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1.
Mol Biol Cell ; 28(12): 1688-1700, 2017 Jun 15.
Article in English | MEDLINE | ID: mdl-28450451

ABSTRACT

Endothelial cells respond to blood vessel injury by the acute release of the procoagulant von Willebrand factor, which is stored in unique secretory granules called Weibel-Palade bodies (WPBs). Stimulated WPB exocytosis critically depends on their proper recruitment to the plasma membrane, but factors involved in WPB-plasma membrane tethering are not known. Here we identify Munc13-4, a protein mutated in familial hemophagocytic lymphohistiocytosis 3, as a WPB-tethering factor. Munc13-4 promotes histamine-evoked WPB exocytosis and is present on WPBs, and secretagogue stimulation triggers an increased recruitment of Munc13-4 to WPBs and a clustering of Munc13-4 at sites of WPB-plasma membrane contact. We also identify the S100A10 subunit of the annexin A2 (AnxA2)-S100A10 protein complex as a novel Munc13-4 interactor and show that AnxA2-S100A10 participates in recruiting Munc13-4 to WPB fusion sites. These findings indicate that Munc13-4 supports acute WPB exocytosis by tethering WPBs to the plasma membrane via AnxA2-S100A10.


Subject(s)
Annexin A2/metabolism , Endothelial Cells/metabolism , Membrane Proteins/metabolism , S100 Proteins/metabolism , Weibel-Palade Bodies/metabolism , Cell Membrane/metabolism , Cells, Cultured , Exocytosis/physiology , Histamine/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Protein Binding , Protein Transport , von Willebrand Factor/metabolism
2.
Commun Integr Biol ; 5(1): 64-7, 2012 Jan 01.
Article in English | MEDLINE | ID: mdl-22482013

ABSTRACT

Natural Killer (NK) cells and Cytotoxic T lymphocytes (CTL) are critical for the immune response against virus infections or transformed cells. They kill target cells via polarized exocytosis of lytic proteins from secretory lysosomes (SL). Rab27a and munc13-4 interact directly and are required for target cell killing. How they cooperate in the intricate degranulation process is not known. We identified critical residues in munc13-4 for rab27 interaction and tested binding mutants in several complementation assays. In a rat mast cell line we replaced endogenous munc13-4 with ectopically expressed munc13-4 constructs. Unlike wild type munc13-4, binding mutants fail to rescue ß-hexosaminidase secretion. In accord, expression of binding mutants in CTL of Familial Hemophagocytic Lymphohistiocytosis type 3 patients, does not rescue CD107 appearance on the plasma membrane. Total Internal Reflection Fluorescence (TIRF) imaging shows that munc13-4*rab27a restricts motility of SL in the subapical cytoplasm. We propose that rab27*munc13-4 tethers SL to the plasma membrane, a requirement for formation of a cognate SNARE complex for fusion.

3.
Blood ; 118(6): 1570-8, 2011 Aug 11.
Article in English | MEDLINE | ID: mdl-21693760

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.


Subject(s)
Lysosomes/metabolism , Membrane Proteins/metabolism , rab GTP-Binding Proteins/metabolism , Amino Acid Sequence , Animals , Binding Sites/genetics , COS Cells , Cell Line, Tumor , Chlorocebus aethiops , Exocytosis , HEK293 Cells , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Lymphohistiocytosis, Hemophagocytic/genetics , Lymphohistiocytosis, Hemophagocytic/metabolism , Lymphohistiocytosis, Hemophagocytic/pathology , Lysosomal-Associated Membrane Protein 1/metabolism , Membrane Proteins/chemistry , Membrane Proteins/genetics , Microscopy, Confocal , Microscopy, Fluorescence , Molecular Sequence Data , Multiprotein Complexes/metabolism , Mutation , Protein Binding , Sequence Homology, Amino Acid , T-Lymphocytes, Cytotoxic/metabolism , rab GTP-Binding Proteins/genetics , rab27 GTP-Binding Proteins
4.
Methods Enzymol ; 438: 185-201, 2008.
Article in English | MEDLINE | ID: mdl-18413249

ABSTRACT

Secretory lysosomes constitute a heterogeneous organelle of hematopoietic cells that combines the properties of regular lysosomes with those of secretory granules. Although secretory lysosomes serve essential functions, such as in the immune system and blood clotting, the mechanisms underlying the release of contents are incompletely understood. It is clear, however, that rab27a and the C2 domain protein munc13-4 serve essential functions. Mutations in these genes lead to immune disorders where the lytic granule function of cytotoxic T cells is jeopardized in humans. We identified munc13-4 as a rab27a binding protein from spleen. Munc13-4 is highly expressed in several hematopoietic cells including cytotoxic T cells and mast cells. We describe the molecular features of the interaction and requirements for localization, and show that munc13-4 is a positive regulator of secretory lysosome exocytosis.


Subject(s)
Membrane Proteins/analysis , rab GTP-Binding Proteins/analysis , Animals , Cytosol/metabolism , Guanylyl Imidodiphosphate/metabolism , Humans , Lysosomes/metabolism , Membrane Proteins/immunology , Membrane Proteins/metabolism , Rabbits , Recombinant Proteins/metabolism , Spleen/cytology , Spleen/physiology , Sus scrofa , Transfection/methods , rab27 GTP-Binding Proteins
5.
Mol Biol Cell ; 16(2): 731-41, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15548590

ABSTRACT

Griscelli syndrome type 2 (GS2) is a genetic disorder in which patients exhibit life-threatening defects of cytotoxic T lymphocytes (CTLs) whose lytic granules fail to dock on the plasma membrane and therefore do not release their contents. The disease is caused by the absence of functional rab27a, but how rab27a controls secretion of lytic granule contents remains elusive. Mutations in Munc13-4 cause familial hemophagocytic lymphohistiocytosis subtype 3 (FHL3), a disease phenotypically related to GS2. We show that Munc13-4 is a direct partner of rab27a. The two proteins are highly expressed in CTLs and mast cells where they colocalize on secretory lysosomes. The region comprising the Munc13 homology domains is essential for the localization of Munc13-4 to secretory lysosomes. The GS2 mutant rab27aW73G strongly reduced binding to Munc13-4, whereas the FHL3 mutant Munc13-4Delta608-611 failed to bind rab27a. Overexpression of Munc13-4 enhanced degranulation of secretory lysosomes in mast cells, showing that it has a positive regulatory role in secretory lysosome fusion. We suggest that the secretion defects seen in GS2 and FHL3 have a common origin, and we propose that the rab27a/Munc13-4 complex is an essential regulator of secretory granule fusion with the plasma membrane in hematopoietic cells. Mutations in either of the two genes prevent formation of this complex and abolish secretion.


Subject(s)
Lysosomes/metabolism , Mast Cells/cytology , Mast Cells/metabolism , Proteins/metabolism , rab GTP-Binding Proteins/metabolism , Animals , Blotting, Western , Cell Line , Glutathione Transferase/metabolism , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Immunohistochemistry , Jurkat Cells , K562 Cells , Mast Cells/ultrastructure , Microscopy, Immunoelectron , Mutation , Protein Binding , Protein Structure, Tertiary , Proteins/chemistry , Proteins/genetics , Proteins/ultrastructure , Rats , Recombinant Proteins/metabolism , Sulfur Radioisotopes/metabolism , T-Lymphocytes, Cytotoxic/metabolism , Transfection , U937 Cells , rab GTP-Binding Proteins/chemistry , rab GTP-Binding Proteins/genetics , rab27 GTP-Binding Proteins
6.
Crit Rev Biochem Mol Biol ; 38(2): 121-42, 2003.
Article in English | MEDLINE | ID: mdl-12749696

ABSTRACT

Membrane flow through the cell is a highly dynamic process in which intracellular compartments communicate via tubulo-vesicular structures shuttling cargo molecules to their destinations. Transport carriers are formed at a donor compartment and navigate through the cytoplasm to the target organelle, on which they subsequently dock and fuse. Many of these events are regulated by the cooperative action of monomeric rab GTPases and their effector proteins. Research in recent years resulted in the identification of many rab effectors, providing first glimpses how the GTPase switch of individual rab proteins is utilized in discrete transport steps.


Subject(s)
Cell Membrane/metabolism , Membrane Transport Proteins/metabolism , rab GTP-Binding Proteins/physiology , Amino Acid Sequence , Animals , GTPase-Activating Proteins/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Humans , Intracellular Membranes/metabolism , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/genetics , Molecular Sequence Data , Signal Transduction/physiology , Transport Vesicles/physiology , rab GTP-Binding Proteins/chemistry , rab GTP-Binding Proteins/genetics
7.
EMBO J ; 22(11): 2645-57, 2003 Jun 02.
Article in English | MEDLINE | ID: mdl-12773381

ABSTRACT

Rab4 regulates recycling from early endosomes. We investigated the role of the rab4 effector rabaptin-5alpha and its putative partner gamma(1)-adaptin in membrane recycling. We found that rabaptin-5alpha forms a ternary complex with the gamma(1)-sigma(1) subcomplex of AP-1, via a direct interaction with the gamma(1)-subunit. The binding site for gamma(1)-adaptin is in the hinge region of rabaptin-5alpha, which is distinct from rab4- and rab5-binding domains. Endogenous or ectopically expressed gamma(1)- adaptin localized to both the trans-Golgi network and endosomes. Co-expressed rabaptin-5alpha and gamma(1)-adaptin, however, co-localized in a rab4-dependent manner on recycling endosomes. Transfection of rabaptin-5alpha caused enlarged endosomes and delayed recycling of transferrin. RNAi of rab4 had an opposing effect on transferrin recycling. Collectively, our data show that rab4-GTP acts as a scaffold for a rabaptin-5alpha- gamma(1)-adaptin complex on recycling endosomes and that interactions between rab4, rabaptin-5alpha and gamma(1)-adaptin regulate membrane recycling.


Subject(s)
Adaptor Protein Complex gamma Subunits/metabolism , Carrier Proteins/metabolism , Membrane Proteins/metabolism , Vesicular Transport Proteins , rab4 GTP-Binding Proteins/metabolism , Adaptor Protein Complex gamma Subunits/chemistry , Animals , Carrier Proteins/chemistry , Carrier Proteins/genetics , Cell Membrane/metabolism , Endosomes/metabolism , HeLa Cells , Humans , Macromolecular Substances , Membrane Proteins/chemistry , Mice , Protein Binding , RNA Interference , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transcription Factor AP-1/chemistry , Transcription Factor AP-1/metabolism , rab4 GTP-Binding Proteins/chemistry , rab4 GTP-Binding Proteins/genetics
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