Isoform and Splice-Variant Specific Functions of Dynamin-2 Revealed by Analysis of Conditional Knock-Out Cells

Abstract

Dynamin (Dyn) is a multifunctional GTPase implicated in several cellular events, including endocytosis, intracellular trafficking, cell signaling, and cytokinesis. The mammalian genome encodes three isoforms, Dyn1, Dyn2, and Dyn3, and several splice variants of each, leading to the suggestion that distinct isoforms and/or distinct splice variants might mediate distinct cellular functions. We generated a conditional Dyn2 KO cell line and performed knockout and reconstitution experiments to explore the isoform-and splice variant specific cellular functions of ubiquitously expressed Dyn2. We find that Dyn2 is required for clathrin-mediated endocytosis (CME), p75 export from the Golgi, and PDGFstimulated macropinocytosis and cytokinesis, but not for other endocytic pathways. Surprisingly, CME and p75 exocytosis were efficiently rescued by reintroduction of Dyn2, but not Dyn1, suggesting that these two isoforms function differentially in vesicular trafficking in nonneuronal cells. Both isoforms rescued macropinocytosis and cytokinesis, suggesting that dynamin function in these processes might be mechanistically distinct from its role in CME. Although all four Dyn2 splice variants could equally restore CME, Dyn2ba and -bb were more effective at restoring p75 exocytosis. This splice variant specificity correlated with their differential targeting to the Golgi. These studies reveal isoform and splice-variant specific functions for Dyn2. INTRODUCTIONDynamin (Dyn) is an ϳ100-kDa multidomain GTPase that was first identified as a microtubule binding and bundling protein (Shpetner and Vallee, 1989). Subsequently, dynamin was found to be the mammalian homologue of the Drosophila protein shibire, mutations in which block endocytosis, including synaptic vesicle recycling (Chen et al., 1991;van der Bliek and Meyerowitz, 1991). Dynamin is conserved throughout higher eukaryotes. There is a single gene in Drosophila and Caenorhabditis elegans, but there are three dynamin isoforms in mammals: Dyn1, which is specifically expressed in neurons; Dyn2, which is ubiquitously expressed; and Dyn3, which is mainly expressed in the brain and testes (Urrutia et al., 1997, Ferguson et al., 2007.The best-studied cellular function of dynamin is its involvement in clathrin-mediated endocytosis (CME; Hinshaw, 2000;Sever et al., 2000;Praefcke and McMahon, 2004). However, dynamin has also been implicated in several other membrane-trafficking events including both caveolae-mediated and clathrin-and caveolin-independent endocytic pathways Oh et al., 1998;Lamaze et al., 2001;Pelkmans et al., 2002), phagocytosis (Gold et al., 1999;Yu et al., 2006), macropinocytosis (Schlunck et al., 2004), and trafficking from the trans-Golgi network (TGN; Jones et al., 1998;Kreitzer et al., 2000;Bonazzi et al., 2005). Because these functions have mostly emerged from studying the effects of overexpression of dominant-negative dynamin mutants, they may reflect indirect or nonspecific effects. Moreover, it is not known whether different dynamin isoforms and/or splice varia...

Dynamin 2 regulates biphasic insulin secretion and plasma glucose homeostasis

“…However, the decay time constant of the average C m trace was much slower in KO cells (τ = 4.3 s, n = 14) than in control cells (τ = 2.7 s, n = 15) ( Figure 6F). Given the critical role of dynamin 2 in multiple forms of endocytosis in nonneuronal cells (32,45,54,58), it is surprising that the membrane retrieval was not blocked by the depletion of dynamin 2, an abundant dynamin isoform in β cells, indicating the presence of dynamin 2 independent endocytosis in these cells.…”

The Dynamin Inhibitors MiTMAB and OcTMAB Induce Cytokinesis Failure and Inhibit Cell Proliferation in Human Cancer Cells

“…It is unknown if dynII is associated with the mitotic centrosome. DynII is also associated with the final stage of mitosis, cytokinesis (21,(23)(24)(25)(26). During cytokinesis, dynamin localizes to the spindle midzone and the intracellular bridge (21,26).…”

LIM Kinase 1 and Cofilin Regulate Actin Filament Population Required for Dynamin-dependent Apical Carrier Fission from theTrans-Golgi Network

“…In none of these cases, however, the specific roles of This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E08 -08 -0891) on November 5, 2008. the actin cytoskeleton in cargo protein exit have been documented. The GTPase dynamin, which associates with the actin cytoskeleton to mediate fission of endocytic vesicles from the PM , also mediates fission from the TGN of transport carriers for the apical protein p75 neurotrophin receptor (p75) in MDCK cells (Kreitzer et al, 2000;Bonazzi et al, 2005;Ya-Wen Liu et al, 2008). Interestingly, dynamin 2 mediates fission from the TGN of vesicular stomatitis virus G protein carriers in a cell-specific manner (Cao et al, 2000(Cao et al, , 2005Bonazzi et al, 2005).…”