David Weisblat 385 Life Sciences Addition Phone 642

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

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

single cell- scanning EM colony of cells 0. 01 mm dish of cell colonies 100 mm

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

cultured neuron extending processes

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

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

Mouse Cell Line Derived from Cell Strain

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

differentiating muscle cell line = C 2 C 12

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: 3 T 3 -21 F • will form tumors in mice 3) from tumor cells • similar to transformed cells • example: He. LA - from cervical carcinoma (Henrietta Lacks, 1951)

normal contact-inhibited transformed by virus pile up, rounded

normal cells scanning EM transformed cells

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”

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

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

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

Actin structures in a fibroblast cell

Microtubules = green DNA = blue interphase mitosis

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 X 100)

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

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

Fluorescence microscope