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MCB 130L Lecture 4. Immunofluorescence of the Cytoskeleton. Cell Biology Module Overview. Inner Life of a Cell Video: http://multimedia.mcb.harvard.edu/media.html. Fluorescence/Immunofluorescence Microscopy--Cytoskeleton Transfection & Vital Staining Respiration Cell Signaling.

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mcb 130l lecture 4

MCB 130L Lecture 4

Immunofluorescence of the Cytoskeleton

cell biology module overview
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
slide3

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

cell culture propagation of cells outside the organism
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
primary cultures
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
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

slide7

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

cytoskeleton
Cytoskeleton

Actin

Microtubules

Nucleus

bovine pulmonary artery endothelial cells, Molecular Probes

actin
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
slide10

Actin cytoskeleton

Fibroblast

Intestinal microvilli

From Lodish

Hair cell stereocilia from ears - Belyantseva et al. (2005) Nat.Cell Biol. 7:148-156

slide11

Actin monomers form actin filaments

http://www.sinauer.com/cooper/4e/animations1201.html

monomer

model:

EM micrograph

Filament

model

From Lodish

slide12

Organization of actin filaments

Intestinal microvilli

platelet cytoskeleton

microtubules
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
slide15

Tubulin dimers form microtubules

http://www.sinauer.com/cooper/4e/animations1203.html

From Lodish

drugs used in lab
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
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
Nocodazole
  • Chemically synthesized
  • Low dose--arrests mitosis
  • High dose--rapidly depolymerizes microtubules
  • actin unaffected

Nocodazole

latrunculin b
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
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
Steps in Cell Staining
  • Fix cells
  • Permeabilize cells
  • Add antibodies or staining reagent
  • Mount coverslips
cell fixation
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
Permeabilization
  • Necessary for staining of intracellular proteins
  • Can expose antigenic epitopes
  • Detergents (Tx-100) or Methanol used to solubilize cell membranes

Triton X-100

slide24
Antibodies

(indirect immunofluorescence)

  • 1o antibody: mouse anti-tubulin
  • 2o antibody: goat anti-mouse (conjugated to a fluorophore or other tag for visualization)
anti tubulin antibody
Anti-tubulin Antibody

1°mouse anti-alpha-tubulin

2° Cy2-goat anti-mouse Fab

NIH/3T3 cells

From http://www.microscopyu.com

phalloidin
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
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
Fluorescence Microscopy

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

slide29

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?

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