Alternative Zippering as an On-Off Switch for SNARE-Mediated Fusion

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102

Complexin (Cpx) is a SNARE-binding protein that regulates neurotransmission by clamping spontaneous synaptic vesicle fusion in the absence of Ca 2+ influx while promoting evoked release in response to an action potential. Previous studies indicated Cpx may cross-link multiple SNARE complexes via a trans interaction to function as a fusion clamp. During Ca 2+ influx, Cpx is predicted to undergo a conformational switch and collapse onto a single SNARE complex in a cis-binding mode to activate vesicle release. To test this model in vivo, we performed structure-function studies of the Cpx protein in Drosophila. Using genetic rescue approaches with cpx mutants that disrupt SNARE cross-linking, we find that manipulations that are predicted to block formation of the trans SNARE array disrupt the clamping function of Cpx. Unexpectedly, these same mutants rescue action potential-triggered release, indicating trans-SNARE cross-linking by Cpx is not a prerequisite for triggering evoked fusion. In contrast, mutations that impair Cpxmediated cis-SNARE interactions that are necessary for transition from an open to closed conformation fail to rescue evoked release defects in cpx mutants, although they clamp spontaneous release normally. Our in vivo genetic manipulations support several predictions made by the Cpx cross-linking model, but unexpected results suggest additional mechanisms are likely to exist that regulate Cpx's effects on SNARE-mediated fusion. Our findings also indicate that the inhibitory and activating functions of Cpx are genetically separable, and can be mapped to distinct molecular mechanisms that differentially regulate the SNARE fusion machinery.synapse | neurotransmitter release | exocytosis

“…Cell-cell fusion assays were performed as previously described (14,24). See SI Materials and Methods for details.…”

Section: Methodsmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

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

Cho

Kümmel

et al. 2014

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102

“…Synaptic vesicle fusion is strictly regulated by neuronal proteins such as Complexin and Synaptotagmin. Evidence suggests that these regulators may act on the half-zippered SNARE (17)(18)(19)(20). Therefore, a kinetic pause between the N-and C-terminal zipperings would be a key factor for organizing the regulation machinery.…”

mentioning

confidence: 99%

Kinetic barriers to SNAREpin assembly in the regulation of membrane docking/priming and fusion

Tiwari

Rothman

et al. 2016

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Neurotransmission is achieved by soluble NSF attachment protein receptor (SNARE)-driven fusion of readily releasable vesicles that are docked and primed at the presynaptic plasma membrane. After neurotransmission, the readily releasable pool of vesicles must be refilled in less than 100 ms for subsequent release. Here we show that the initial association of SNARE complexes, SNAREpins, is far too slow to support this rapid refilling owing to an inherently high activation energy barrier. Our data suggest that acceleration of this process, i.e., lowering of the barrier, is physiologically necessary and can be achieved by molecular factors. Furthermore, under zero force, a low second energy barrier transiently traps SNAREpins in a half-zippered state similar to the partial assembly that engages calcium-sensitive regulatory machinery. This result suggests that the barrier must be actively raised in vivo to generate a sufficient pause in the zippering process for the regulators to set in place. We show that the heights of the activation energy barriers can be selectively changed by molecular factors. Thus, it is possible to modify, both in vitro and in vivo, the lifespan of each metastable state. This controllability provides a simple model in which vesicle docking/ priming, an intrinsically slow process, can be substantially accelerated. It also explains how the machinery that regulates vesicle fusion can be set in place while SNAREpins are trapped in a halfzippered state.SNAREpin assembly | fusion regulation | RRP refilling | Tomosyn | fluorescence anisotropy

“…Although the accessory domain is required for regulating spontaneous release, mutations of this domain do not affect the activating function of Cpx compared with wild-type neurons in rescue experiments with Cpx knockdown (28,29), and it can be entirely eliminated while still maintaining the activating function of Cpx in a reconstituted system (30). The α-helical central domain (amino acid residues 49-70) binds to the SNARE complex (31,32) and is essential for all functions of Cpx in all species studied to date, including priming (16,28,29,33), inhibiting spontaneous fusion (12,19,24,25,(34)(35)(36), and activation of Ca 2+ -triggered fusion (12-14, 19, 34, 36-38).…”

mentioning

confidence: 99%

“…The C-terminal domain of worm complexin preferentially interacts with curved membranes via a membrane-binding motif (20,21). The accessory domain (amino acid residues 28-48) regulates spontaneous fusion in neurons and it suppresses Ca 2+ -independent fusion in reconstituted systems (12,14,19,(22)(23)(24)(25)(26)(27). Although the accessory domain is required for regulating spontaneous release, mutations of this domain do not affect the activating function of Cpx compared with wild-type neurons in rescue experiments with Cpx knockdown (28,29), and it can be entirely eliminated while still maintaining the activating function of Cpx in a reconstituted system (30).…”

mentioning

confidence: 99%

C-terminal domain of mammalian complexin-1 localizes to highly curved membranes

Gong

Lai

et al. 2016

In presynaptic nerve terminals, complexin regulates spontaneous "mini" neurotransmitter release and activates Ca 2+ -triggered synchronized neurotransmitter release. We studied the role of the C-terminal domain of mammalian complexin in these processes using singleparticle optical imaging and electrophysiology. The C-terminal domain is important for regulating spontaneous release in neuronal cultures and suppressing Ca 2+ -independent fusion in vitro, but it is not essential for evoked release in neuronal cultures and in vitro. This domain interacts with membranes in a curvature-dependent fashion similar to a previous study with worm complexin [Snead D, Wragg RT, Dittman JS, Eliezer D (2014) Membrane curvature sensing by the C-terminal domain of complexin. Nat Commun 5:4955]. The curvature-sensing value of the C-terminal domain is comparable to that of α-synuclein. Upon replacement of the C-terminal domain with membrane-localizing elements, preferential localization to the synaptic vesicle membrane, but not to the plasma membrane, results in suppression of spontaneous release in neurons. Membrane localization had no measurable effect on evoked postsynaptic currents of AMPA-type glutamate receptors, but mislocalization to the plasma membrane increases both the variability and the mean of the synchronous decay time constant of NMDA-type glutamate receptor evoked postsynaptic currents.