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MCB 130L Lecture 4
Immunofluorescence of the Cytoskeleton
Cell Biology ModuleOverview
1. Fluorescence/Immunofluorescence Microscopy--Cytoskeleton
2. Transfection & Vital Staining
3. Respiration
4. Cell Signaling
Inner Life of a Cell Video: http://multimedia.mcb.harvard.edu/media.html
Actinrequired for cell morphology & motility
TubulinTubulinforms microtubule “tracks” that enable chromosomes & forms microtubule “tracks” that enable chromosomes & vesicles to move within cellsvesicles to move within cells
Purpose: 1. to stain cells to observe the cytoskeleton2. to observe and record the effects of different drugs
on cytoskeletal components and cell morphology
Lab: Immunofluorescence of the Cytoskeleton
Benefits:
1) Cellular environment easily observed and manipulateda. 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
Cell Culturepropagation of cells outside the organism
Primary cultures
Cells collected directly from tissue (Harrison, 1907) Advantage: cells have been minimally modified Disadvantage:
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
Characterized by “immortality”
A subset of cultured cells become “transformed” spontaneously
Transformation of cells by expression of certain genes
Derived from tumor cells (in vivo)
Cell lines from different cell types have been derived
HeLa cells, 1951Human cervical cancer cells
Bsc-1 cells, 1961African Green Monkey kidney cells
dish of cell colonies
100 mm
single cell (scanning EM)
>0.01 mm
colony of cells
1 mm
How big are animal cells?
~10,000,000 HeLa cells in a 100 mm dish
Cytoskeleton
ActinMicrotubulesNucleus
bovine pulmonary artery endothelial cells, Molecular Probes
Actin
Structure Cell morphology and polarity Specialized cell structures such as epithelial microvilli, hair
cell stereocilia, filopodia
Tracks for myosin motors Cell motility Endocytosis, transport (protein, vesicles, organelles)
Cytokinesis
Muscle contraction
Hair cell stereocilia from ears - Belyantseva et al. (2005) Nat.Cell Biol. 7:148-156
Actin cytoskeleton
Fibroblast
Intestinal microvilli
From Lodish
Actin monomers form actin filamentshttp://www.sinauer.com/cooper/4e/animations1201.html
monomermodel: EM micrograph
Filamentmodel
From Lodish
Organization of actin filaments
Intestinal microvilli platelet cytoskeleton
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
Microtubules
Microtubule cytoskeleton
From Lodish
Tubulin dimers form microtubuleshttp://www.sinauer.com/cooper/4e/animations1203.html
From Lodish
Drugs used in lab
Taxol* Nocodazole* Latrunculin B* Tumor promoter (TPA or PMA)
*alter the equilibrium between subunits and polymers of actin or tubulin
Taxol
Isolated from pacific yew Binds and stabilizes microtubules
Promotes lateral interactions between protofilaments Low dose- blocks mitosis High dose- increases polymerization
actin unaffected
Taxol
Nocodazole
Chemically synthesized Low dose--arrests mitosis High dose--rapidly depolymerizes microtubules
actin unaffected
Nocodazole
Latrunculin B
Isolated from red sea sponge Binds actin monomers and inhibits polymerization Causes loss of actin fibers (collapse onto nucleus) due to
continued disassembly
Alters microtubule morphology as well
Latrunculin B
Phorbol Myristate Acetate
Tumor promoter
Increases frequency w/ which certain chemicals cause cancer
Mimics 1,2-diacylglycerol (DAG)--activates protein kinase C
Changes in cell growth, cell shape, and the cytoskeleton
Affects actin cytoskeleton
Steps in Cell Staining
1. Fix cells
2. Permeabilize cells
3. Add antibodies or staining reagent
4. Mount coverslips
Cell Fixation
Aldehydes (formaldehyde, glutaraldehyde) Cross-links amino groups Preserves cell structure (+) Can block antibody access (-)
Alcohols (methanol, ethanol) Removes lipids, dehydrates cells, precipitates proteins Fast and easy (+) Poor morphology (-)
*Both may result in denatured antigen
Permeabilization
Necessary for staining of intracellular proteins
Can expose antigenic epitopes
Detergents (Tx-100) or Methanol used to
solubilize cell membranes
Triton X-100
Antibodies (indirect immunofluorescence)
1o antibody: mouse anti-tubulin
2o antibody: goat anti-mouse (conjugated to a fluorophore or other tag for visualization)
1°mouse anti-alpha-tubulin2° Cy2-goat anti-mouse Fab
Anti-tubulin Antibody
NIH/3T3 cells
From http://www.microscopyu.com
Phalloidin
Phallotoxin from Amanita phalloides
mushroom (“Death cap”)
Binds filamentous actin only
Directly conjugated to fluorophore
(i.e. rhodamine) for visualization
Membrane impermeable
DAPI and Hoechst
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
BSC-1 cells in culture
Drug X Stain treated and untreatedcontrol cells for actin/microtubules
Examine cells by fluorescence microscopy
How does drug tmt affect the overall cell morphology?Does drug tmt affect the actin and/or microtubule cytoskeleton?
Microbe Astronomer