Core Tools of Cell Biology

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(from last slide set) ArfGAP1 dynamics and its role in COPI coat assembly on Golgi membranes of living cells "
" Wei Liu1, Rainer Duden3, Robert D. Phair2, and Jennifer Lippincott-Schwartz1

Secretory protein trafcking relies on the COPI coat, which by assembling into a lattice on Golgi membranes concentrates cargo at specic sites and deforms the membranes at these sites into coated buds and carriers. The GTPase-activating protein (GAP) responsible for catalyzing Arf1 GTP hydrolysis is an important part of this system, but the mechanism whereby ArfGAP is recruited to the coat, its stability within the coat, and its role in maintenance of the coat are unclear. Here, we use FRAP to monitor the membrane turnover of GFP-tagged versions of ArfGAP1, Arf1, and coatomer in living cells. ArfGAP1 underwent fast cytosol/Golgi exchange with 40% of the exchange dependent on engagement of ArfGAP1 with coatomer and Arf1, and affected by secretory cargo load. Permanent activation of Arf1 resulted in ArfGAP1 being trapped on the Golgi in a coatomer-dependent manner. These data suggest that ArfGAP1, coatomer and Arf1 play interdependent roles in the assemblydisassembly cycle of the COPI coat in vivo."

Why is it so hard to gure out how cells work? Don t we have the power to observe cellular processes in incredible detail?

The reality is far less glamorous

What are the limits to what can be seen with a light microscope?

Light Microscopes 1: Transmitted light

Light microscopes magnify a specimen using one or more lenses
Cells are mostly transparent. They neither reect nor absorb much light (unless colored, e.g. chloroplasts) or stained)
To bring out detail (contrast), we need to use constructive or destructive interference
Contrast= the difference in intensity between an object and the adjacent background

Techniques for enhancing contrast (9-8)

Brightfield (transmitted light only) Chemical stain

Phase contrast Differential Interference Contrast (DIC)

For more, see http://micro.magnet.fsu.edu/primer/techniques/index.html

Two ways to enhance the contrast of cellular structures

Modulate Intensity Modulate Phase

Magnication and Resolution

Magnication is dependent upon the lens used and the amount you blow up the initial image
The resolution is dependent upon the properties of light and how it interacts with the specimen

Resolution = how far apart two objects have to be to be seen as two separate objects
It is not related to magnication
It is determined by:

The wavelength of light

Shorter wavelength=better resolution

The angles of light collected by the lens

Greater angle=better resolution

Magnication

is not the same as resolution

Actual structure Image seen

Unresolved structures

Resolved structures

Microscope Resolution

Basic properties of light and the lens determine resolution

http://micro.magnet.fsu.edu/primer/java/imageformation/airyna/index.html

Limits of Resolution

You can only get so close to increase the angle and then you run out of space
You can only go so low in wavelength and then you can t see it or you destroy your eyes while looking
Best resolution for optical microscope = ~200 nm for visible light
About the width of a mitochondrion
This doesn t mean you can see the mitochondria in the microscope (that depends on contrast), only that you can resolve two objects that distance apart if you can see them.

What all this means practically

An object 200 nm or smaller will appear to be 200 nm regardless of its actual size If you see a 200 nm spot in a microscope, it could be: 1 200 nm object 2 100 nm objects close together 4 50 nm objects close together

100

200

300

400

500

Actual object size (nm)

Epiuorescence Microscopy dramatically improves contrast and allows specic structures to be labeled

Fluorescence - a molecule absorbs light of one wavelength and then re-emits it at a longer wavelength
Fluorophores (uors) an be linked to various chemicals or biological molecules to label structures specically

http://www.shsu.edu/~chemistry/chemiluminescence/JABLONSKI.html

Green- Fluorescent protein Yellow- antibody Red- Fluorescent protein Purple- antibody Blue- DNA binding dye

Immunocytochemistry uses antibodies to visualize proteins in cells

Obtain pure protein
Make an antibody to it by injecting it into a rabbit or mouse (primary antibody)
Fix and permeabilize the cell
Fix: add chemicals that cross-link everything in the cell to nearby molecules
Permeabilize: add a detergent to remove some of the membrane so antibodies can get in
Add the antibody to the xed cell and allow it to bind its target
Use a uorescent secondary antibody (anti-rabbit or mouse) to bind to the primary antibody (Indirect)

Antibodies are all around useful molecules

In addition to Immunocytochemistry, antibodies are also useful for: Immunoblotting Immunoprecipitation Immunoisolation There are monoclonal antibodies and polyclonal antisera

Immunoblotting or Western blotting

Immunoisolation

Green Fluorescent Protein (GFP)- A genetically encoded uorescence marker

N-

GFP

Tubulin

-C

GFP-tubulin movie

Amoeba cells expressing GFP-Coronin fusion protein (green) phagocytosing (engulng and eating) yeast (red)

Transfection of cells is a useful cell biology tool

Transfection: to infect a cell with foreign DNA; to cause a foreign protein to be expressed in a cell
In addition to GFP-tagged versions of molecules present in a cell, you can express (either tagged or not):

Molecules that aren t normally present in a cell
Mutant molecules that are constitutively active- i.e. active all the time
Mutant molecules that are dominant negative- i.e. don t function right and block the function of the cell s own version of the molecule

Confocal microscopy deblurs images by removing out of focus light from above and below the focal plane of interest

Laser-scanning confocal microscope uses pinholes to deblur

Digital deconvolution uses computational methods to deblur

The resolution limit of light microscopy can been broken with some really fancy ($$) optics
STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosisKatrin I. Willig, Silvio O. Rizzoli, Volker Westphal, Reinhard Jahn andStefan W. HellNature 440, 935-939 (13 April 2006)doi:10.1038/ nature04592

Scale bar =500 nm

How do most people get around the fundamental limits of resolution for light microscopy?

Use electron microscopes
Electron beams have a shorter wavelength and thus a higher resolution
Transmission electron microscope (TEM)- images electrons that pass through a thin specimen


Resolution=.6/NA


(wavelength) of electron = 0.004 nm (as opposed to 400-500nm

for light)
Actual resolution = 0.1-2 nm (100-1000x better than light microscope)
If lenses were as good as optical ones, resolution would be 0.002 nm (100,000x better than light) but NA of magnetic lenses is much worse

Scanning Electron microscope (SEM)- images electrons scattered by an intact object. Depth of focus gives images a three-dimensional quality.

Resolution of SEM is about 5 nm

The transmission electron microscope: just like the light microscopeexcept with electrons!!

Sample Preparation for TEM

EM must be done in a vacuum for electron gun to work
Can t have wet samples
Dry tissue does not have enough density to scatter electrons so you have to replace it with something dense-
Bind metals like osmium and platinum to membranes and proteins
Now the material is too dense for electrons to penetrate
Section material into very thin slices
Procedure
Fix Tissue (glutaraldehyde or osmium)
Stain with Osmium, lead etc. or make metal replica
Dehydrate and embed with plastic
For TEM- Cut thin slices (sections) (0.02-0.1m thick)- sample must be thin otherwise electrons don t get through
What you see is the scattering of electrons by the metal. There is no biological material left!

TEM of a plant cell (9-25)

Immuno-electron microscopy

You can t see antibodies in the EM, but you can attach dense particles to antibodies to make them visible in the EM
Allows you to visualize the localization of specic proteins in the EM
Very difcult technique!

Scanning Electron Microscope

Used to look at surfaces of structures
Samples are xed, passed through series of alcohols, and dried. Surface of sample is coated with a layer of metal.
The electron beam is scanned across the surface and the reection of electrons at each point measured

Compare light, TEM and SEM microscopy (9-30)

Cells can be grown articially in culture

Transformed (i.e. cancerous cells) can be grown continuously in culture- HeLa cells (cervical cancer cells) for example have been growing continuously since the 1950s.
Many of these cell lines duplicate key features of normal cells and can be used to study important processes
Primary cells (non-cancerous, non-transformed) can also be cultured, usually more laboriously

Organelles can be isolated from fractionated cells