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      Rab8a regulates the exocyst-mediated kiss-and-run discharge of the Dictyostelium contractile vacuole

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          Abstract

          A molecular dissection of contractile vacuole (CV) discharge shows that Rab8a is recruited to the CV a few seconds before the exocyst. Together they tether it to the plasma membrane and commit it to fusion. GTP hydrolysis is necessary for vacuole detethering, a process in which LvsA, a protein of the Chédiak–Higashi family, plays a crucial role.

          Abstract

          Water expulsion by the contractile vacuole (CV) in Dictyostelium is carried out by a giant kiss-and-run focal exocytic event during which the two membranes are only transiently connected but do not completely merge. We present a molecular dissection of the GTPase Rab8a and the exocyst complex in tethering of the contractile vacuole to the plasma membrane, fusion, and final detachment. Right before discharge, the contractile vacuole bladder sequentially recruits Drainin, a Rab11a effector, Rab8a, the exocyst complex, and LvsA, a protein of the Chédiak–Higashi family. Rab8a recruitment precedes the nucleotide-dependent arrival of the exocyst to the bladder by a few seconds. A dominant-negative mutant of Rab8a strongly binds to the exocyst and prevents recruitment to the bladder, suggesting that a Rab8a guanine nucleotide exchange factor activity is associated with the complex. Absence of Drainin leads to overtethering and blocks fusion, whereas expression of constitutively active Rab8a allows fusion but blocks vacuole detachment from the plasma membrane, inducing complete fragmentation of tethered vacuoles. An indistinguishable phenotype is generated in cells lacking LvsA, implicating this protein in postfusion detethering. Of interest, overexpression of a constitutively active Rab8a mutant reverses the lvsA-null CV phenotype.

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          Most cited references71

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          The exocyst is an effector for Sec4p, targeting secretory vesicles to sites of exocytosis.

          Polarized secretion requires proper targeting of secretory vesicles to specific sites on the plasma membrane. Here we report that the exocyst complex plays a key role in vesicle targeting. Sec15p, an exocyst component, can associate with secretory vesicles and interact specifically with the rab GTPase, Sec4p, in its GTP-bound form. A chain of protein-protein interactions leads from Sec4p and Sec15p on the vesicle, through various subunits of the exocyst, to Sec3p, which marks the sites of exocytosis on the plasma membrane. Sec4p may control the assembly of the exocyst. The exocyst may therefore function as a rab effector system for targeted secretion.
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            Coordinated protein sorting, targeting and distribution in polarized cells.

            The polarized distribution of functions in polarized cells requires the coordinated interaction of three machineries that modify the basic mechanisms of intracellular protein trafficking and distribution. First, intrinsic protein-sorting signals and cellular decoding machineries regulate protein trafficking to plasma membrane domains; second, intracellular signalling complexes define the plasma membrane domains to which proteins are delivered; and third, proteins that are involved in cell-cell and cell-substrate adhesion orientate the three-dimensional distribution of intracellular signalling complexes and, accordingly, the direction of membrane traffic. The integration of these mechanisms into a complex and dynamic network is crucial for normal tissue function and is often defective in disease states.
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              Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells.

              During clathrin-mediated endocytosis, membrane scission marks the isolation of a cargo-laden clathrin-coated pit (CCP) from the cell exterior. Here we used live-cell imaging of a pH-sensitive cargo to visualize the formation of clathrin-coated vesicles (CCVs) at single CCPs with a time resolution of seconds. We show that CCPs are highly dynamic and can produce multiple vesicles in succession. Using alternating evanescent field and epifluorescence illumination, we show that CCP invagination and scission are tightly coupled, with scission coinciding with maximal displacement of CCPs from the plasma membrane and with peak recruitment of cortactin-DsRed, a dynamin and F-actin binding protein. Finally, perturbing actin polymerization with latrunculin-B drastically reduces the efficiency of membrane scission and affects many aspects of CCP dynamics. We propose that CCP invagination, actin polymerization, and CCV formation are highly coordinated for efficient endocytosis.
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                Author and article information

                Contributors
                Role: Monitoring Editor
                Journal
                Mol Biol Cell
                Mol. Biol. Cell
                molbiolcell
                mbc
                Mol. Bio. Cell
                Molecular Biology of the Cell
                The American Society for Cell Biology
                1059-1524
                1939-4586
                01 April 2012
                : 23
                : 7
                : 1267-1282
                Affiliations
                [1] aDépartment de Biochimie, Faculté des Sciences, Université de Genève, Sciences II, CH-1211-Geneva 4, Switzerland
                [2] bMRC Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom
                University of North Carolina
                Author notes

                *Present address: Unité de Biologie des Interactions Cellulaires, Institut Pasteur, F-75015 Paris, France.

                1Address correspondence to: Thierry Soldati ( thierry.soldati@ 123456unige.ch ).
                Article
                E11-06-0576
                10.1091/mbc.E11-06-0576
                3315810
                22323285
                fcd9a3d5-7daf-4a55-a0f9-dfb0594619c4
                © 2012 Essid et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License ( http://creativecommons.org/licenses/by-nc-sa/3.0).

                “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology.

                History
                : 27 June 2011
                : 09 January 2012
                : 03 February 2012
                Categories
                Articles
                Membrane Trafficking
                A Highlights from MBoC Selection

                Molecular biology
                Molecular biology

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