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Munc13-4*rab27 complex tethers secretory lysosomes at the plasma membrane

Edo D Elstak, Maaike Neeft, Nadine T Nehme

    Communicative & Integrative Biology
    |April 7, 2012
    PubMed

    Insights

    Natural Killer (NK) cells and Cytotoxic T lymphocytes (CTL) use secretory lysosomes (SL) to kill target cells. Rab27a and munc13-4 proteins cooperate to tether SL, enabling immune responses.

    Area of Science:

    • Immunology
    • Cell Biology
    • Molecular Biology

    Background:

    • Natural Killer (NK) cells and Cytotoxic T lymphocytes (CTL) are crucial for eliminating virus-infected or transformed cells.
    • These immune cells utilize polarized exocytosis of lytic proteins from secretory lysosomes (SL) to eliminate target cells.
    • The proteins Rab27a and munc13-4 directly interact and are essential for target cell killing, but their cooperative mechanism remains unclear.

    Purpose of the Study:

    • To elucidate the cooperative mechanism between Rab27a and munc13-4 in the degranulation process of immune cells.
    • To identify critical residues in munc13-4 essential for its interaction with Rab27a.
    • To investigate the functional consequences of disrupting the Rab27a-munc13-4 interaction on secretory lysosome (SL) exocytosis.

    Main Methods:

    • Site-directed mutagenesis was used to identify critical residues in munc13-4 for Rab27a binding.
    • Complementation assays were performed in a rat mast cell line by replacing endogenous munc13-4 with wild-type or mutant constructs.
    • Functional rescue assays were conducted in CTLs from Familial Hemophagocytic Lymphohistiocytosis type 3 patients.
    • Total Internal Reflection Fluorescence (TIRF) microscopy was employed to visualize SL motility.

    Main Results:

    • Mutagenesis identified specific residues in munc13-4 crucial for Rab27a interaction.
    • Expression of munc13-4 binding mutants failed to rescue β-hexosaminidase secretion in mast cells.
    • These binding mutants did not rescue CD107a (degranulation marker) appearance on the plasma membrane of patient-derived CTLs.
    • TIRF imaging revealed that the Rab27a-munc13-4 complex restricts SL motility in the subapical cytoplasm.

    Conclusions:

    • Rab27a and munc13-4 directly interact via specific residues in munc13-4.
    • This interaction is essential for the proper tethering of secretory lysosomes (SL) to the plasma membrane.
    • The tethering function of Rab27a-munc13-4 is a prerequisite for SNARE complex formation and subsequent membrane fusion during immune cell degranulation.
    Keywords:
    Degranulationcytotoxic T cellmast cellmunc13-4rab27secretory lysosomestethering

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