1. MCB 130L Lecture 4 Immunofluorescence of the Cytoskeleton

2. Cell Biology Module Overview 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

3. Actin required for cell morphology & motilityTubulin forms microtubule “tracks” that enable chromosomes & vesicles to move within cells 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 Lab: Immunofluorescence of the Cytoskeleton

4. 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 Cell Culture propagation of cells outside the organism

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 cellsMacrophage 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. 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

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

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 microvilliplatelet cytoskeleton

13. 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

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 1. Fix cells 2. Permeabilize cells 3. Add antibodies or staining reagent 4. 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) 1 o antibody: mouse anti-tubulin 2 o antibody: goat anti-mouse (conjugated to a fluorophore or other tag for visualization)

25. 1°mouse anti-alpha-tubulin 2° Cy2-goat anti-mouse Fab Anti-tubulin Antibody 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 BSC-1 cells in culture Drug X Stain treated and untreated control 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?

30. Microbe Astronomer