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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
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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
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