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Related Experiment Videos

Occluding junctions in cultured epithelial monolayers

M Cereijido, I Meza, A Martínez-Palomo

    The American Journal of Physiology
    |March 1, 1981
    PubMed
    Summary
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    Madin-Darby canine kidney (MDCK) cell tight junctions limit paracellular routes and require protein synthesis and extracellular calcium for sealing. Cytoskeletal disruption, particularly actin microfilaments, impairs junction formation and resealing.

    Area of Science:

    • Cell Biology
    • Epithelial Biology
    • Membrane Biophysics

    Background:

    • Madin-Darby canine kidney (MDCK) cells form polarized monolayers with functional tight junctions.
    • Tight junctions regulate paracellular permeability, a critical barrier function in epithelial tissues.
    • Understanding tight junction assembly and regulation is vital for comprehending epithelial integrity.

    Purpose of the Study:

    • To investigate the molecular requirements for tight junction assembly, sealing, and maintenance in MDCK cell monolayers.
    • To elucidate the role of protein synthesis, RNA synthesis, and extracellular calcium in tight junction formation.
    • To examine the involvement of the cytoskeleton, specifically actin microfilaments and microtubules, in tight junction dynamics.

    Main Methods:

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  • Culturing MDCK cells in monolayers to form tight junctions.
  • Utilizing inhibitors of protein and RNA synthesis to assess their impact on junction formation.
  • Measuring transepithelial electrical resistance (TER) to quantify junctional sealing.
  • Employing freeze-fracture electron microscopy to visualize junctional strand morphology.
  • Assessing ion selectivity and macromolecular tracer permeability.
  • Manipulating extracellular calcium concentrations and intracellular calcium levels.
  • Using immunofluorescence staining to visualize cytoskeletal components and treating cells with cytochalasin B.

    • Main Results:

    • Tight junction assembly and sealing are dependent on protein synthesis but not RNA synthesis.
    • Established tight junctions confer significant electrical resistance (80-600 omega . cm2) to the monolayer.
    • Junctional strand networks exhibit microscopic heterogeneity (1-10 strands).
    • Tight junctions are impermeable to macromolecules and exhibit specific ion selectivity (9:1 Na+/Cl- discrimination, K+ > Na+ > Rb+ > Cs+ > Li+).
    • Extracellular calcium is essential for sealing, while increased intracellular calcium disrupts junctions.
    • Disruption of actin microfilaments by cytochalasin B leads to junction opening, decreased electrical resistance, and inhibited resealing.

    Conclusions:

    • Tight junction formation and maintenance in MDCK cells are protein-dependent processes.
    • The structural integrity and barrier function of tight junctions are modulated by extracellular and intracellular calcium.
    • The actin cytoskeleton plays a crucial role in regulating tight junction assembly, stability, and resealing.