Cell and Molecular Biology

Lecture 9

The Cytoskeleton
and Cell Motility

John Donnie A. Ramos, Ph.D.

Department of Biological Sciences
College of Science
University of Santo Tomas

Cytoskeleton
€ The “skeletal system” of cells
€ Composed of filamentous structures
 Microtubules – rigid tubes
€ tubulin
 Microfilaments – solid, thin structures
€ actin
 Intermediate filaments – tough, ropelike fibers
€ keratin, vimentin, desmin, Lamin etc.

€ Highly dynamic structures

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Functions of the Cytoskeleton

Microtubules and Peroxisomes

Peroxisomes are labeled with GFP while microtubules are labeled

with antibodies fused with a red fluorescent dye.

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 Studying the Cytoskeleton
€ Fluorescence microscopy
 Real-time study
 Ideal for studying the dynamics of cytoskeleton
 Uses antibodies and fluorescent dyes/proteins
 Identifying the location proteins in small amounts

centrin

Studying the Cytoskeleton

€ Video microscopy
 Monitoring of cell movement (real-time)
 In vitro motility assay
 Used to study motor proteins
 Made possible with the development of
nanotechnology

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 Studying the Cytoskeleton
€ Genetically engineered cells
 Based on recombinant DNA technology
 Uses modified proteins (site directed mutagenesis)
 Knockout animals
 Overexpression of mutant proteins (transfection)

Normal pigment cell with pigment Mutant cell conatining modified

granules equally distributed throughout of kinesin II resulting in aggregation of
the cell with the help of kinesin II pigment granules

Microtubules
Plus (+) end
€ Hollow, tubular structures
€ Found in nearly all eukaryotic cell
€ Form mitotic spindle, flagella, cilia
€ Outer diameter: 24 nm; thickness: 5 nm
€ Composed of 13 protofilaments (globular proteins)

Minus (-) end

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Microtubule-Associated Proteins
€ MAPs are found only in brain tissue
€ With a domain attached to
microtubule and a domain
extending outward as a filament
€ Form cross bridges connecting
microtubules
€ Alter rigidity or influence the rate of
assembly
€ Phosphorylated proteins
€ Mutation of a MAP protein (tau)
resulting to overphosphorylation
causes dementia (formation of
neurofibrillar tangles)

Microtubule Function
€ Structural Support and Organizers
 Serve as mechanical support
Axon
 Determine cell shape
 Tracks for organelles
 Maintains internal organization of cells

Lysosome
Vesicles

Microtubules of culture mouse cell

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 Microtubule Function
€ Agents of intracellular motility
 Axonal transport (movement of
proteins and neurotransmitters along
the axon)
 Rate of movement: 5 µm/sec (400
mm/day)
 Both anterograde and retrograde
direction
 Microtubules are the main tracks

Motor Proteins
 Utilizes ATP – conversion of chemical energy to mechanical energy
 Types of motor proteins:
€ Myosins – move along microfilaments
€ Kinesins – move along microtubules
€ Dyneins – move along microtubules
 Move in unidirectional manner along a track
 During movement, motor proteins undergo mechanical and
chemical cycle (series of conformational changes providing the
necessary fuel for movement)
 Steps:
€ ATP binding to motor protein
€ ATP hydrolysis
€ Release of products (ADP and Pi)

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 Kinesins
€ First isolated in 1985 from squid giant
axons
€ Tetramer (2 identical heavy chains and
2 identical light chains)
€ Globular head – force generating
“engine”
€ Tail region – binds to cargo (vesicle)
€ Movement is a coordinated activity
between the 2 heads)
€ Movement towards + end
€ Belong to a family of proteins called
KLPs (Kinesin-like Proteins) or KRPs
(Kinesin-related Proteins)
€ XKCM1 – KLP that is incapable of
movement (destabilize microtubules)

Kinesin-Mediated Organelle Transport

Microtubules

Mitochondria

Normal 9.5 day mouse KIF5B kinesin-deficient 9.5

embryo cell day mouse embryo cell

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 Cytoplasmic Dynein
€ First isolated in 1963 from cilia and
flagella (cytoplasmic form was
discovered in 1983)
€ Molecular mass: 1.5 million D
€ 2 identical heavy chain and variety of
intermediate and light chains
€ Movement towards minus end
€ Functions:
 Spindle positioning and chromosome
movement during cell division
 Minus end-directed positioning of GC
 Movement of vesicles and organelles in
the cytoplasm
€ Dynactin – regulate dynein activity and
binds dynein to microtubules

Model of Motor-Mediated Transport

Kinesin: Dynein:
anterograde retrograde and
movement anterograde
movement

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 Microtubule-Organizing Centers
€ Specialized structures involved in microtubule nucleation and
organization
€ Microtubules – assembly of αβ-tubulin dimers
€ Stages of microtubule assembly:
 Nucleation – slower phase (initial formation of a part of microtubule)
 Elongation – rapid phase (formation of the entire organelle)

Centrosomes
€ A complex of 2 centrioles surrounded by amorphous,
electron dense pericentriolar material (PCM)
€ Site of microtubule nucleation
€ Centrioles
 9 triplets of tubules
 In pairs arranged at right angle
€ Minus end of microtubule is associated with centrosome
while plus end (growing end) is situated on the other side.

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 MTOC of Plant Cells
€ Plant cells lack both centrosomes and centrioles
€ Microtubule-organizing center originates near the nuclear envelope
€ Microtubule nucleation throughout the plant cell cortex

Localization of microtubules Origin of nucleation on the

nuclear envelope

Microtubule Nucleation
€ Tubulin – protein component of all MTOCs
€ Types of tubulin:
 γ tubulin
€ the main protein involved in microtubule nucleation
€ 0.005 % of the total cell protein content
 β tubulin
 α tubulin

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 Dynamic Nature of Microtubules

€ Microtubules assemble and disassemble regularly

€ Result of polymerization and depolymerization

Interphase: Pre-prophase: Mitosis: microtubules Late Telophase:

microtubules microtubules form a single form spindle fibers microtubules form
distributed throughout transverse band (mitotic spindles) phragmoplast (involved in
the cell cortex (preprophase band)- site cell wall formation
of future division plane between daughter cells)

Microtubules Assemble In Vitro

€ First performed by Richard Weisenberg in 1972
using brain cell homogenate.
€ Assembly occurs at 37°C but disassembles at
lower or higher temperatures
€ Requires GTP for assembly (after binding of
tubulin dimer to a growing microtubule)
€ GTP is hydrolyzed to GDP
€ Structure cap model of dynamic instability (a
process of assembling and disassembling
microtubules)

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 Microtubules Assemble In Vivo

Dynamic Instability -
ability to grow and
shrink (assemble
and disassemble)
in vivo

Eukaryotic Cilia
€ Locomotory organs
€ Contains microtubules with dynein arms
€ Responsible for basic vertebrate body plan
(sidedness of some organs)
€ Cells of the embryonic node during
gastrulation stage contain cilia responsible for
the movement of cells
€ Abnormal cilia causes “situs inversus”

Cilia on the surface of the

Protozoan cilia in metachronal waves fimbrium of mouse oviduct

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 Eukaryotic Flagella
€ Locomotory organs
€ Contains microtubules with dynein
arms
€ Fewer but longer (compared to cilia)

Chlamydomonas reinhardtii

“Breast stroke”-like movement

Ciliary or Flagellar Axoneme

€ Axoneme is the central core of cilia

or flagella containing 9 doublets and
1 central pair of microtubules (9+2
array structure).
€ Microtubules are of same polarity (-
end at the base and + end at the
tip)

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 Ciliary or Flagellar Axoneme

Longitudinal view of axoneme Basal bodies and axoneme

Ciliary Dynein
€ Protein responsible for the
conversion of ATP into mechanical
energy of ciliary locomotion
€ Discovered by Ian Gibbons using an
experiment involving the “chemical
dissection of cilia from the protozoan
Tetrahymena

Heavy chains

Intermediate
chains

Light chains

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 Ciliary Motility
€ Causes motility by sliding mechanism
(microtubule sliding)
€ Dynein arms act as swinging cross-bridges
between A and B tubules

Intermediate Filaments
€ Size between microtubules and
microfilaments
€ Solid, smooth-surfaced,
unbranched
€ Average diameter: 10 nm
€ Found around cytoplasm
€ Heterogenous group of
cytoskeletons encoded by at least
50 different genes in humans
€ Basic unit pattern: tetramer
(antiparallel, staggered dimers)
€ Tetramer lacks polarity
€ Dimers exist as homodimers or
heterodimers

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 Major Mammalian IFs

Keratins
€ Type I keratins
 Acidic keratins
€ Type II keratins
 Basic or neutral keratins
€ Keratin heterodimers are
combinations of different
types
€ Originate on the nuclear
membrane, radiate
Cultured skin cells (keratinocytes)
around the cytoplasm and
terminate in desmosomes
and hemidesmosomes

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 Neurofilaments
€ Fibers are parallel to
nerve cell axon
€ Type IV proteins: NF-L,
NF-H, NF-M
€ Main supporting
cytoskeleton of neurons
(axons) as they increase
in diameter

Microfilaments
€ Major contractile proteins of muscle
cells
€ Composed of actin
€ Also called “actin filament” or “F actin”
€ 8 nm in diameter (smallest among
cytoskeletons)
€ Responsible for cell and organelle
motility
€ Actin monomer composed three
subunits (each subunit with 4
subdomains)
€ Can form double helical structure
(dimer)
€ Highly conserved gene
€ Interacts with myosin (motor protein)

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 Actin Assembly
€ Actin is an ATPase (binds ATP)
€ Polymerization and depolymerization of
actin filaments can be simulated in vitro
€ Addition (assembly) of actin subunits
occurs at the + end (fast growing end)
€ Disassembly occurs at the – end
€ Microfilament assembly and
disassembly can be inhibited by
cytochalasins (promotes
depolymerization of microfilaments)
and phalloidin ( increase microfilament
stability)

Actin Polymerization
€ Force-generating
mechanism for cell motility
without the use of motor
proteins
€ Examples:
 Acrosomal Reaction (rapid
extension of the
acrosomal filament to the
antierior tip of
spermatozoon)
 Propulsion of of Listeria
monocytogenes to the
cytoplasm of an infected
cell

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 Myosin
€ Molecular motor of actin
filaments
€ 2 globular heads
 Actin binding site
 Catalytic site (hydrolyses ATP)
€ 2 neck regions (α helix) – heavy
chain
 2 essential light chains
 2 regulatory light chains
€ Tail region – intertwined heavy
chains (coiled coil protein)

In Vitro Motility Assay for Myosin

€ Myosin heads immobilized on cover slip

€ Allowed to react with actin extracts
€ Observed for movement of actin
€ Video microcopy

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 Bipolar Myosin II Filament

€ Myosin heads (globular)

on opposite ends while
tails overlap at the center
of the filament
€ Plays a structural role in
muscle cells (stable
componant of the
contractile apparatus)
€ Results in a bipolar
filament

Uncoventional Myosins
€ Myosin I
 With single head
 Cannot assemble into filaments
(in vitro)
 Can move actin (motor protein)
 Localized on plasma membrane
(generates forces on cell
surface)
 First isolated in Acanthamoeba
€ Myosin V
 Involved in the transport of
pigment cells in humans
 Abnormality results in partial
albisms
€ Myosin VII
 Locallized in hair cells of cochlea
of the inner ear
 Mutation results deafness

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 Muscle Fibers
€ Results of myoblast fusion
(multinucleated)
€ 10-100 µm thick
€ Composed of myofibrils with contractile
units called sarcomeres
€ Sarcomere composed of overlapping
actin (thin) and myosin (thick)
filaments
€ Each sarcomere separated by a Z line

Contractile Machinery of Sarcomere

M Line

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 Sliding Filament Model
€ Explains the mechanism of
muscle contraction
€ Decreased width of the I band
and H zone

Associated Proteins
€ Tropomyosin
 Rod-shaped protein
associated with 7actin
filaments

€ Troponin
 Binding site of Ca+ during
muscle contaction

€ Titin
 Prevents overstretching of
sarcomere

€ Nebulin
 Regulates the number of
actin monomers assembling
into thin filaments

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Actinomyosin Contractile Cycle

1. ATP-binding (actin
detaches from myosin)
2. Hydrolysis of ATP
3. Weak interaction of
actin with myosin
4. Release of Pi (Power
Stroke)
5. Release of ADP
(attachment of myosin
to actin)

Functional Anatomy of Muscle Fiber

1. Arrival of nerve impulse

causes the release of Ca+
from SR
2. Ca+ binds to troponin
3. Conformational change of
troponin subunit
4. Movement of trpomyosin
5. Myosin binding site is
exposed thus myosin
interacts with actin

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Actin-Binding Proteins

Actin-Binding Proteins

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Nonmuscle Motility and Contractility

€Cytokinesis
€Phagocytosis
Stress fibers
€Cytoplasmic streaming
€Vesicle trafficking
€Blood platelet activation
€Lateral movement of integral
membrane proteins
€Cell-substratum interactions
€Cell locomotion
€Axonal outgrowths
€Changes in cell shape

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