MCB 130L Lecture 4
Immunofluorescence of the Cytoskeleton
Inner Life of a Cell Video: http://multimedia.mcb.harvard.edu/media.html
Cell Biology Module
Overview
1.
Fluorescence/Immunofluorescence
Microscopy--Cytoskeleton
2.
Transfection & Vital Staining
3.
Respiration
4.
Cell Signaling
Lab: Immunofluorescence of the Cytoskeleton
Purpose:
1. to stain cells to observe the cytoskeleton
2. to observe and record the effects of different drugs
on cytoskeletal components and cell morphology
Actin
required for cell morphology & motility
Tubulin
forms microtubule “tracks” that enable chromosomes &
vesicles to move within cells
Cell Culture
propagation of cells outside the organism
Benefits:
1) Cellular environment easily observed and manipulated
a. Pharmaceutical manipulation
b. Genetic manipulation (transfection, RNAi)
c. Fluorescent tracers (live or fixed cells)
2) Homogeneous cells
3) Large quantities of cells
4) Investigation of diverse cellular functions
5) Noninvasive way to study mammalian cells
Drawbacks:
1) Requires care and $$$
2) May not demonstrate real cellular physiology
3) Easy to contaminate
Primary cultures
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Cells collected directly from tissue (Harrison, 1907)
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Advantage: cells have been minimally modified
Disadvantage:
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requires sacrifice of animal
mortal; must be generated for each experiment
heterogeneous cell population
Rat neurons and glial cells
Macrophage phagocytosis of E. coli
Cell lines
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Characterized by “immortality”
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A subset of cultured cells become “transformed” spontaneously
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Transformation of cells by expression of certain genes
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Derived from tumor cells (in vivo)
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Cell lines from different cell types have been derived
HeLa cells, 1951
Human cervical cancer cells
Bsc-1 cells, 1961
African Green Monkey kidney cells
How big are animal cells?
single cell (scanning EM)
colony of cells
>0.01 mm
1 mm
dish of
cell colonies
~10,000,000 HeLa cells
in a 100 mm dish
100 mm
Cytoskeleton
Actin
Microtubules
Nucleus
bovine pulmonary artery endothelial cells, Molecular Probes
Actin
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Structure
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Cell morphology and polarity
Specialized cell structures such as epithelial microvilli, hair
cell stereocilia, filopodia
Tracks for myosin motors
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Cell motility
Endocytosis, transport (protein, vesicles, organelles)
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Cytokinesis
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Muscle contraction
Actin cytoskeleton
Fibroblast
Intestinal microvilli
Hair cell stereocilia from ears - Belyantseva et al. (2005) Nat.Cell Biol. 7:148-156
From Lodish
Actin monomers form actin filaments
http://www.sinauer.com/cooper/4e/animations1201.html
monomer
model:
EM micrograph
Filament
model
From Lodish
Organization of actin filaments
Intestinal microvilli
platelet cytoskeleton
Microtubules
Structural
 cell morphology and polarity
 subcellular localization of organelles
Tracks for kinesin and dynein motors
 intracellular transport (protein, vesicles, organelles)
Motility
 cilia and flagella (specialized structures)
Mitosis
 Mitotic spindle
Microtubule cytoskeleton
From Lodish
Tubulin dimers form microtubules
http://www.sinauer.com/cooper/4e/animations1203.html
From Lodish
Drugs used in lab
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Taxol*
Nocodazole*
Latrunculin B*
Tumor promoter (TPA or PMA)
*alter the equilibrium between subunits and polymers
of actin or tubulin
Taxol

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Isolated from pacific yew
Binds and stabilizes microtubules
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Promotes lateral interactions between protofilaments
Low dose- blocks mitosis
High dose- increases polymerization
Taxol
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actin unaffected
Nocodazole
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Chemically synthesized
Low dose--arrests mitosis
High dose--rapidly depolymerizes microtubules
Nocodazole
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actin unaffected
Latrunculin B
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Isolated from red sea sponge
Binds actin monomers and inhibits polymerization
Causes loss of actin fibers (collapse onto nucleus) due to
continued disassembly
Latrunculin B
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Alters microtubule morphology as well
Phorbol Myristate Acetate
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Tumor promoter
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Increases frequency w/ which certain chemicals cause cancer
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Mimics 1,2-diacylglycerol (DAG)--activates protein kinase C
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Changes in cell growth, cell shape, and the cytoskeleton
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Affects actin cytoskeleton
Steps in Cell Staining
1.
Fix cells
2.
Permeabilize cells
3.
Add antibodies or staining reagent
4.
Mount coverslips
Cell Fixation
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Aldehydes (formaldehyde, glutaraldehyde)
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Cross-links amino groups
Preserves cell structure (+)
Can block antibody access (-)
Alcohols (methanol, ethanol)
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Removes lipids, dehydrates cells, precipitates proteins
Fast and easy (+)
Poor morphology (-)
*Both may result in denatured antigen
Permeabilization
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Necessary for staining of intracellular proteins
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Can expose antigenic epitopes
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Detergents (Tx-100) or Methanol used to
solubilize cell membranes
Triton X-100
Antibodies
(indirect immunofluorescence)
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1o antibody: mouse anti-tubulin
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2o antibody: goat anti-mouse
(conjugated to a fluorophore or
other tag for visualization)
Anti-tubulin Antibody
1°mouse anti-alpha-tubulin
2° Cy2-goat anti-mouse Fab
NIH/3T3 cells
From http://www.microscopyu.com
Phalloidin
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Phallotoxin from Amanita phalloides
mushroom (“Death cap”)
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Binds filamentous actin only
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Directly conjugated to fluorophore
(i.e. rhodamine) for visualization
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Membrane impermeable
DAPI and Hoechst
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Fluorescent molecules that emit blue under UV
Bind directly to DNA
Allow visualization of the nucleus
Membrane permeable
Hoechst
Anti-tubulin
Fluorescence Microscopy
Video: http://probes.invitrogen.com/resources/education/tutorials/1Introduction/player.html
Experiment
Drug X
Stain treated and untreated
control cells for actin/microtubules
Examine cells by
fluorescence microscopy
BSC-1 cells in culture
How does drug tmt affect the overall cell morphology?
Does drug tmt affect the actin and/or microtubule cytoskeleton?
Microbe Astronomer