Mcb 130l lecture 4
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MCB 130L Lecture 4. Immunofluorescence of the Cytoskeleton. Cell Biology Module Overview. Inner Life of a Cell Video: Fluorescence/Immunofluorescence Microscopy--Cytoskeleton Transfection & Vital Staining Respiration Cell Signaling.

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MCB 130L Lecture 4

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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:

  • Fluorescence/Immunofluorescence Microscopy--Cytoskeleton

  • Transfection & Vital Staining

  • Respiration

  • Cell Signaling

Mcb 130l lecture 4

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


required for cell morphology & motility


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

    Mcb 130l lecture 4

    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






    bovine pulmonary artery endothelial cells, Molecular Probes



    • 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

    Mcb 130l lecture 4

    Actin cytoskeleton


    Intestinal microvilli

    From Lodish

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

    Mcb 130l lecture 4

    Actin monomers form actin filaments



    EM micrograph



    From Lodish

    Mcb 130l lecture 4

    Organization of actin filaments

    Intestinal microvilli

    platelet cytoskeleton




    • cell morphology and polarity

    • subcellular localization of organelles

      Tracks for kinesin and dynein motors

    • intracellular transport (protein, vesicles, organelles)


    • cilia and flagella (specialized structures)


    • Mitotic spindle

    Mcb 130l lecture 4

    Microtubule cytoskeleton

    From Lodish

    Mcb 130l lecture 4

    Tubulin dimers form microtubules

    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



    • Isolated from pacific yew

    • Binds and stabilizes microtubules

      • Promotes lateral interactions between protofilaments

    • Low dose- blocks mitosis

    • High dose- increases polymerization

    • actin unaffected




    • Chemically synthesized

    • Low dose--arrests mitosis

    • High dose--rapidly depolymerizes microtubules

    • actin unaffected


    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



    • Necessary for staining of intracellular proteins

    • Can expose antigenic epitopes

    • Detergents (Tx-100) or Methanol used to solubilize cell membranes

    Triton X-100

    Mcb 130l lecture 4


    (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




    • 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



    Fluorescence microscopy

    Fluorescence Microscopy


    Mcb 130l lecture 4


    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?

    Microbe astronomer

    Microbe Astronomer

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