title: The munc13-4-rab27 complex is specifically required for tethering secretory lysosomes at the plasma membrane.
abstract: Cytotoxic T lymphocytes (CTLs) kill target cells through the polarized release of lytic molecules from secretory lysosomes. Loss of munc13-4 function inhibits this process and causes familial hemophagocytic lymphohistiocytosis type 3 (FHL3). munc13-4 binds rab27a, but the necessity of the complex remains enigmatic, because studies in knockout models suggest separate functions. In the present study, we describe a noncanonical rab27a-binding motif in the N-terminus of munc13-4. Point mutants in this sequence have severely impaired rab27a binding, allowing dissection of rab27a requirements in munc13-4 function. The munc13-4-rab27a complex is not needed for secretory lysosome maturation, as shown by complementation in CTLs from FHL3 patients and in a mast cell line silenced for munc13-4. In contrast, fusion of secretory lysosomes with, and content release at the plasma membrane during degranulation, strictly required the munc13-4-rab27a complex. Total internal reflection fluorescence microscopy imaging revealed that the complex corrals motile secretory lysosomes beneath the plasma membrane during degranulation and controls their docking. The propensity to stall motility of secretory lysosomes is lost in cells expressing munc13-4 point mutants that do not bind rab27. In summary, these results uncovered a mechanism for tethering secretory lysosomes to the plasma membrane that is essential for degranulation in immune cells.
authors.affiliation: Department of Cell Biology, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Edo D
authors.initials: ED
authors.lastname: Elstak
authors.forename: Maaike
authors.initials: M
authors.lastname: Neeft
authors.forename: Nadine T
authors.initials: NT
authors.lastname: Nehme
authors.forename: Jarno
authors.initials: J
authors.lastname: Voortman
authors.forename: Marc
authors.initials: M
authors.lastname: Cheung
authors.forename: Monireh
authors.initials: M
authors.lastname: Goodarzifard
authors.forename: Hans C
authors.initials: HC
authors.lastname: Gerritsen
authors.forename: Paul M P
authors.initials: PM
authors.lastname: van Bergen En Henegouwen
authors.forename: Isabelle
authors.initials: I
authors.lastname: Callebaut
authors.forename: Geneviève
authors.initials: G
authors.lastname: de Saint Basile
authors.forename: Peter
authors.initials: P
authors.lastname: van der Sluijs
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D005089:Exocytosis; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051359:Lymphohistiocytosis, Hemophagocytic / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D052118:Lysosomal-Associated Membrane Protein 1 / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013602:T-Lymphocytes, Cytotoxic / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000075745:rab27 GTP-Binding Proteins
chemical_list: D008164:Luminescent Proteins; D052118:Lysosomal-Associated Membrane Protein 1; D008565:Membrane Proteins; D046912:Multiprotein Complexes; C507969:UNC13D protein, human; D000075745:rab27 GTP-Binding Proteins; C066694:RAB27A protein, human; D020691:rab GTP-Binding Proteins