title: PIP2-dependent regulation of Munc13-4 endocytic recycling: impact on the cytolytic secretory pathway.
abstract: Cytotoxic lymphocytes clear infected and transformed cells by releasing the content of lytic granules at cytolytic synapses, and the ability of cytolytic effectors to kill in an iterative manner has been documented previously. Although bidirectional trafficking of cytolytic machinery components along the endosomal pathway has begun to be elucidated, the molecular mechanisms coordinating granule retrieval remain completely unexplored. In the present study, we focus on the lytic granule priming factor Munc13-4, the mutation of which in familial hemophagocytic lymphohistiocytosis type 3 results in a profound defect of cytotoxic function. We addressed the role of phosphatidylinositol (4,5)-bisphosphate (PIP2) in the regulation of Munc13-4 compartmentalization. We observed that in human natural killer cells, PIP2 is highly enriched in membrane rafts. Granule secretion triggering induces a transient Munc13-4 raft recruitment, followed by AP-2/clathrin-dependent internalization. Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) γ gene silencing leads to the impairment of granule secretion associated with increased levels of raft-associated Munc13-4, which is attributable to a defect in AP-2 membrane recruitment. In such conditions, the ability to subsequently kill multiple targets was significantly impaired. These observations indicate that Munc13-4 reinternalization is required for the maintenance of an intracellular pool that is functional to guarantee the serial killing potential.
authors.affiliation: Department of Experimental Medicine, Istituto Pasteur–Fondazione Cenci-Bolognetti, Fondazione Eleonora Lorillard Spencer Cenci, Sapienza University, Rome, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Capuano
authors.forename: Rossella
authors.initials: R
authors.lastname: Paolini
authors.forename: Rosa
authors.initials: R
authors.lastname: Molfetta
authors.forename: Luigi
authors.initials: L
authors.lastname: Frati
authors.forename: Angela
authors.initials: A
authors.lastname: Santoni
authors.forename: Ricciarda
authors.initials: R
authors.lastname: Galandrini
mesh_terms: D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003594:Cytoplasmic Granules / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D020014:K562 Cells; D007694:Killer Cells, Natural / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009154:Mutation; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D055571:Secretory Pathway / Q000502:physiology*
chemical_list: D008565:Membrane Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; C507969:UNC13D protein, human; D049452:Green Fluorescent Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase