2-Methods Cell Biol 2011

8/4/2019 2-Methods Cell Biol 2011

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Core Tools of Cell Biology"


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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 trafficking relies on the COPI coat, which by assembling intoa lattice on Golgi membranes concentrates cargo at specific sites and

deforms the membranes at these sites into coated buds and carriers. TheGTPase-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 coatomerin 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 inArfGAP1 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."

(from last slide set)


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Why is it so hard to figure out how cells work? Dont we have the

power to observe cellular processes in incredible detail?


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What are the

limits to what

can be seen

with a lightmicroscope?


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Light Microscopes 1: Transmitted light Light microscopes

magnify a specimen

using one or morelenses Cells are mostly

transparent. Theyneither reflect norabsorb much light

(unless colored, e.g.chloroplasts) orstained)

To bring out detail(contrast), we need touse constructive or

destructiveinterference Contrast= the

difference inintensity betweenan object and the

adjacentbackground


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Techniques for enhancing contrast (9-8)Brightfield

(transmitted light only)

Differential InterferenceContrast (DIC)

Phase contrast

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

Chemical stain


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Two ways to enhance the contrast of cellular structures

Modulate Intensity Modulate Phase


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Magnification and Resolution Magnification is dependent upon the lens used and

the amount you blow up the initial image

The resolution is dependent upon the properties oflight 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 magnification It is determined by:

The wavelength of light Shorter wavelength=better resolution

The angles of light collected by the lens

Greater angle=better resolution


10/36Unresolved structures Resolved structures

Actual structure Image seen

Magnification

is not the same as resolution


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Microscope Resolution


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Basic properties of light and the lens determine resolution

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


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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

cant 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 doesnt mean you can see the mitochondria inthe microscope (that depends on contrast), only that

you can resolve two objects that distance apart if youcan see them.


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What all this means practically

100 200 300 400 500

Actual object size (nm)

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 closetogether

4 50 nm objects close

together

E ifl Mi d ti ll i


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Epifluorescence Microscopy dramatically improves

contrast and allows specific structures to be labeledFluorescence - amolecule absorbs

light of one

wavelength and

then re-emits it ata longer

wavelengthFluorophores(fluors) an be

linked to various

chemicals or

biological

molecules to label

structuresspecifically

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


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Green- Fluorescent

protein

Yellow- antibody

Red- Fluorescent

protein

Purple- antibody

Blue- DNA binding

dye


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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 fixed cell and allow it to bind its target Use a fluorescent secondary antibody (anti-rabbit or mouse) to bind to the

primary antibody (Indirect)


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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


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Immunoblotting

or Western blotting


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Immunoisolation

G P i (GFP)


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Green Fluorescent Protein (GFP)-

A genetically encodedfluorescence marker

GFP Tubulin N- -C


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GFP-tubulin movie


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Amoeba cells expressing GFP-Coronin fusion

protein (green) phagocytosing (engulfing and

eating) yeast (red)


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Transfection of cells is a useful

cell biology toolTransfection: to infect a cell with foreign DNA; to

cause a foreign protein to be expressed in a cellIn addition to GFP-tagged versions of moleculespresent in a cell, you can express (either tagged or

not):Molecules that arent normally present in a cellMutant molecules that are constitutively active- i.e. activeall the timeMutant molecules that are dominant negative- i.e. dont

function right and block the function of the cells ownversion of the molecule

Confocal microscopy deblurs images by removing out of focus light from


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Confocal microscopy deblurs images by removing out of focus light from

above and below the focal plane of interestLaser-scanning

confocalmicroscope

uses pinholes to

deblur

Digital

deconvolutionuses

computational

methods to

deblur

Th l ti


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Scale bar =500 nm

STED microscopy reveals thatsynaptotagmin remains clustered

after synaptic vesicle

exocytosisKatrin I. Willig, SilvioO. Rizzoli, Volker Westphal,Reinhard Jahn andStefan W.HellNature 440, 935-939 (13April 2006)doi:10.1038/nature04592

The resolutionlimit of lightmicroscopy can been

broken with somereally fancy ($$) optics

H d t l t d th f d t l li it


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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 resolutionTransmission 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 ismuch 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

Th t i i l t i j t


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The transmission electron microscope: just

like the light microscopeexcept with

electrons!!


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Sample Preparation for TEM EM must be done in a vacuum for electron gun to work

Cant 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 dont get through What you see is the scattering of electrons by the metal. There is no

biological material left!

TEM f l t ll (9 25)


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TEM of a plant cell (9-25)


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Immuno-electron microscopy You cant 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

specific proteins in the EM Very difficult technique!


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l


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Scanning Electron Microscope Used to look at surfaces

of structures Samples are fixed,

passed through series ofalcohols, and dried.

Surface of sample iscoated with a layer ofmetal.

The electron beam isscanned across thesurface and thereflection of electrons ateach point measured

C li ht TEM d SEM i (9 30)


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


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Cells can be grown artificially in

culture Transformed (i.e. cancerous cells) can be growncontinuously in culture- HeLa cells (cervical

cancer cells) for example have been growingcontinuously 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


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Organelles can be isolated from

fractionated cells

2-Methods Cell Biol 2011

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