David Weisblat 385 Life Sciences Addition Phone 642-8309 Email: weisblat@berkeley.edu

1. David Weisblat 385 Life Sciences Addition Phone 642-8309 Email: weisblat@berkeley.edu Office hours by appointment

2. Cell culture = propagation of cells outside the organism Advantages: 1) cellular environment can be manipulated 2) cell type well defined 3) large quantities of cells can be obtained 4) many cellular functions can be investigated Caveat: How well does what we see in the dish correspond to what goes on in the animal? Three types of cultured cells: 1) primary cultures 2) cell strains 3) cell lines

3. colony of cells single cell- scanning EM 0.01 mm 1 mm dish of cell colonies 100 mm

4. 1 day spinal cord explant + lymphatic fluid axons grow in culture! Primary Cultures • Cells derived directly from tissues First developed in 1907:

5. cultured neuron extending processes

6. dissociate cells media plate cells tissue explant cells dish • Grow and divide while adhering to plastic dishes • Require media for growth • Cells die after 50-100 divisions Cell Strains • Derived from primary cultures

7. Cell Lines Can be derived from several different sources: • cell strains • transformed cells • tumor cells 1) from cell strains (“normal cells”) • rare genetic changes generate variant cells that • can grow indefinitely (immortal) • example: BSC-1 cell line (derived from African Green Monkey kidney) • grow until contacting neighboring cells, then exit • cell cycle (experience contact inhibition) • will not form tumors when injected into mice

8. Mouse Cell Line Derived from Cell Strain

9. Some Cell Lines: Retain properties of precursor cells: e.g. liver cell lines T-cell lines Can differentiate in culture: e.g. epithelial cell lines muscle cell lines

10. differentiating muscle cell line = C2C12

11. Cell lines 2) from “transformed” cells • further genetic changes: • can be caused by radiation, chemical carcinogens, tumor viruses • see changed morphology, loss of growth control, loss of • contact inhibition • example: 3T3-21F • will form tumors in mice 3) from tumor cells • similar to transformed cells • example: HeLA - from cervical carcinoma • (Henrietta Lacks, 1951)

12. normal transformed by virus contact-inhibited pile up, rounded

13. normal cells scanning EM transformed cells

14. Properties of Cancer Cells 1. Lack normal growth controls a) self-sufficiency in growth signals b) insensitivity to anti-growth signals c) evade programmed cell death (apoptosis) d) unlimited replicative potential 2. Able to invade tissues and metastasize a) loss of dependency on anchorage for growth b) loss of contact inhibition 3. Able to develop vasculature - blood supply “angiogenesis”

15. tumor formation carcinogen + - tumor promoter carcinogen + tumor promoter +++ Tumor Promoters • Enhance tumor formation when combined with • carcinogens, but are not themselves carcinogenic

16. TPA = phorbal myristate acetate = PMA Phorbol ester • mimics 1, 2 diacylglycerol (DAG) • DAG + Ca++ activates protein kinase C (PKC) • causes phosphorylation of PKC substrates • changes in cell growth, cell shape and the cytoskeleton

18. Actin Microtubules Examining cell architecture using fluorescence microscopy? • can visualize and localize individual proteins • within a cell. Two cytoskeletal elements examined: Test the effects of different drugs on the cytoskeleton and cell shape TPA/PMA Latrunculin Taxol Nocodazole

19. Actin structures in a fibroblast cell

20. Microtubules = green DNA = blue interphase mitosis

21. Visualizing the cytoskeleton using fluorescence microscopy 1) Prepare samples: Fixation - kills and immobilizes cells A. aldehydes - cross-link amino groups in proteins (formaldehyde, glutaraldehyde) B. alcohols - denature proteins, precipitate in place (methanol) Permeabilization - detergents make proteins accessible to staining reagents (Triton X100)

22. Microtubules - immunofluorescence 1o ab: rabbit anti-tubulin; 2o ab: fluorescein anti-rabbit 2) Staining Actin - phalloidin covalently linked to rhodamine (red) - binds to filamentous actin only

23. excitation ex em emission intensity fluorescent molecule wavelength 3) Fluorescence microscopy

24. Fluorescence microscope