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MCB 130L Lecture 4

MCB 130L Lecture 4

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MCB 130L Lecture 4

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  1. MCB 130L Lecture 4 Immunofluorescence of the Cytoskeleton

  2. Cell Biology ModuleOverview Inner Life of a Cell Video: http://multimedia.mcb.harvard.edu/media.html • Fluorescence/Immunofluorescence Microscopy--Cytoskeleton • Transfection & Vital Staining • Respiration • Cell Signaling

  3. Lab: Immunofluorescence of the Cytoskeleton • Purpose: • to stain cells to observe the cytoskeleton • 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

  4. Cell Culturepropagation of cells outside the organism • Benefits: • Cellular environment easily observed and manipulated • Pharmaceutical manipulation • Genetic manipulation (transfection, RNAi) • Fluorescent tracers (live or fixed cells) • Homogeneous cells • Large quantities of cells • Investigation of diverse cellular functions • Noninvasive way to study mammalian cells • Drawbacks: • Requires care and $$$ • May not demonstrate real cellular physiology • Easy to contaminate

  5. 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

  6. 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, 1951 Human cervical cancer cells Bsc-1 cells, 1961 African Green Monkey kidney cells

  7. single cell (scanning EM) colony of cells dish of cell colonies 100 mm >0.01 mm 1 mm How big are animal cells? ~10,000,000 HeLa cells in a 100 mm dish

  8. Cytoskeleton Actin Microtubules Nucleus bovine pulmonary artery endothelial cells, Molecular Probes

  9. 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

  10. Actin cytoskeleton Fibroblast Intestinal microvilli From Lodish Hair cell stereocilia from ears - Belyantseva et al. (2005) Nat.Cell Biol. 7:148-156

  11. Actin monomers form actin filaments http://www.sinauer.com/cooper/4e/animations1201.html monomer model: EM micrograph Filament model From Lodish

  12. Organization of actin filaments Intestinal microvilli platelet cytoskeleton

  13. 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

  14. Microtubule cytoskeleton From Lodish

  15. Tubulin dimers form microtubules http://www.sinauer.com/cooper/4e/animations1203.html From Lodish

  16. Drugs used in lab • Taxol* • Nocodazole* • Latrunculin B* • Tumor promoter (TPA or PMA) *alter the equilibrium between subunits and polymers of actin or tubulin

  17. 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

  18. Nocodazole • Chemically synthesized • Low dose--arrests mitosis • High dose--rapidly depolymerizes microtubules • actin unaffected Nocodazole

  19. 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

  20. 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

  21. Steps in Cell Staining • Fix cells • Permeabilize cells • Add antibodies or staining reagent • Mount coverslips

  22. 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

  23. Permeabilization • Necessary for staining of intracellular proteins • Can expose antigenic epitopes • Detergents (Tx-100) or Methanol used to solubilize cell membranes Triton X-100

  24. Antibodies (indirect immunofluorescence) • 1o antibody: mouse anti-tubulin • 2o antibody: goat anti-mouse (conjugated to a fluorophore or other tag for visualization)

  25. Anti-tubulin Antibody 1°mouse anti-alpha-tubulin 2° Cy2-goat anti-mouse Fab NIH/3T3 cells From http://www.microscopyu.com

  26. Phalloidin • Phallotoxin from Amanita phalloides mushroom (“Death cap”) • Binds filamentous actin only • Directly conjugated to fluorophore (i.e. rhodamine) for visualization • Membrane impermeable

  27. DAPI and Hoechst • Fluorescent molecules that emit blue under UV • Bind directly to DNA • Allow visualization of the nucleus • Membrane permeable Hoechst Anti-tubulin

  28. Fluorescence Microscopy Video: http://probes.invitrogen.com/resources/education/tutorials/1Introduction/player.html

  29. 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?

  30. Microbe Astronomer