

9.2.2.1. The First Maturation Division (Meiosis).


The meiosis interests the first degree spermatocytes and ovocytes; their cell cycle
is developed in two stages:
I. The metabolic stage or interphase is similar to that of somatic cells with the
single difference that chromosomal DNA and histones self-replication are not
achieved in this stage but in meiosis prophase.
II. The distributive stage or meiosis is characterised by reducing at a half the
chromosome number. In this way it is avoided the doubling of the number of
chromosomes from egg or zygote. It has four stages: prophase, metaphase, telophase
and anaphase.
The prophase is very long and has 5 successive stages:
a. Leptotene is characterised by nucleus volume growth, DNA self-replication
and the synthesis of histone proteins. Chromosomes become visible and have a
filamentous aspect. Sex chromosomes X and Y are found in a condensed form (the
sex granule or vesicle on the internal face of the nuclear cover).
b. Zygotene is characterised by the longitudinal stage attaching of the
homologue chromosomes (each paternal autosome attaches to the homologue
maternal autosome). This phenomenon is called synapse. They are bound through a
protein network called synaptolemal complex. It achieves a perfect alignment of the
two chromosomes. Such a pair is called bivalent chromosome or dyad. At the end of
this stage in the nucleus there are only 22 dyads (bivalent chromosomes), therefore it
was achieved the reducing at a half of the diploid chromosomal set. Sex chromosomes
are found in the condensed form.
c. Pachytene - at the beginning of this stage, the connection between
chromosomes is very tight. Then, at the level of each bivalent a longitudinal cleavage
occurs so that the dyad will have 4 chromatids, forming a tetrad.
d. Diplotene - the cleavage of the chromatids is very obvious. It is the period
where is made an exchange of genetic material through exchanges of chromatids
fragments between the homologous chromosomes of the dyad. This recombination of
the genetic material is called crossing-over.
e. Diakinesis is characterised by movements or migrations of the bivalents
toward cell periphery. The two sex chromosomes X and Y differentiate from the sex
vesicle. The nucleoli and the nuclear cover start disappearing.
The last 3 stages are similar to those of a somatic mitosis.
In prometaphase the nucleoli and the nuclear cover disappear, the centrioles
move away and the bivalents direct over the cell equator.
In metaphase the bivalents form the equatorial plaque.
In anaphase each bivalent separates into two halves, each of them migrating
toward one of the cell poles.
The telophase is similar to that of the somatic mitosis. It marks the beginning of
the separation of the two daughter cells (of the second degree spermatocytes and
ovocytes). They have the chromosomal equipment reduced at a half from zygotene
stage of the prophase.

9.2.2.2. The second maturation division.


To become mature, the second degree spermatocytes and ovocytes undergoes a
second maturation division, that do not modify the number of chromosomes.
Through this division are formed the gametes - spermatozoon and ovule - with
a haploid number of chromosomes that, by their structural and functional
characteristics are able to fecundate.

9.2.3. Direct Division or Amitosis.


Amitosis or direct division is simple and rarely found in human beings. It is
characterised by a cleavage that interests both the nucleus and the cytoplasm. It was
described by Regaud in Sertoli cells from testis, by Bucher (1959) in fibroblast
cultures and by Grundmann and Bach (1960) in mammalian liver after partial
hepatectomy.
Amitosis significance is not well explained. It is found in old cells, near the end
of their life. In cell cultures it may be found when living conditions are bad.
Recent researches with histophotometry techniques showed that amitosis is
preceded by DNA self-replication.
Figure 45a. Comparison of meiosis and normal cell
division.
CHAPTER X

CELL DIFFERENTIATION

10.1. Introduction.

Cell differentiation is the process that obtains cells with a high degree of
structural and functional organisation, leaving for a primitive, not-differentiated, but
multipotent cell. The multipotent cell contains the genetic information necessary for
the evolution.
Cell differentiation begins in the pre-embryo period when in the gonads of parent
organisms, the primitive germ cells (ovogonia and spermatogonia) pass through
differentiation mitosis that leads to gametes realisation. It is a necessary stage for
fecundating and for keeping the morpho-functional characters of the species. Through
the differentiation process, there are achieved:
„ species perpetuity
„ organism growing and development
„ species evolution and organism adaptation to the medium
The ideal material for cell differentiation study is the embryo, although such
processes take place during whole life, toward the death of the individual.

10.2. Embryo Differentiation.

During the fecundation process, the two pronuclei (male and female) with a
haploid number of chromosomes get near one to another without fusing, achieving the
cariogamy or amphimixy. This stage marks the end of the fecundating phenomena
where the zygote or fecundated egg is obtained. Before approaching, the DNA
replication takes place inside the two pronucleoli, and the DNA amount doubles. In
the fecundating process are achieved:
• the activation of the egg recovers the division capacity (the unfecundated ovule
degenerates);
• the remake of the diploid chromosomal equipment;
• sex determination: X or Y chromosomes;
• it is formed the hereditary dowry of the new individual through the fusion in the
same cell of the paternal and maternal genes. After the zygote forming, it begins a
new stage of the differentiation. This stage is called segmentation. Immediately
after amphimixy, the maternal and paternal chromosomes (46 chromosomes in the
fecundated egg) are longitudinal cleaved and then separated, forming 92
chromosomes. Half of their number is directed toward a pole of the cell and the
other half migrates toward the opposite pole. During the migration, it begins the
cytodierhesis. Finally there will be separated two cells (blastomeres) from the
zygote. Each blastomere has a diploid number of chromosomes: 44+XX or
44+XY.
The fecundated egg and the first four blastomeres born from the first two
segmentation mitoses can form through the differentiation process all cell types of an
adult organism. That is why they are called pluripotent cells. In these blastomeres that
have the same size and shape, all the genes from the nucleus are depressed (are
active) so that every message received by them is accepted. In this way, the cells have
a very different evolution (figure 46).
Figure 46. Cell differentiation in

In the stage of morula appear differences in size and position: small blastomeres
appear. They are named micromeres and occupy the animal pole. Large blastomeres
also appear and they occupy the vegetative pole. The difference in size and position is
due to some extrinsic factors.
In the second week of embryonic life, the differences become more evident: it is
formed the blastula, the embryonic button appears. Its cell will form the first two
embryonic layers: the endoblast formed by small cells and the ectoblast formed by
large cells. The cells in these two layers are obliged to follow a certain evolution and
are named determinate cells. This state is determined by the action of some repressor
agents that inactivate a certain number of genes from cell’s genetic dowry. During the
evolution are repressed a variable number of genes, always others, depending on the
specialized cell type that has to evolve.
The factors that direct the differentiation process toward a certain specialized cell
type are called inductors. The target cells must be permissive (permeable to the
inductor) so that the inductors can act. This property appears as a result of the
appearance of receptor zones located in cell membrane that bind the inductors,
because the activity of some genes. During the differentiation process, the permisivity
for a certain type of inductor is limited in time. The periods of action for the first
inductor and the moment of the first determination are essential for realizing a normal
individual.
In the developing process of the human being (in ontogenesis) the number of the
successive determinations is equal to the number of inductors. These elements finally
create stem cells. From these cells will form different types of specialized cells. For
example, from stem cells of hematogenous bone marrow, there will be differentiated
the leukocytes, red blood cells and platelets.
In an adult organism, there are not pluripotent cells, but each tissue has stores of
stem cells except the heart muscle and the nervous tissue. The differentiation process
is characterised by the appearance of some biochemical, morphological and functional
changes. From biochemical point of view, cells differentiation is connected with
specific protein synthesis during the embryo development. From functional point of
view, the differentiation process is characterised by the appearance of some
characteristic function for each type of new formed cells. For example, muscle cells
are able to contract, because they are synthesising contractile proteins - actin and
myosin.

10.3. The Nucleus and Cytoplasm roles in Cell Differentiation.

The nature of the inducting factors, of the mechanisms that control cell
differentiation represents problems of modern biology. Egg fecundation is quickly
followed by more segmentation mitosis. The blastomeres become smaller and smaller
and will form the morula, which dimensions are equal with that of the fecundated egg.
Between the central blastomeres of the morula that is formed by 64 cells, there appear
spaces. These spaces will grow and will form a cavity: central blastomeres are
localised at one of the poles of this cavity. A single layer of blastomeres forming the
embryonic button delimits the cavity. It is the blastula stage.
In this stage, the cytoplasm role is more important than the nucleus’s.
In amphibian eggs, there are two different peripheral zones:
• the animal pole that is dark-brown and
• the vegetative pole that is white.
Immediately after fecundation, on zygote surface appears a zone of intermediate
pigmentation, localised in the subequatorial region called gris semiluna. From this
structure will develop the dorsal part of the frog. If the superficial layer of this zone is
destroyed (dorsal cortex) the development of axial organs does not take place
anymore. The cortex of gris semiluna that appears at a short time after fecundation is
pure cytoplasmic.
The germinative plasma is another cytoplasmic territory localised at the
vegetative pole of the amphibian fecundated egg. It contains big granules of
ribonucleoproteins. Destroying this territory with UV rays gives the appearance of the
individual without gonads. If it is injected in such an irradiated egg, cytoplasm from
the vegetative pole of an non-irradiated egg, the obtained individual is normal. It
results that in the first development stages, all blastomeres nuclei are equipotents, but
the cytoplasm plays an important role in the differentiation process.
In the gastrula stage, the interaction between nucleus and cytoplasm has an
important role in differentiation. In this stage, the cell continues to multiply but two
new events appear: the embryo growth and cell grouping in distinct assemblies. T.H.
MORGAN gave the nucleo-cytoplasmic interaction theory in 1934. He showed that in
this stage, under the cytoplasm influence, in some nuclei, one or more genes would be
derepressed, modifying the biochemical cytoplasm structure that will become more
heterogeneous.
The nucleus transplant techniques in non-nucleated cells with different
differentiation degrees were used to demonstrate an important phenomenon in the
differentiation process. In non-nucleated Xenopus eggs were transplanted nuclei from
different adult cell types, perfect differentiated. There were obtained perfect
developed tadpoles. This experiment demonstrates that the nuclei of adult cells are
identical with that of fecundated cells. They have conserved all the genetic
information contained in ovules and spermatozoon nuclei. The genes from the nuclei
of adult cells are identical with that of the nuclei of fecundated eggs. This
demonstrates that nuclei are not irreversible differentiated during the individual
evolution and that their genes are made active or repressed because of the nucleus-
cytoplasm interaction.
In the gastrulation period, from embryo button are formed three layers:
• the endoblast formed by small cells, localised between the segmentation cavity and
the embryo button;
• the ectoblast formed by big cells, localised between the endoblast and
cytotrofoblast;
• the cordomesoblast is differentiated from the posterior zone of the primary
ectoblast and lies between the endoblast and cytotrofoblast.
The gastrulation marks the beginning of the derepression or of the repression of
some genes through:
• cytoplasm-nucleus interaction
• interaction between cells (induction)
• the control messengers from the surrounding medium.

10.4. The Induction in Differentiation.

From the three embryo layers will be formed all organs of an adult individual.
Cells of these layers are “obliged” to follow a certain evolution before to be obliged
or determined, they are competent, in the sense that their development depends on
their answer to different stimuli. When they answer to a stimulus, they become
determined cells.
In the developing embryo, the induction term names the effect of a surrounding
influence that determines the competent cells to undergo a certain differentiation
degree through which they arrive to a certain determination degree. The induction has
a very important role during the development of an embryo and on it depends the
tissue development in comparison with the surrounding tissues.

10.5. The Differentiation Biochemistry.

In spermatogenesis and ovogenesis take place important biochemical phenomena


that influence embryo development after fecundating process. In this period take place
the DNA transcription that releases the cell synthesis processes. The ovule
accumulates the substances synthesised by other cell types and produces its own
nucleic acids and proteins. Germ cells synthesise protein-enzymes that probably are
not useful but which will be used when their substratum appears, some days after
fecundation. The ovocyte synthesises a large number of ribosomal RNA molecules
and messenger RNA.
The maturation period is characterised by an intense protein synthesis directed by
the pre-existent mRNA. It is supposed that these proteins migrate into the nuclei
during the segmentation and they play a role in the regulation of the genetic activity.
Spermatozoon roles resume only at its genetic content.
Fecundation has more important biological consequences: chromosome number
becomes diploid, the zygote contains hereditary maternal and paternal characters and
is established the sex of the future adult. The definitive orientation of the two poles is
produced. Into the nucleus of the fecundated eggs, RNA synthesis does not take place
while DNA replication is very fast.
In most animal species, fecundation is followed by an intense stimulation of cell
respiration and of protein synthesis determined by mRNA and rRNA molecules
synthesised during ovogenesis. The fecundation destroys and inhibiting protein that
exists in the virgin egg and that brakes the ribosome coupling with mRNA.
During the segmentation the genes are repressed. In this period takes place a very
fast DNA auto-replication because segmentary mitosis is very fast. There is no protein
synthesis, although there are ribosomes and mRNA. The new-formed blastomeres will
have a different biochemical content in proteins, mRNA, tRNA and rRNA.
In pre-gastrulation are synthesised new messengers that did not exist before but
which are necessary for the gastrulation. This is possible through the derepression of
some genes. F. Crick showed that in morphogenesis the inducting factors are specific
molecules. They can not pass easily through all cell membranes but only through
those of interested tissues (through junctions).

10.6. Cell Differentiation in Adult.

In mammalian adult organisms there are no pluripotent cell, but only stem cells
both for somatic and sex cells. In adult these differentiation processes are called
regenerative processes and take place in the same conditions as in embryo
(hematogeneous bone marrow, gonads etc.).
CHAPTER XI

APOPTOSIS

11.1. Introduction

11.1.1 General Characteristics. History. Programmed Cell Death Concept.


Circumstances of Apparition.
Complex forms of life exist because it appeared well-defined mechanisms that regulate
proliferation, differentiation, ageing and homeostasis in the cell. At the beginning, researches
were focused only on proliferation mechanisms and cell differentiation understanding. Cell
death was considered a chaotic process. The two forms of cell death encountered in living
organisms are necrosis and apoptosis (see table 1).

Comparison between the essential death characteristics by necrosis and apoptosis


CHARACTERISTIC NECROSIS APOPTOSIS
Death condition Catastrophic Programmed
Way Passive Active (ATP-dependent)
Cell volume Increased Decreased
Cell density Decreased Increased
Ca2+ concentration Decreased Increased
Plasma membrane lysis First step Last step
ADN hydrolysis Last step First step
Speed Fast Slow
Cell organelles Lysis Compact
Scar type Fibrous scar Without scar
Table 1.

Necrosis means pathologic cell death and is induced when major cell alterations are
present. Abrupt cell confrontation with extreme non-physiologic conditions guides to the loss
of control on ionic flux, fast water penetration in the cell, followed by swelling and membrane
rupture. Organelles volume increasing, of mitochondria especially, is characteristic for this
type of cell death. At the same time necrosis is characterised by lysosome destruction and
hydrolytic enzyme overflowing in cytoplasm. Necrosis is a passive process that does not need
active cell participation, cell that finally dies.
Apoptosis or programmed cell death is a regulating physiologic phenomenon that
equilibrates cells number in a normal tissue. It is a part of the normal cell life, like
differentiation or cell division; it is also an active process that engages cell metabolism. The
latter observation leaded to the term of programmed cell death or cell suicide. The cell
participates actively to a self-destruction program similar to a suicide. Apoptosis is
irreversible, and it is not toxic for neighbour cells. It may appear in physiological conditions
(embryo development, normal turnover in the tissues) and also in pathological ones
(malignacy).
The inducing mechanism disturbances of apoptosis may cause various pathological
processes (table 2).
Glucksmann described cell death during normal organism development in 1951 then by
Kerr in 1965 and Sannders in 1966.
Their experiments demonstrate that human embryonic cell or from insects during
metamorphosis carry the information necessary for time delayed self-destruction.
Consequently, it appeared the idea that cells contain a suicidal program controlled by a
biological watch.

Physiologic situations for apoptosis disturbances


Malignacy and Pre-malignacy
- Solid tumors
- B-cell lymphoma
- Chronic lymphocyte leukemia
- Prostate hypertrophy
- Hepatic preneoplasic sites
- Chemiotherapy resistance
Neurological Disturbances
- Alzheimer disease
- Prionic diseases
- Telangiectasic ataxis
Heart diseases
- Ischemia
- Drug induced myocardial suppression
Immune system disturbances
- AIDS
- Type I diabetes
- Lupus
- Sjogren syndrome
- Glomerulonephritis
Intestinal disturbances
- Dysentery
- Inflammatory gut diseases
- HIV or X-ray induced diarrhea
Kidney diseases
- Polycystic disease
- Anemia/Erythropoesis
Table 2.

11.1.2. The Circumstances of Apoptosis Apparition


a. Apoptosis during embryonal, fetal and postnatal development
Apoptosis frequently appears during normal histogenesis and organogenesis. Even from
the first stages of the development, part of blastocyst cells die by apoptosis. During the
embryonal period, male or female germinate cells and also numerous neurons, disappear by
apoptosis. Neuronal death by apoptosis was observed even in latter stages during fetal and
postnatal period (e.g. at the vertebrate nervous sister, more than 50% by normal neurons dies
in short time after they realize synaptic connections with target cells). Also motoneurons,
simpatic system and retinal neurons can disappear by apoptosis. Duodenal and cord
morphogenesis, interdigital membrane resurgence, Muller canals disappearance, fusion of
palatine processes in mammals, uses apoptosis as a mechanism. Is remarkable the fact that
apoptosis appear in the embryonal tissue, always in the same place and in well determined
moments of the development. In this period, it affects a big number of cells, which is
contrariwise with the observations in adults, when is present at one cell or a small group level.
b. Apoptosis during cellular renewal from normal tissue in adults
Apoptosis contribution to the cell turnover from normal tissues is one of the less studied
aspects of cell physiology. In adults, in numerous organs, mature cells are regular renewal,
tissues are in that way into dynamic equilibrium: stem cells are dividing and then are
differentiated in mature cells, which after a certain period of time disappear, programmed
cellular death being responsible by this turnover. Today is known the fact that the apoptosis is
presented in thymus corticosuprarenal, prostate, intestine, liver, lymphatic ganglions,
mammalian gland and ovary, but the importance of the phenomenon was rarely morphologic
quantified: at the liver level, in humans and mouse and at the corticosuprarenal level in mice.
In the first case was constituted that 80-90% from apoptotic cells was localized around the
center-lobular hepatic veins and in the second case, the number of apoptotic cells was
estimated to 6% (table 3).

Physiologic and pathologic events associated to apoptosis


1. Normal cell turnover
2. Embryogenesis/metamorphosis
3. Decreased trophic hormones, cytokines, other growth factors
4. Radiations
5. Steroids
6. Cell mediated immunity
7. Viral infections
8. Auto-immune diseases
9. Anticancer drugs, other chemicals
10. Oxidating agents
11. Thermal stimuli
12. Aging
13. Neoplasia
Table 4.
c. Systemic or hormone-dependent apoptosis
It is well known for a long time that a decreasing in blood concentration of the certain
hormones is accompanied by an atrophy of target organs. This atrophy acts with a high level
of apoptosis: suppression of the ACTH secretion, which drives at the apoptosis rate, induce
increasing in corticosuprarenal and suppression of testosterone secretion increase apoptosis in
ventral lob of prostate. The endometrium, the ovarian follicle makes an involution from
apoptosis in the normal ovarian cycle as the mammal gland after the lactation stopping. Is also
known that the reducing of the blood flux can conduce to initiation of apoptosis at the hepatic
renal, cerebral or cardiac level.
d. Apoptosis in immunity system
The apoptosis was studied in immune system where it looks like it plays a very important
place in autoreactive lymphocyte elimination, and cytotoxic timocites, the K cells and NK,
which induces apoptosis in target cells.
During embryonic development, apoptosis appears in many situations: e.g. in vertebrate
nervous system more than 50% from normal neurons die in short time after realizing synaptic
connections with target cells. The releasing signal is usually chemical, a hormone for example
or a growth factor that has no effect on neighbor cells.
Hormone dependence of apoptosis was emphasized in the adrenal gland cells after ACTH
secretion suppression and also in prostate cells after testosterone secretion suppression.
In many organs from adults, mature cells are replaced from time to time, tissues being in
a dynamic equilibrium: stem cells are dividing then are differentiating in mature cells that
disappear after a time; cell death is responsible for this turnover.
Apoptosis was also found during keratinocyte formation, prostate atrophy after gelding,
regression of secretor cells in mammary gland after lactation, involution of the ovarian follicle
etc. (Table 4).
Apoptosis was intensively observed in the immune system where it appears to play an
important role to eliminate self-reactive lymphocytes; cytotoxic thymocytes, K (killer) and
NK (natural killer) cells induce apoptosis in target cells.
In 1971, Kerr et al. described the ultrastructure of apoptotic cells, proposing the term
apoptosis; this term is derived from the ancient Greek - hipocratic corpus meaning leaf
falling. Other terms also used for apoptosis are: normal cell death, programmed cell death,
physiologic cell death, cell suicide.

11.2. Molecular Components of Apoptosis

11.2.1. Tanatogenes
First of all, cell death programming involves the existence of specific genes, the
tanatogenes, that coding for proteins with lethal effect on a specified cell. Molecular analyses
lead to the discovery of two genes Ced-3 and Ced-4 that become functional in cells that enter
the apoptosis.
If these genes are inactivated, apoptosis do not occur. Both genes act autonomous to
induce programmed cell death. Ced-3 codes for a new protein with phosphorylation sites
while Ced-4 codes for a new protein with two Ca+-binding sites; however,it is not well known
the way of interaction with other proteins or the way by which other proteins are inducing
apoptosis. At the same time, it was shown that other genes are essential to induce apoptosis in
mammalian cells: proto-oncogenes, c-myc - that normally stimulates cell proliferation is
involved in apoptosis induction, P53 - tumor gene suppressor, is another gene associated to
apoptosis; high levels of P53 are associated to programmed cell death.

11.2.2. Surviving Genes.


Surviving genes represent another class of genes that impedes apoptosis induction; they
are also called anti-apoptotic genes. If these genes are inactivated, many cells that should
normally survive, enter the apoptosis. In this way, Ced-9 is a gene of special interest because
it acts like an antagonist for suicidal program. If its function is activated following mutations,
cell death do not appear anymore.
Bcl-2, a protein of the inner mitochondrial membrane, may inhibit apoptosis when it is
overexpressed. It inhibits apoptosis induction by c-myc, making cells less sensible to radiation
and cytotoxic drugs.
Consequently, activation of many genes is essential for apoptosis; this activity was
conserved during evolution from worms to human, confirming that apoptosis is an important
process for animal cells.
Tanatogenes can not be considered, however, killer genes, while their products are not
lonely responsible for cell death induction.

11.3. Induction of Cell programmed Death.

Physiologic cell death starts when a cell in the organism dies by a mechanism controlled
by proteins coded by the host genome. The purpose of this process is to eliminate undesired
cells; it becomes active in three situations:
• development and homeostasis;
• apoptosis mechanism;
• aging.
A large variety of stimuli are able to induce apoptosis for a specific cell type; for
example: hormones, growth factors, monoclonal antibodies directed over a surface molecule,
etc.
At the same time, stimulus missing - physical agent, metal, biological agent, xenobiotic
agent, antitumoral agent, may have the same consequence. These agents act on extremely
various cell types (Table 4).
As we have seen, apoptosis can be induced by all factors that induce necrosis, but at a
lower concentration or dose. Kerr demonstrates that a reduced degree of hypoxia induces
apoptosis while severe hypoxia provokes infarcts and necrosis; hyperthermia induces
apoptosis or necrosis depending on quantity of heat absorbed, depending also on cell type.

11.4. Molecular Mechanisms in Apoptosis

Generally, physiological cell death process was divided in following steps:


1. Early period when acts the stimulus or stimuli that lead to apoptotic answer;
2. Reception and transduction of the signal - transduction ways will transmit the message to
the effector mechanism of cell death; tanatogenes activation takes place and it is followed
by macromolecule synthesis.
3. Effector period that includes protease action, their positive or negative regulators action,
endonuclease and transglutaminase action; the effect is nuclear fragmentation (chromatin
condensation and DNA degradation), followed by cell fragmentation and apoptotic bodies
formation.
4. Post-mortem period when apoptotic bodies are phagocytized.
The answer to apoptotic inducting factors action is few studied. It appears that an abrupt
increase in intracellular Ca++ concentration plays the second messenger role. From this point
of view, the second stage was divided in 2 phases: pre-engaging (that is still reversible) and is
characterized by increasing concentrations of second messengers: Ca++, IP3 and/or cAMP;
engaging supposes endonuclease activation, mRNA and protein synthesis stimulation.
Inhibition of these syntheses can impede cell death, fact that emphasizes their importance
during this process.

INDUCING SIGNAL

TANATOGENE ACTIVATION

Increased intracellular Ca++ Protein-kinase activation, protein


concentration, second messenger phosphorylation,
synthesis c-fos, c-jun, P53, c-myc expression

MACROMOLECULAR SYNTHESIS

Cell or nuclear receptors Endonucleases, proteases,


transglutaminases

NUCLEAR FRAGMENTATION

Chromatin condensation DNA fragmentation

CELL FRAGMENTATION

Cytoskeleton destruction, cytoplasmic Apoptotic bodies formation


protein cross-linkage

APOPTOTIC BODIES DEGRADATION AND PHAGOCYTOSIS

Figure 47. Molecular events in apoptosis

The role of second messengers


During transduction of inducing apoptosis signal, many second messengers play an
important role. For example, cAMP increasing concentration stimulates DNA fragmentation
and seems to be responsible for intracellular Ca++ increasing concentration. Increased cAMP
concentration evolves without C-protein-kinase activation that could impede cell suicide,
while A-protein-kinase have an opposite effect. In apoptotic macrophages it was found an IP3
increase that provokes Ca++ delivery from stocks. Calcium plays a major role: if we impede
the increase of its cytoplasmic concentration, endonuclease activation is impeded while the
usage of a Ca++ ionophore engage DNA fragmentation. At the same time, Ca++-channel
antagonists - verapamil and nifedipin - delays apoptosis. Cell targets for Ca++ ions in
apoptosis initiation are very diverse. These targets can be endogenous endonucleases that are
Ca++ dependent in most cell types, transcription activation for some genes that code for
surface receptors for apoptosis inducing signals (e.g. Fas receptor involved in autocrin suicide
of mature T cells). Recently, it was identified another target for Ca++ ions, a calcium-
dependent nuclear serine-protease that realise H1-histone cleavage in thymocytes, CLL
lymphocytes and melanoma cell lines, whose inhibitors block endonuclease activation,
demonstrating that proteases control endonuclease activation directly or indirectly.

11.5. Morphologic and Molecular Characteristics of Apoptotic Cells.

11.5.1. Morphologic characteristics.


From morphologic point of view, apoptosis evolves in 3 steps:
First step is characterised by abrupt apparition of nuclear and cytoplasmic alterations. In
nucleus, the chromatin is condensed and appears a chromatin ring that encircles almost
completely the nucleus, giving a characteristic aspect. Nucleolar fibres and granules are
dispersed in the nucleoplasm.
In cytoplasm, organelles are approaching, agglomerate, especially mitochondria,
cytoskeleton filaments and free ribosomes. Cells win a round shape while desmosomes are
destroyed and intercellular spaces are enlarged. An essential aspect of this step is that
endoplasmic reticulum and mitochondria (especially mitochondrial DNA) remain intact by
difference to necrosis where we can see fast installed morphologic alterations of synthesis and
energetic apparatus of the cell. In this step, lysosomes are not altered and the plasma
membrane is not permeable to vital dyeing colouring matters.
Second step is characterized by nuclear membrane indentation that announces nuclear and
cytoplasmic fragmentation that leads to cell fragments forming more or less large, still
bounded between them by cytoplasmic bridges. In these fragments we can see nuclear
residues, apparently non-affected organelles or a mixture of nuclear and cytoplasmic residues,
easy or difficult to be seen even in electron microscopy. In this stage, cell volume decreases
and cell density increases. Examined in scanning electron microscopy, cell surface presents
large cytoplasmic bubbles that deform the plasma membrane and suggest the important
changes at cytoskeleton level. Cell fragments or apoptotic bodies are separating one from
another and from the tissue in order to be phagocytized by macrophages or by neighbor cells.
Neutrophils have no action in this process.
Third step is marked by apoptotic bodies’ degradation. This degradation provokes membrane
lesions that have as consequence the increased permeability to vital dyeing colouring matters
and the destruction of cytoplasmic and nuclear residues by macrophages. The disappearance
of apoptotic bodies is not accompanied by any lymphoid inflammatory reaction or by fibrosis
reaction that make the difference between apoptosis and necrosis. These phenomena occur
fast, with an average duration of 6 hours; the extreme periods extend from few minutes to few
days (for example, hepatocyte apoptosis take 3 hours).

11.5.2. Molecular characteristics.


Nuclear changes.
From molecular point of view, chromatin condensation becomes evident by a deep DNA
reshuffling: gel electrophoresis shows DNA fragments with molecular weight corresponding
to a nucleosome - 180 base pairs. The emphasising of a regular DNA fragmentation
represents an essential marker for molecular confirmation of chromatin morphologic changes.
Also, nuclear matrix proteins leaves apoptotic nucleus, being detectable in culture medium.
DNA fragmentation is realized by a Ca++ and Mg++-dependent endonuclease that is active on
neutral pH and is inactivated by Zn++. However, the endonuclease responsible for this
fragmentation was not yet characterized.
Arends and Wyllie extracted a 130 kDa protein from apoptotic thymocyte nucleus with
endonucleasic activity that could be one of the topoisomerase II subunits; this enzyme plays
an important role in DNA loops organization and its activity could induce DNA segmentation.
There were revealed also endonucleases with higher molecular weight like NUC-18 protein,
DNAase I and DNAase II. Precise identification of endonucleases that act during apoptosis is
essential for understanding apoptosis mechanisms.
Cytoplasmic changes.
The transglutaminases are Ca++-dependent enzymes, with cytoplasmic localization that
are activated in most apoptotic cells. They catalyses protein polymerization with bridge
formation between them could be responsible for ultrastructural changes in cytoplasm. We
consider that cell organelle agglomeration in first two steps of apoptosis impedes cell
macromolecules to move out from cell, so explaining the absence of inflammatory reaction.
Plasma membrane changes.
Apoptotic cell recognition during fragmentation by macrophages suggests that important
change in membrane proteins and lipids occur. There are, up to now, three hypotheses to
explain this phenomenon:
• The first and well known is based on the existence of changes in glycoproteins and
glycolipids in the membrane (e.g. sialic acid loss). In this case not only macrophages
but also other cells that have receptors for asialoglycoproteins - like hepatocytes -
participate to phagocytosis of apoptotic cells. In this context, it was demonstrated that
hepatocyte apoptosis is accompanied by an asialoglycoprotein receptor overexpression
in neighbor hepatocytes and galactose receptor overexpression in Kuppfer cells.
• The second possibility involves the possible role of vitronectin receptor; vitronectin is
an adhesive protein from integrin family that interacts with CD36 ligand in
macrophages.
• The third possibility could be those related to apoptotic cell membrane modification
that could lead to an exposure in the outer monolayer of phosphatidyl-serine.

11.6. Apoptosis and Cell Senescence.

During aging we can observe the diminishing of cell number in most organs, considering
that apoptosis is inducing agent. Apoptosis induction in senescent cells is due to the lack in
growth factors, anomalies in intercellular and intracellular signal transmission, and
modifications of the cell cycle. Apoptosis acts probably to eliminate altered senescent cells
and its perturbation may involve elimination of healthy cells with the exception of abnormal
one.
Depriving of growth factors and/or surviving factors represents a possibility to induce
apoptosis: a cell can activate the self-destruction program because it did not received
surviving signal. So it is explained fast regression by apoptosis of experimental hyperplasia in
various organs (kidney, liver, pancreas). During aging we can see similar phenomena of
homeostatic mechanism disregulation in order to maintain cell number in organism. Growth
factor and/or surviving factor synthesis decreases with age inducing apoptosis mechanism
activation. This phenomenon was revealed during some organs aging: mammary gland and
uterus involution during menopause happens by apoptotic mechanisms and reported to
deprivation of sexual hormones; cutaneo-mucous modifications are reported to estrogens
deficit and are based on apoptosis mechanisms activation; diminishing of androgen synthesis
with age is partially responsible for some organic atrophy, especially in muscle mass melting
in older man. Muscle and cutaneous atrophy observed during senescence could by of
apoptotic origin and linked to diminished production of growth and surviving factors, like
growth hormone and insulin-like growth hormone (IGF-1). The observation that a hormone
treatment for 6 months with growth hormones leads to a significant increase in muscle mass,
of epidermal thickness and of vertebral bones density in persons over 60 years of age with
IGF-1 deficit, pleads for apoptotic origin or organic atrophy during senescence. Apoptosis
induction by lacking of surviving factors is well demonstrated in nervous system, where
neurones die when neuronal growth factor produced by target cell lacks.
Defects in transmembrane signalling.
Death by apoptosis can occur while signalling ways that lead to cell division are
interrupted or disturbed. C-protein-kinase (PKC) plays an important role for cell option for
death or proliferation. PKC activation inhibits apoptosis while PKC inhibitors have the
reverse effect. Tirosin-kinases are involved in apoptotic processes and their dysfunction can
provoke the arrest of cell cycle and death by apoptosis. During ageing, membrane receptor
bound tyrosin-kinase activity diminishes, leading to alterations in the transmission of
proliferative signals.
Cell cycle alterations.
After ontogenesis, a multicellular organism must realise the equilibrium between
proliferation and cell death for maintaining a constant volume. No doubt that these genes are
involved in the control of cell cycle and also in apoptosis control. During aging, it is possible
that the modification of gene expression that controls proliferation mechanisms to move the
equilibrium death-proliferation over apoptosis. For example, p53 is a cell cycle regulator but,
in some special conditions it can induce apoptosis. Its expression is induced especially by
some events susceptible to alter DNA but also by deprivation of growth factors.
Concluding, apoptosis mechanism that acts during organogenesis and then during adult
life, can be activated during aging processes. During aging, apoptosis eliminates non-
functional cells but it could represent an abusive phenomenon; in other situations apoptosis
control mechanisms are affected by aging and their activation leads to elimination of normal
cells - aberrant apoptosis.
CHAPTER XII

CELL AGING

12.1. Introduction.

Cellular aging was defined as a progressive loss of the physiological skills of an


organism that finally drive to death. Most of the physiological functions do decline
differently with aging.

Figure 48. Decline of different parameters to human species.

A biological definition of aging is that of structural and functional changes that


happen in an organism following the development and maturation periods, called
usually post-maturity period. The last period is characterized by an increased
vulnerability to medium conditions and demands, provoked and enclosed with a
decline of physiological and adaptive functions of the organism, due in turn to
morphological and molecular changes at cellular level.

12.2. Cellular Senescence.

12.2.1. Generalities.
The life of the majority of cell population from human organisms can be divided
in four phases: normal functioning, aging (senescence) agony and cellular death. The
most studied model of cellular aging is represented by cultured human fibroblasts.
More than 25 years ago, Hayflick demonstrated that normal diploid fibroblasts have a
limited life span in culture.
The normal human fibroblasts can be multiplied (by duplication) up to 60 times
after what the cells stop dividing.
The proliferate potential can be slightly modified by adding some compounds al
glucocorticoids and growth factors. It was observed a limited time span too for other
cell types as: keratinocytes, chondrocytes, glial cells, smooth muscle cells, granulosa
cells, adrenal cortical cells, vascular endothelial cells and mammary epithelial cells.
Then it became clear that the most important parameter of cellular aging is not the
survival chronological time in culture but the number of complete cell divisions,
because it is known that one of the essential events linked to cellular aging is the
diminishing cell proliferate potential followed by a decreasing cell number in most
organs.
From the morphological and biochemical changes that characterizes cellular
aging in senescent cultured fibroblasts and in other cells in senescent tissues we are
mentioning: the increased number and dimensions of the lysosomes, chromosomal
abnormalities, accumulation of altered forms of structural proteins that drives to the
perturbation of cell functionality (e.g. the accumulation of α-DNA-polymerize is
partial responsible for restraining the proliferate capacity of senescent fibroblasts),
reducing the transcription and translation rate, reducing the protein degradation,
decreasing response to a wide variety of growth factors and hormones, due to the
decreased number of receptors according to aging or to the altered ways of signal
transmission in target cells.

Characteristic Cultured Liver Brain Skeletal Cardiac


fibroblasts muscle muscle
Decreased transcription + + + + +
Decreased translation O + + + +
Decreased proteolysis + + + + +
Increased abnormal proteins + + O + +
Increased lipofuscin + + + + +
Increased number of + + O O O
abnormal nuclei
Increased number and size + + + + +
of lysosomes
Increased number of + + + + +
chromosomal abnormalities
Reduced response to + + + + +
hormones
Table 5

During aging, in the vascular system appears the “aging vasculopathy” – a result
of the adaptation mechanisms in senescent cells – and that represents a distinct
phenomenon from atherosclerosis but it is close to it by the susceptibility of aged
vessels to develop atherosclerotic lesions.
This susceptibility is due to morphological, architectural and molecular
modifications that appears in vessels, especially in senescent endothelial cells; vessels
dimensions are increasing, also increases the thickness of medium and internal layer,
of the extracellular matrix that is more rich in glycosaminoglycans and collagen, the
cell volume, elastin fibers are progressively disorganized and become fragmented,
transendothelial traansport is altered favoring the entrance of plasmatic molecules in
subendothelial space, retaining substances as LDL or AGEs that accelerates
atherosclerosis processes.
The endothelium permeability is increasing by age while the ability to produce
vasoactive substances is decreasing.
At the same time, aging is associated with the accumulation of some AGEs
(high end-glycated products) that produces particular cellular events like: oxidative
stress, leukocyte adhesion molecules expression, transendothelial migration of
monocytes, all being considerated important prelesional events in atherogenesis
process.
Aging affects the ability of endothelial cells to proliferate and to repopulate
nude zones resulted by vascular injuries. Chang and Harley observed that the length
of telomeres in endothelial cells taken from donors of different ages decreases with
aging.
Aging modifies the properties of smooth muscle cells from blood vessels walls
(migration and proliferation), the structure and the composition of extracellular matrix
that increases the susceptibility of the vessels to atherosclerosis. So, at aorta’s level it
was observed a decreasing in the concentration of heparan-sulphate associated to the
endothelial basement membrane while the the quantity of condroitin and dermatan-
sulphate in coronaries increases with aging. These changes in the percent of
glycosaminoglycans are similar to those observed in atherosclerotic coronaries and
are close linked with the accumulation of esterificated cholesterol.

12.2. The causes of cellular aging.

Recent studies show the involvement in this complex phenomenon of two kind of
factors:
⊇ extrinsic factors (influence of medium and of life style)
⊄ intrinsic factors (primary aging processes genetically induced)
According to these two categories of factors, it appeared some theories regarding
the causes of cellular aging, classified as follows. (table 6)
There are two major types of hypotheses to explain cellular aging.
The first proposes that aging is provoked by the passive accumulation of errors in
cellular constituents as DNA, RNA, protein and lipid due to a variety of
environmental insults coupled with imperfect repair mechanisms.
The second considers aging to be an active, genetically programmed event.

Theory Description
Error theories
Rate of living Fixed metabolic units
Waste product Build up of a toxic substance
Somatic mutation Mutations in somatic cells
Cross linking Cross-linked macromolecules
Free radical Hyperactive oxygen radicals
Error catastrophe Exponential errors in protein synthesis
mistakes
Altered proteolysis Changes in protein breakdown

Program theories
Codon restriction Switching codon preferences in early
development restricts codons available
later in life
Repetitive DNA loss Multiple gene copies sequentially
inactivated or lost
Telomere shortening Altered telomerase activity
Terminal diferentiation Growth simulators not expressed or
growth inhibitors overexpressed.
Table 6. Cellular theories of aging

A. Error theories.
The different error theories of aging focus on somatic mutations, faulty proteins,
damaged lipids or a combination of damaged molecules.
1. Rate of living.
2. Waste product accumulation.
3. Somatic mutations.
4. Cross-linking.
5. Free radicals.
6. Error catastrophe
7. Altered protein degradation
B. Program theories.
Preponents of the idea that aging is actively programmed often cite examples of
programmed cell death in early development.
1. Codon restriction
2. Inactivation of multicopy deoxyribonucleic acid sequences
3. Shortening of telomeres
4. Terminal differentiation
In fact, two events are always linked to senescence: diminishing of cells
proliferate potential and diminishing of cell mass.
This progressive cell rarefaction affects the most organs, being an evidence
especially in skin, muscles, kidney, brain and blood; at the same time is associated
with an increased DNA concentration in plasma blood in aged persons. We consider
today that this decreasing in cell mass in senescent organs is probably of apoptotic
reason. Recent progresses in the understanding of biological basics of cellular
senescence drive to the hypothesis that the mechanism of apoptosis induction (cell
programmed death) are activated by physic-pathological modifications characteristics
for cellular aging. The connection between apoptosis and cellular aging is somehow
still intuitive and incomplete explored.

Perspectives.
Numerous deficits underlie age changes. Replacement of these deficient factors,
such as estrogen and growth hormone, is being actively investigated in an effort to
thwart age-related changes in bone and muscle, body composition and other
functions.
With a better defined causes of aging and its molecular bases there is a real
opportunity to develop approaches for reducing age-associated deficits and thus
reduce the incidence of age associated diseases. Notable in this regard is the increase
in longevity via long-term diet restriction in animals, an increase associated with a
reduction in AGE glycation products, reduced oxidative damage and a reduction in
degenerative diseases.
CHAPTER XIII

EXTRACELLULAR MATRIX

The tissues do not contain only the cells in their structure but they have also an
extracellular space filled up with specific substances called extracellular matrix. The
extracellular matrix is a complex network of proteins and polysaccharides between the
intercells space where we find many types of molecules secreted by cells. In addition to
serving as universal biological glue, they also form highly specialized structures such as
cartilage, tendons, basal laminae, and bone and teeth with some forms of calcium phosphate
crystals. The quantitative variations of the different types of the matrix macromolecules
determine the diversity of the matrix shapes, very enclose with the tissue functions. Thus, in
the epithelial tissues the extracellular matrix is limited in volume, but in the connective
tissues its volume is bigger than the cells.
The matrix has an active and a complex role: influencing the cells in their development,
migration, and proliferation and stabilizes the shapes and the metabolic functions. In the
differentiation process the cells need specific components. The cells morphogenesis is very
close dependent of the matrix fibers. The matrix components can bind growth factors and
hormones giving signal abundance for present cells.

13.1. The Constitutive Macromolecules of the Extracellular Matrix

Since the different cells secrete varied components of extracellular matrix we can group
them in two types:
1. Polysacharride chains of glycosaminoglycans, which have a covalent bind at a
protein forming proteoglycans.
2. Fibrilar proteins, which could be:
- structural such as colagenes and elastins
- adhesive such as fibronectin and laminin
The glycosaminoglycans and proteoglycans molecules from connective tissues form a
highly hydrated gel-like essential substance in which collagen fibers are embedded. These
collagen fibers strengthen and help to organize the matrix and the polysaccharide gel offer
resistance to the pressure. Also, the aqueous phase of the polysaccharide gel permits a rapid
diffusion of nutrients, metabolites, and hormones between the blood and the tissue cells. In
the same time the elastin fibers give to the matrix the specific elasticity, and some proteins as
fibronectin are responsible with the attaching of the fibroblasts and other cells to
extracellular matrix in the connective tissues. For the epithelial cells laminin realizes them
connecting to basal membrane.
There is a diversity of the presented components due to the multiple gene sets that are
responsible of the collagen IV coding, and the laminin A and B1 chains. Many of these
molecules and their isoforms have a limited tissue distribution; their presence can be
described in specific stages of tissue growth.

13.1.1. The Glycosaminoglycans


The Glycosaminoglycans (GAGs), formerly known as mucopolysaccharides are
constituted by long unbranched polysaccharide chains having disaccharide as the repeating
units. The name glycosaminoglycans come because one of two sugars residues in the
repeating disaccharide is an amino sugar, usually N-acetylglucosamine or N-
acetylgalactosamine. Due to the presence of sulfate or carboxyl groups glycosaminoglycans
are highly negatively charged. According to their sugar residues, the type of linkage between
these residues, and the location of sulfate groups have been distinguished the following four
GAGs groups:
- hyaluronic acid
- chondroitin sulfate
- heparan sulfate or heparin
- keratan sulfate

13.1.2. Hyaluronic acid


Hyaluronic acid also called hyaluronate is simplest GAGs. It is the major component,
which surrounds the proliferative and migratory cells, particularly the embryonic tissues. It
can be also found in the extracellular matrix of the cartilage as the major components of the
proteoglycans. Hyaluronic acid is non-typical glycosaminoglycans because:
- the others tend to contain a number of different disaccharide units arranged in
more complex sequences
- the others have very much shorter chains, consisting usually of fewer than 300
sugar residues
- other glycosaminoglycans are covalently linked to protein to form proteoglycan
molecules called mucoproteins.
A hyaluronic acid molecule is formed from 50,000 repeating units of a disaccharide. Its
shape is that of a long and rigid stick. One single molecule may have around 20 μm.
The most important function of the hyaluronic acid is linked about the cells’ migration.
The hyaluronate assure the movement and the proliferation freedom of cells. The stopping of
cell movement and initiation of the intercellular attachment would be linked with the
diminishing of hyaluronic acid concentration. When the cellular migration is stopped, a
specific enzyme called hyaluronidase degrades the hyaluronic acid molecules.

13.1.3. Proteoglycans
Proteoglycans are macromolecules find in the all connective tissues. They are formed
from a proteinic core covalently linked with one or more glycosaminoglycans’ molecule.
The polypeptidic chain or proteinic chain of the proteoglycans is synthesized on the
ribosomes of rough endoplasmic reticulum membranes and introduced into their lumen. The
polysaccharide chains are usually assembled in the Golgi complex.
Proteoglycans are classified depending on the repeating disaccharide structure unit of
their glycosaminoglycan component. Sometimes, the proteoglycan molecules are not
exclusively components of the extracellular matrix.
Proteoglycans from cartilages are the best known molecules. It size can be more than 4
μm and a volume bigger than bacterian cell. These proteoglycans confer to the cartilage its
characteristic properties: resistance on the strength and consistence like a gel. A long
hyaluronic acid molecule forms the central component of proteoglycans. At it is linked the
core proteins of proteoglycans to chondroitin sulfate. A linker protein assures the proteins’
linkage on the hyaluronic acid. On each proteinic core is attached multiple chondroitin
sulfate and keratan sulfate chains.

13.2. Structural Fibrilar Proteins


Up to here we discussed about the specific molecules found in extracellular matrix. But
the extracellular matrix needs also other structural elements. From them it is distinguishes a
very important class given by fibrilar proteins, especially collagen.
13.2.1. Collagen
Collagen is the most found fibrilar protein from extracellular matrix. It is known
twelve types of collagen whom four are the most common. The types I, II, and III form the
fibrils with the similar structure. The type IV does not form fibrils, but bidimensional
reticular structures. The type IV is the mainly components of the basal lamina.

Schematic drawing of the various intracellular and extracellular events involved in the
formation of a collagen fibril. While the extension peptide cleavage and fibril formation
are shown occurring after secretion, there is some evidence that cleavage of the amino-
terminal peptides and some aggregation of collagen molecules occurs just prior to
secretion from the cell. Because the larger extracellular aggregates of collagen
molecules are stabilized by covalent cross-links as an example of how the collagen
fibrils can form ordered arrays in the extracellular space, they are shown further
assembling into large collagen fibers that are visible in the light microscope.
The structural properties of each collagen fibril assure their specific functions.
The structural unit of the collagen is long protein, with the length about 300nm and the
thickness around 1.5 nm, formed by three polypeptidic chains, triple-stranded: two chains of
α1, and one chain α2. Each chain is formed by 1050 aminoacids, and a α chain contains 3
aminoacid per each complete helix. Characteristic for collagen is its great contents of glycine
and proline as well as hydroxyproline and hydroxylysine. Glycine is the single small
aminoacid to occupy the space from the interior of the triple-stranded. Hydroxyproline and
hydroxylysine realize the hydrogen bindings between the α chains stabilizing the molecule.
The collagen synthesis has place in the ribosomes attached to the endoplasmic reticulum as
pro-α-chains, then being hydroxylated and glycosylated. Of them, three associates to form
triple-stranded procollagen molecules in the endoplasmic reticulum and the Golgi complex.
After the secretion off the cell, some peptides from the end of the molecules will be cut
forming the microfibrils through the covalent bindings. The microfibrils are grouped in
bundles forming the collagen fibers observed on the light microscope.
This structure has a great mechanical resistance especially on traction.

Schematic diagram of the covalent intramolecular and intermolecular cross-links


formed between modified lysine side chains within a collagen fibril.

13.2.2. Elastin
Elastin is other protein rich also in proline and glycine, but un-hydroxylated. Secreted
in the extracellular space, elongated elastin molecules are linked into a network. Each
molecule being stranded as random-coil in the network, as fibers or layers, has the rubber
elasticity because the “eyes” of the network modifies its shape according with the force
direction.
Elastic fibers are not composed solely of elastin, however; they also contain a
glycoprotein that is usually distributed as microfibrils on the surface of the elastic fibers. In
developing elastic tissues, these glycoprotein microfibrils often appear before elastin does
and may serve to organize the secreted elastin molecules into the fibers and sheets which
they later form.
It would be found in skin, lung and blood vessels.
Therefore, elastin is a cross-linked, random-coil protein that gives tissues their
elasticity.

Elastin molecules are joined together by Schematic drawing of an elastin


covalent bonds to generate an extensive molecule in various “random-coil”
cross-linked network. Because each conformations. Unlike most proteins, the
elastin molecule in the network can elastin molecule does not adopt a unique
expand and contract as a random coil, the structure but oscillates between a variety
entire network can stretch and recoil like of partially extended, random
a rubber band. conformations, as illustrated.

13.3. The Adhesive Proteins

Except proteoglycans and collagens, the most important components of the


extracellular matrix are represented by other special proteins that have binding sites with the
receptors of the cellular surface. They realize matrix components organization, determine
cellular shape and regulate both the cells attachment on the matrix and cells’ migration.

13.3.1. Laminin
Laminin is a molecule with a specific shape having 70nm the length. The molecule is
formed from three polypeptides and contains sites with a good linkage affinity for other
components of basal lamina. Basal laminae are thin layers of specialized extracellular matrix
that underlie all epithelial cell sheets and tubes. They also surround individual muscle cells,
and fat cells. The basal lamina thus separates these cells and cell sheets from underlying or
surrounding connective tissue.

The basal lamina is synthesized by the cells that rest on it (see the figure below).
Although the precise composition varies from tissue to tissue and even from the region
within the same lamina, a major component of basal laminae is type IV collagen. Type IV
pro-α-chains is unusual in having extra-long extension peptides that are probably not cleaved
after secretion.
In addition to proteoglycans and fibronectin, which are important constituents of basal
laminae, the large glycoprotein laminin has been shown to be a major component of all basal
laminae studied so far, together with entactin, perlecan and type IV collagen. It consists of at
least two subunits that are disulfide-bonded to each other. Basal laminae undoubtedly
contain many other proteins yet to be identified. The detailed molecular organization of basal
laminae is unknown, although there is some evidence that laminin and proteoglycan
molecules are concentrated along the inner and outer surfaces of the basal lamina, with
collagen molecules sandwiched in the middle.

13.3.2. Fibronectin
The fibronectin form a major class of glycoproteins from the matrix. Fibronectin
molecules assure the cells attachment on different extracellular matrix types. The presence
on the surface of un-transformed culture cells and the absence on transformed tumoral cells
of fibronectin confirm us the fibronectin implication in cellular adhesive. In this way,
fibronectin regulates the cell shape and the cytoskeleton organization. In embryogenesis, in
the cellular migration and the differentiation process fibronectin is found.
The fibronectin is a dimmer molecule formed from 2 similar peptides, which are
bounded with disulfuric links. Each chain is embedded in many globular domains, separated
by linker polypeptide segments. The individual domains are specialized for specific binding
on other different molecules from matrix. Thus, fibronectin has binding sites on the surface
receptors, collagen and sulfate proteoglycans.

13.3.3. Integrin
Between cells and matrix the complex relations are established. Due to multiple
interactions achieved on the level is very difficult to precisely establish where the membrane
components are finished and where the matrix begins. Sometimes, the cellular matrix
components are linked on the cells’ surface through the specific glycoproteic receptors.
The most known receptor for matrix is fibronectin receptor found on the surface of
mammalian cells. This receptor has 2 polypeptides called α and β, it acts as transmembranar
linker which mediates the interaction between intracellular skeleton (formed through actin)
and fibronectin (found in extracellular matrix).
There fore integrins are the mainly cell receptors to link the extracellular matrix, but
integrins are also connectors between matrix and cytoskeleton.

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