Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion

Diao, Jiajie; Grob, Patricia; Cipriano, Daniel J.; Kyoung, Minjoung; Zhang, Yunxiang; Shah, Sachi; Nguyen, Amie; Padolina, Mark; Srivastava, Ankita; Vrljic, Marija; Shah, Ankita; Nogales, Eva; Chu, Steven; Brunger, Axel T.

doi:10.7554/elife.00109

Cited by 174 publications

(324 citation statements)

References 71 publications

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“…Though EPR data agreed with this model of membrane bridging (24), and increasing evidence supports the notion that the membrane-bridging activity of synaptotagmin-1 is critical for its function (7,(25)(26)(27)(28)(29)(30)(31), a recent study concluded that the bridging occurs by a fundamentally different mechanism that requires trans interactions between synaptotagmin-1 oligomers bound to each membrane, without binding of the bottom of the C 2 B domain to the lipids (29). This mechanism, which we refer to as the oligomerization model, is compared in Fig.…”

mentioning

confidence: 78%

Prevalent mechanism of membrane bridging by synaptotagmin-1

Seven¹,

Brewer²,

Shi

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Synaptotagmin-1 functions as a Ca 2+ sensor in neurotransmitter release through its two C 2 domains (the C 2 A and C 2 B domain). The ability of synaptotagmin-1 to bridge two membranes is likely crucial for its function, enabling cooperation with the soluble Nethylmaleimide sensitive factor adaptor protein receptors (SNAREs) in membrane fusion, but two bridging mechanisms have been proposed. A highly soluble synaptotagmin-1 fragment containing both domains (C 2 AB) was shown to bind simultaneously to two membranes via the Ca 2+ -binding loops at the top of both domains and basic residues at the bottom of the C 2 B domain (direct bridging mechanism). In contrast, a longer fragment including a linker sequence (lnC 2 AB) was found to aggregate in solution and was proposed to bridge membranes through trans interactions between lnC 2 AB oligomers bound to each membrane via the Ca 2+ -binding loops, with no contact of the bottom of the C 2 B domain with the membranes. We now show that lnC 2 AB containing impurities indeed aggregates in solution, but properly purified lnC 2 AB is highly soluble. Moreover, cryo-EM images reveal that a majority of lnC 2 AB molecules bridge membranes directly. Fluorescence spectroscopy indicates that the bottom of the C 2 B domain contacts the membrane in a sizeable population of molecules of both membranebound C 2 AB and membrane-bound lnC 2 AB. NMR data on nanodiscs show that a fraction of C 2 AB molecules bind to membranes with antiparallel orientations of the C 2 domains. Together with previous studies, these results show that direct bridging constitutes the prevalent mechanism of membrane bridging by both C 2 AB and lnC 2 AB, suggesting that this mechanism underlies the function of synaptotagmin-1 in neurotransmitter release.membrane bending | Ca2+ sensing | exocytosis | synaptic transmission N eurotransmitter release is a key event in interneuronal communication that is acutely triggered by Ca 2+ influx into a presynaptic terminal (1). The synaptic vesicle protein synaptotagmin-1 acts as a Ca 2+ sensor in fast release through the two C 2 domains that form most of its cytoplasmic region (the C 2 A and C 2 B domains) (2-5). This function is coupled to membrane fusion through the neuronal soluble N-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) proteins (6, 7), which bring the synaptic vesicle and plasma membranes together by forming SNARE complexes (2, 3). Synaptotagmin-1 function also depends on a tight interplay with complexins (8-10) and other key proteins of the release machinery (11, 12). The synaptotagmin-1 C 2 domains bind three or two Ca 2+ ions through loops at the top of β-sandwich structures (13-15) (Fig. 1A). These top loops also mediate Ca 2+ -dependent phospholipid binding (15-17), which is crucial for synaptotagmin-1 function (5, 18). Moreover, the synaptotagmin-1 cytoplasmic region clusters chromaffin granules and liposomes (19), and a fragment containing its two C 2 domains was shown by cryo-EM to bind simultaneously to two membranes, bringing them i...

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mentioning

confidence: 78%

Prevalent mechanism of membrane bridging by synaptotagmin-1

Seven¹,

Brewer²,

Shi

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…To study the molecular mechanisms of Cpx, several reconstituted systems have been developed (19,(39)(40)(41)(42). As we reported previously, the C-terminal domain is important for suppression of Ca 2+ -independent fusion in a single-vesicle content mixing assay with reconstituted full-length neuronal SNAREs, and synaptotagmin-1 (19,43,44).…”

Section: Resultsmentioning

confidence: 99%

“…Subsequently, Ca 2+ -solution was injected into the flow chamber consisting of 500 μM Ca 2+ , 500 nM Cy5 dye molecules (used as an indicator for the arrival of Ca 2+ in the evanescent field), and Cpx, Cpx fragments, or Cpx mutant at 2-μM concentration in buffer V. Ca 2+ -triggered fusion events were monitored within the same field-of-view upon injection for a 1-min period. The injection was performed at a speed of 66 μL/s by a motorized syringe pump (Harvard Apparatus) using a withdrawal method similar to the one described previously (19,39). All experiments were carried out at ambient temperature (25°C).…”

Section: Methodsmentioning

confidence: 99%

“…The surface of the quartz slides was passivated by coating the surface with PEG molecules, which alleviated nonspecific binding of vesicles. The same protocol and quality controls (surface coverage and nonspecific binding) were used as described previously (39,44), except that PEG-SVA (Laysan Bio) instead of mPEG-SCM (Laysan Bio) was used because it has a longer half-life. The surface was functionalized by inclusion of biotin-PEG (Laysan Bio) during pegylation.…”

Section: Methodsmentioning

confidence: 99%

“…The size distributions of the SV and PM vesicles were analyzed by cryo-EM, as described previously (39). Single-vesicle content-mixing assay.…”

Section: Methodsmentioning

confidence: 99%

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

Gong

Lai

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Protein–lipid interactions critical to replication of the influenza A virus

Chlanda

Zimmerberg

2016

FEBS Letters

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Influenza A virus (IAV) assembles on the plasma membrane where viral proteins localize to form a bud encompassing the viral genome, which ultimately pinches off to give rise to newly formed infectious virions. Upon entry, the virus faces the opposite task—fusion with the endosomal membrane and disassembly to deliver the viral genome to the cytoplasm. There are at least four influenza proteins—hemagglutinin (HA), neuraminidase (NA), matrix 1 protein (M1), and the M2 ion channel—that are known to directly interact with the cellular membrane and modify membrane curvature in order to both assemble and disassemble membrane-enveloped virions. Here, we summarize and discuss current knowledge of the interactions of lipids and membrane proteins involved in the IAV replication cycle.

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Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion

Cited by 174 publications

References 71 publications

Prevalent mechanism of membrane bridging by synaptotagmin-1

Prevalent mechanism of membrane bridging by synaptotagmin-1

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

Protein–lipid interactions critical to replication of the influenza A virus

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