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Related Experiment Videos

Isolation of epidermal desmosomes.

C J Skerrow, A G Matoltsy

    The Journal of Cell Biology
    |November 1, 1974
    PubMed
    Summary

    Researchers developed a new method to isolate desmosomes, key cell adhesion structures, using a citric acid-sodium citrate buffer and sucrose gradient centrifugation. This technique yields pure desmosomes for biochemical studies and reveals a filamentous layer involved in cell attachment.

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    Area of Science:

    • Cell Biology
    • Biochemistry
    • Dermatology

    Background:

    • Desmosomes are crucial intercellular junctions in stratified epithelia, essential for tissue integrity.
    • Previous methods for desmosome isolation yielded low purity or damaged structures, hindering biochemical analysis.

    Purpose of the Study:

    • To develop a high-yield, high-purity method for isolating desmosomes suitable for biochemical analysis.
    • To characterize the ultrastructure of isolated desmosomes and identify components involved in filament attachment.

    Main Methods:

    • Selective solubilization of non-cornified epidermal layers using citric acid-sodium citrate (CASC) buffer (pH 2.6).
    • Discontinuous sucrose density gradient centrifugation to isolate desmosomes.
    • Electron microscopy (thin sections and spread preparations) for structural analysis.

    Main Results:

    • Desmosomes were isolated with high yield and purity, predominantly at the 55-60% sucrose interface.
    • Isolated desmosomes retained their characteristic structure, including midline, unit membranes, and dense plaques.
    • A finely filamentous layer, consistent with the proposed 'connecting component', was observed on the cytoplasmic plaque surface.

    Conclusions:

    • The CASC buffer and sucrose gradient method provides an effective means for isolating pure, structurally intact desmosomes.
    • The identified filamentous layer supports the role of the 'connecting component' in anchoring epidermal filament bundles to desmosomes.
    • This isolation technique facilitates future biochemical investigations into desmosome function and regulation.

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