scholarly journals A synaptotagmin suppressor screen indicates SNARE binding controls the timing and Ca2+ cooperativity of vesicle fusion

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Zhuo Guan ◽  
Maria Bykhovskaia ◽  
Ramon A Jorquera ◽  
Roger Bryan Sutton ◽  
Yulia Akbergenova ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Binding Pocket ◽  
Vesicle Fusion ◽  
Snare Complex ◽  
Structural Basis ◽  
C2 Domains ◽  
Vesicle Cycling ◽  
Key Residues ◽  
Synaptic Vesicle Fusion

The synaptic vesicle Ca2+ sensor Synaptotagmin binds Ca2+ through its two C2 domains to trigger membrane interactions. Beyond membrane insertion by the C2 domains, other requirements for Synaptotagmin activity are still being elucidated. To identify key residues within Synaptotagmin required for vesicle cycling, we took advantage of observations that mutations in the C2B domain Ca2+-binding pocket dominantly disrupt release from invertebrates to humans. We performed an intragenic screen for suppressors of lethality induced by expression of Synaptotagmin C2B Ca2+-binding mutants in Drosophila. This screen uncovered essential residues within Synaptotagmin that suggest a structural basis for several activities required for fusion, including a C2B surface implicated in SNARE complex interaction that is required for rapid synchronization and Ca2+ cooperativity of vesicle release. Using electrophysiological, morphological and computational characterization of these mutants, we propose a sequence of molecular interactions mediated by Synaptotagmin that promote Ca2+ activation of the synaptic vesicle fusion machinery.

scholarly journals Synaptotagmin 1 clamps synaptic vesicle fusion in mammalian neurons independent of complexin

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Nicholas A. Courtney ◽  
Huan Bao ◽  
Joseph S. Briguglio ◽  
Edwin R. Chapman
Keyword(s):  
Synaptic Vesicle ◽  
Vesicle Fusion ◽  
Snare Proteins ◽  
Major Focus ◽  
C2 Domains ◽  
Releasable Pool ◽  
Readily Releasable Pool ◽  
Synaptotagmin 1 ◽  
Synaptic Vesicle Fusion ◽  
C2a Domain

Abstract Synaptic vesicle (SV) exocytosis is mediated by SNARE proteins. Reconstituted SNAREs are constitutively active, so a major focus has been to identify fusion clamps that regulate their activity in synapses: the primary candidates are synaptotagmin (syt) 1 and complexin I/II. Syt1 is a Ca2+ sensor for SV release that binds Ca2+ via tandem C2-domains, C2A and C2B. Here, we first determined whether these C2-domains execute distinct functions. Remarkably, the C2B domain profoundly clamped all forms of SV fusion, despite synchronizing residual evoked release and rescuing the readily-releasable pool. Release was strongly enhanced by an adjacent C2A domain, and by the concurrent binding of complexin to trans-SNARE complexes. Knockdown of complexin had no impact on C2B-mediated clamping of fusion. We postulate that the C2B domain of syt1, independent of complexin, is the molecular clamp that arrests SVs prior to Ca2+-triggered fusion.

scholarly journals SNARE-complex disassembly by NSF follows synaptic-vesicle fusion

2001 ◽  
Vol 98 (21) ◽  
pp. 12233-12238 ◽  
Author(s):  
J. T. Littleton ◽  
R. J. O. Barnard ◽  
S. A. Titus ◽  
J. Slind ◽  
E. R. Chapman ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Vesicle Fusion ◽  
Snare Complex ◽  
Synaptic Vesicle Fusion

scholarly journals Alpha-Synuclein and Calpains Disrupt SNARE-Mediated Synaptic Vesicle Fusion During Manganese Exposure in SH-SY5Y Cells

Cells ◽  
2018 ◽  
Vol 7 (12) ◽  
pp. 258 ◽  
Author(s):  
Can Wang ◽  
Zhuo Ma ◽  
Dong-Ying Yan ◽  
Chang Liu ◽  
Yu Deng ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Vesicle Fusion ◽  
Alpha Synuclein ◽  
Convincing Evidence ◽  
Snare Complex ◽  
Attachment Protein ◽  
Over Expression ◽  
Synaptic Vesicle Fusion

Synaptic vesicle fusion is mediated by an assembly of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs), composed of syntaxin 1, soluble NSF-attachment protein (SNAP)-25, and synaptobrevin-2/VAMP-2. Previous studies have suggested that over-exposure to manganese (Mn) could disrupt synaptic vesicle fusion by influencing SNARE complex formation, both in vitro and in vivo. However, the mechanisms underlying this effect remain unclear. Here we employed calpeptin, an inhibitor of calpains, along with a lentivirus vector containing alpha-synuclein (α-Syn) shRNA, to examine whether specific SNAP-25 cleavage and the over-expression of α-Syn disturbed the formation of the SNARE complex in SH-SY5Y cells. After cells were treated with Mn for 24 h, fragments of SNAP-25-N-terminal protein began to appear; however, this effect was reduced in the group of cells which were pre-treated with calpeptin. FM1-43-labeled synaptic vesicle fusion decreased with Mn treatment, which was consistent with the formation of SNARE complexes. The interaction of VAMP-2 and α-Syn increased significantly in normal cells in response to 100 μM Mn treatment, but decreased in LV-α-Syn shRNA cells treated with 100 μM Mn; similar results were observed in terms of the formation of SNARE complexes and FM1-43-labeled synaptic vesicle fusion. Our data suggested that Mn treatment could increase [Ca2+]i, leading to abnormally excessive calpains activity, which disrupted the SNARE complex by cleaving SNAP-25. Our data also provided convincing evidence that Mn could induce the over-expression of α-Syn; when combined with VAMP-2, α-Syn prevented VAMP-2 from joining the SNARE complex cycle.

scholarly journals Cell-Specific Loss of SNAP25 from Cortical Projection Neurons Allows Normal Development but Causes Subsequent Neurodegeneration

2018 ◽  
Vol 29 (5) ◽  
pp. 2148-2159 ◽  
Author(s):  
Anna Hoerder-Suabedissen ◽  
Kim V Korrell ◽  
Shuichi Hayashi ◽  
Alexander Jeans ◽  
Denise M O Ramirez ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Hippocampal Neurons ◽  
Vesicle Fusion ◽  
Conditional Knockout ◽  
Snare Complex ◽  
Projection Neurons ◽  
Cortical Projection ◽  
Markers Of Inflammation ◽  
Synaptic Vesicle Fusion

Abstract Synaptosomal associated protein 25 kDa (SNAP25) is an essential component of the SNARE complex regulating synaptic vesicle fusion. SNAP25 deficiency has been implicated in a variety of cognitive disorders. We ablated SNAP25 from selected neuronal populations by generating a transgenic mouse (B6-Snap25tm3mcw (Snap25-flox)) with LoxP sites flanking exon5a/5b. In the presence of Cre-recombinase, Snap25-flox is recombined to a truncated transcript. Evoked synaptic vesicle release is severely reduced in Snap25 conditional knockout (cKO) neurons as shown by live cell imaging of synaptic vesicle fusion and whole cell patch clamp recordings in cultured hippocampal neurons. We studied Snap25 cKO in subsets of cortical projection neurons in vivo (L5—Rbp4-Cre; L6—Ntsr1-Cre; L6b—Drd1a-Cre). cKO neurons develop normal axonal projections, but axons are not maintained appropriately, showing signs of swelling, fragmentation and eventually complete absence. Onset and progression of degeneration are dependent on the neuron type, with L5 cells showing the earliest and most severe axonal loss. Ultrastructural examination revealed that cKO neurites contain autophagosome/lysosome-like structures. Markers of inflammation such as Iba1 and lipofuscin are increased only in adult cKO cortex. Snap25 cKO can provide a model to study genetic interactions with environmental influences in several disorders.

Sign in / Sign up

Export Citation Format

Share Document