Complexin Controls the Force Transfer from SNARE Complexes to Membranes in Fusion

Abstract

Trans-SNARE complexes catalyze fast synaptic vesicle fusion and bind complexin, but the function of complexin binding to SNARE complexes remains unclear. Here we show that in neuronal synapses, complexin simultaneously suppressed spontaneous fusion and activated fast Ca 2+ -evoked fusion. The dual function of complexin required SNARE binding, and additionally involved distinct N-terminal sequences of complexin that localize to the point where trans-SNARE complexes insert into the fusing membranes, suggesting that complexin controls the force that trans-SNARE complexes apply onto the fusing membranes. Consistent with this hypothesis, a mutation in the membrane insertion sequence of the v-SNARE synaptobrevin/VAMP phenocopied the complexin loss-of-function state without impairing complexin-binding to SNARE complexes. Thus, complexin probably activates and clamps the force-transfer from assembled trans-SNARE complexes onto fusing membranes.Synaptic vesicle fusion is driven by assembly of trans-SNARE complexes (or SNAREpins) from syntaxin-1 and SNAP-25 on the plasma membrane, and synaptobrevin/ VAMP on the vesicle membrane [1][2][3]. Ca 2+ then triggers fast synchronous synaptic vesicle fusion by binding to the Ca 2+ -sensor synaptotagmin [4][5][6]. Besides SNARE proteins and synaptotagmin, fast Ca 2+ -triggered fusion requires complexin [7]. Complexin is composed of short N-and C-terminal sequences and two central α-helices. Complexin binds to SNARE complexes via its central α-helix, which inserts in an anti-parallel orientation into a groove formed by synaptobrevin/VAMP and syntaxin-1 [8,9]. Although multiple approaches have revealed an essential role of complexin in synaptic fusion [7,[10][11][12][13][14][15], the nature of this role remains unclear. In vertebrate autapses, deletion of complexin selectively impairs fast synchronous neurotransmitter release without changing asynchronous or spontaneous release [7,10]. In in vitro fusion assays, conversely, addition of complexin causes a general block of SNARE-dependent fusion, indicating that complexin is a SNARE clamp [11][12][13][14]. In Drosophila neuromuscular synapses, deletion of complexin produces a >20-fold increase in spontaneous release but only a small decrease in evoked release [15]. Thus, the role of complexin in fusion is unclear. Moreover, even the importance of complexin SNARE- [16,17]. Here we addressed these questions with two complementary approaches -RNAi-dependent knockdown of complexin with rescue, and replacing wild-type synaptobrevin with specific mutants using synaptobrevin knockout (KO) mice [18].We knocked down complexin expression in cultured cortical neurons using an shRNA that targets both complexin-1 and -2, the only complexin isoforms significantly expressed in these neurons [19]. For this purpose, we used lentiviruses that simultaneously synthesize the complexin shRNA and either GFP, wild-type complexin-1, or 4M-mutant complexin-1 that is unable to bind to SNARE complexes (19,20). Lentivirus expressing GFP without the shRNA ...

Genetic analysis of the Complexin trans-clamping model for cross-linking SNARE complexes in vivo

“…In addition to its role as a fusion clamp, Cpx also promotes Ca 2+ -triggered release in both Drosophila and mammals (16,18,21,29,30). cpx null mutants exhibited reduced evoked release, measured as evoked junctional potentials (EJPs) in both low and high Ca 2+ concentrations (16) (Fig.…”

“…In addition to enhanced minis, cpx mutants have reduced evoked release. Together with data from other genetic systems (17)(18)(19)(20)(21), an emerging model is that Cpx plays a dual role in release, acting as a vesicle fusion clamp that prevents spurious fusion of vesicles at some synapses, while simultaneously promoting synchronous release in response to an action potential at all synapses (16,17,(19)(20)(21)(22). Structurally, these dual activities are likely to require an α-helix motif found within Cpx that is divided into an accessory and central helix region (Fig.…”

A molecular machine for neurotransmitter release: synaptotagmin and beyond

“…One of the a-helices is bound to the SNARE complex and is essential for all complexin function 18 . The second a-helix is required only for the clamping, and not for the activating function of complexin 17 npg calcium channels and a loss of the tight coupling of a presynaptic action potential to release. RIMs perform their functions in a direct complex with the calcium channels, with other active zone proteins such as RIM-BPs (which, in turn, also bind calcium channels) and with Rab3/ Rab27 GTPases on synaptic vesicles (Fig.…”

“…triggered fusion 17 . Complexin-deficient neurons exhibit a phenotype milder than that of Syt1-deficient neurons, with a selective suppression of fast synchronous exocytosis and an increase in spontaneous exocytosis, which suggests that complexin and synaptotagmins are functionally interdependent.…”

Minimal membrane docking requirements revealed by reconstitution of Rab GTPase-dependent membrane fusion from purified components

“…Regulation of SNARE-dependent membrane fusion by the SNARE-interacting protein Sec1/Munc18 (SM) (14,15) and the SNARE-binding proteins synaptotagmin and complexin (16)(17)(18)(19)(20)(21)(22)(23)(24)(25) has also been reconstituted. We have recently reported reconstituted membrane fusion that requires yeast vacuolar SNAREs, regulatory lipids, and the homotypic vacuole fusion and protein sorting (HOPS)/class C vacuole protein sorting (class C Vps) complex, a six-subunit effector and GEF for the yeast vacuolar Rab GTPase Ypt7p (26)(27)(28).…”