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 manipulated

a. Pharmaceutical manipulationb. Genetic manipulation (transfection, RNAi)c. Fluorescent tracers (live or fixed cells)

2) Homogeneous cells3) Large quantities of cells4) Investigation of diverse cellular functions5) Noninvasive way to study mammalian cells

Drawbacks:1) Requires care and $$$2) May not demonstrate real cellular physiology3) 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