Architecture of the synaptotagmin–SNARE machinery for neuronal exocytosis

Zhou, Qiuhong; Lai, Ying; Bacaj, Taulant; Zhao, Minglei; Lyubimov, A.Y.; Uervirojnangkoorn, Monarin; Zeldin, Oliver B.; Brewster, Aaron S.; Sauter, Nicholas K.; Cohen, Aina E.; Soltis, S. Michael; Alonso-Mori, Roberto; Chollet, Matthieu; Lemke, Henrik T.; Pfuetzner, Richard A.; Choi, Ucheor B.; Weis, William I.; Diao, Jiajie; Südhof, Thomas C.; Brunger, Axel T.

doi:10.1038/nature14975

Cited by 318 publications

(702 citation statements)

References 86 publications

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“…Subsequently, Ca 2+ -independent fusion is monitored for a 1-min period, followed by injection of 500 μM Ca 2+ , and Ca 2+ -triggered fusion was monitored for an additional 1-min period (for more details, see Methods). Our system qualitatively reproduces the effects of synaptotagmin-1, Cpx, and their mutations, on both spontaneous release and evoked release that have been observed in cortical neuronal cultures (19,45).…”

Section: Resultsmentioning

confidence: 57%

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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Section: Resultsmentioning

confidence: 57%

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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“…52 Ca 2+ -independent role of SytI is also supported by its ability to facilitate close membrane apposition in vitro even in the absence of calcium binding [24][25][26] and the recent identification of Ca 2+ -independent SNARE-binding motifs. 53 Syt XI may similarly facilitate lysosome docking to the cell membrane. Such a mode of action of Syt XI would recapitulate our recent finding with an unrelated C2 domain containing protein called dysferlin, which facilitated injury-triggered lysosome exocytosis by tethering lysosomes to the cell membrane.…”

Section: Discussionmentioning

confidence: 99%

Injured astrocytes are repaired by Synaptotagmin XI-regulated lysosome exocytosis

et al. 2015

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Astrocytes are known to facilitate repair following brain injury; however, little is known about how injured astrocytes repair themselves. Repair of cell membrane injury requires Ca 2+ -triggered vesicle exocytosis. In astrocytes, lysosomes are the main Ca 2+ -regulated exocytic vesicles. Here we show that astrocyte cell membrane injury results in a large and rapid calcium increase. This triggers robust lysosome exocytosis where the fusing lysosomes release all luminal contents and merge fully with the plasma membrane. In contrast to this, receptor stimulation produces a small sustained calcium increase, which is associated with partial release of the lysosomal luminal content, and the lysosome membrane does not merge into the plasma membrane. In most cells, lysosomes express the synaptotagmin (Syt) isoform Syt VII; however, this isoform is not present on astrocyte lysosomes and exogenous expression of Syt VII on lysosome inhibits their exocytosis. Deletion of one of the most abundant Syt isoform in astrocyte -Syt XI -suppresses astrocyte lysosome exocytosis. This identifies lysosome as Syt XI-regulated exocytic vesicle in astrocytes. Further, inhibition of lysosome exocytosis (by Syt XI depletion or Syt VII expression) prevents repair of injured astrocytes. These results identify the lysosomes and Syt XI as the sub-cellular and molecular regulators, respectively of astrocyte cell membrane repair.

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“…However, recent studies using high-resolution NMR (37) and crystallography (38) suggest that the C2 domains of Syt1 mediate SNARE binding, making it unlikely that PKC-dependent changes in the linker affect SNARE binding directly. However, it was recently suggested that the linker undergoes a structural change (39) and might become sterically able to engage the SNAREs or membranes without any change in the bimolecular affinity as such.…”

Section: Potentiation Of Spontaneous Release Is Mediated By Multiple mentioning

confidence: 99%

Phosphorylation of synaptotagmin-1 controls a post-priming step in PKC-dependent presynaptic plasticity

Jong

Meijer

Saarloos

et al. 2016

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

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Presynaptic activation of the diacylglycerol (DAG)/protein kinase C (PKC) pathway is a central event in short-term synaptic plasticity. Two substrates, Munc13-1 and Munc18-1, are essential for DAGinduced potentiation of vesicle priming, but the role of most presynaptic PKC substrates is not understood. Here, we show that a mutation in synaptotagmin-1 (Syt1 T112A ), which prevents its PKCdependent phosphorylation, abolishes DAG-induced potentiation of synaptic transmission in hippocampal neurons. This mutant also reduces potentiation of spontaneous release, but only if alternative Ca 2+ sensors, Doc2A/B proteins, are absent. However, unlike mutations in Munc13-1 or Munc18-1 that prevent DAG-induced potentiation, the synaptotagmin-1 mutation does not affect pairedpulse facilitation. Furthermore, experiments to probe vesicle priming (recovery after train stimulation and dual application of hypertonic solutions) also reveal no abnormalities. Expression of synaptotagmin-2, which lacks a seven amino acid sequence that contains the phosphorylation site in synaptotagmin-1, or a synaptotagmin-1 variant with these seven residues removed (Syt1 Δ109-116 ), supports normal DAG-induced potentiation. These data suggest that this seven residue sequence in synaptotagmin-1 situated in the linker between the transmembrane and C2A domains is inhibitory in the unphosphorylated state and becomes permissive of potentiation upon phosphorylation. We conclude that synaptotagmin-1 phosphorylation is an essential step in PKC-dependent potentiation of synaptic transmission, acting downstream of the two other essential DAG/PKC substrates, Munc13-1 and Munc18-1.P resynaptic strength changes rapidly during repetitive stimulation [short-term plasticity (STP)] and activation of intracellular signal transduction pathways (1, 2). The diacylglycerol (DAG)/protein kinase C (PKC) cascade is one of the most potent pathways at the presynaptic terminal. Its activation leads to 50-100% potentiation of spontaneous and action potential (AP)-evoked release (3-5), and is critical for multiple forms of presynaptic plasticity (6-8). DAG directly activates the vesicle priming factor Munc13-1 (9, 10) and indirectly activates downstream effectors via PKC. Activation of both Munc13-1 and PKC is essential for this pathway to operate (Fig. 1A) (8, 11). We previously identified Munc18-1 as an essential PKC substrate, because a nonphosphorylatable Munc18-1 mutant completely inhibits PKC-dependent STP (8, 12). Importantly, a phosphomimetic mutation of Munc18-1 cannot fully bypass the requirement for PKC activation, indicating that other PKC substrates must contribute to this form of plasticity (8). These substrates have not been identified to date.Among other presynaptic PKC substrates is synaptotagmin-1 (Syt1, ref. 13). Syt1 is the vesicular Ca 2+ sensor that mediates fast AP-evoked release in the hippocampus (14) and drives a large fraction of spontaneous release (15). In the latter case, Syt1 competes with alternative sensors, in particular Doc2s, for SNARE binding ...

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Architecture of the synaptotagmin–SNARE machinery for neuronal exocytosis

Cited by 318 publications

References 86 publications

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

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

Injured astrocytes are repaired by Synaptotagmin XI-regulated lysosome exocytosis

Phosphorylation of synaptotagmin-1 controls a post-priming step in PKC-dependent presynaptic plasticity

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