scholarly journals Molecular Determinants of Complexin Clamping in Reconstituted Single-Vesicle Fusion

2021 ◽  
Author(s):  
Manindra Bera ◽  
Sathish Ramakrishnan ◽  
Jeff Coleman ◽  
Shyam S Krishnakumar ◽  
James E Rothman

Previously we reported that Synaptotagmin-1 and Complexin synergistically clamp the SNARE assembly process to generate and maintain a pool of docked vesicles that fuse rapidly and synchronously upon Ca2+ influx (Ramakrishnan et al. 2020). Here using the same in vitro single-vesicle fusion assay, we establish the molecular details of the Complexin clamp and its physiological relevance. We find that a delay in fusion kinetics, likely imparted by Synaptotagmin-1, is needed for Complexin to block fusion. Systematic truncation/mutational analyses reveal that continuous alpha-helical accessory-central domains of Complexin are essential for its inhibitory function and specific interaction of the accessory helix with the SNAREpins, analogous to the trans clamping model, enhances this functionality. The c-terminal domain promotes clamping by locally elevating Complexin concentration through interactions with the membrane. Further, we find that Complexin likely contributes to rapid Ca2+-synchronized vesicular release by preventing un-initiated fusion rather than by directly facilitating vesicle fusion.

2012 ◽  
Vol 7 (5) ◽  
pp. 921-934 ◽  
Author(s):  
Jiajie Diao ◽  
Yuji Ishitsuka ◽  
Hanki Lee ◽  
Chirlmin Joo ◽  
Zengliu Su ◽  
...  

2017 ◽  
Author(s):  
Yulia Akbergenova ◽  
Yao V. Zhang ◽  
Shirley Weiss-Sharabi ◽  
Karen L. Cunningham ◽  
J. Troy Littleton

AbstractNeurons communicate through neurotransmitter release at specialized synaptic regions known as active zones (AZs). Using transgenic biosensors to image postsynaptic glutamate receptor activation following single vesicle fusion events at Drosophila neuromuscular junctions, we analyzed release probability (Pr) maps for a defined connection with ~300 AZs between synaptic partners. Although Pr was very heterogeneous, it represented a stable and unique feature of each AZ. Pr heterogeneity was not abolished in mutants lacking Synaptotagmin 1, suggesting the AZ itself is likely to harbor a key determinant(s). Indeed, AZ Pr was strongly correlated with presynaptic Ca2+ channel density and Ca2+ influx at single release sites. In addition, Pr variability was reflected in the postsynaptic compartment, as high Pr AZs displayed a distinct pattern of glutamate receptor clustering. Developmental analysis suggests that high Pr sites emerge from earlier formed AZs, with a temporal maturation in transmission strength occurring over several days.


eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Ying Lai ◽  
Jiajie Diao ◽  
Daniel J Cipriano ◽  
Yunxiang Zhang ◽  
Richard A Pfuetzner ◽  
...  

Previously we showed that fast Ca2+-triggered vesicle fusion with reconstituted neuronal SNAREs and synaptotagmin-1 begins from an initial hemifusion-free membrane point contact, rather than a hemifusion diaphragm, using a single vesicle–vesicle lipid/content mixing assay (<xref ref-type="bibr" rid="bib5">Diao et al., 2012</xref>). When complexin-1 was included, a more pronounced Ca2+-triggered fusion burst was observed, effectively synchronizing the process. Here we show that complexin-1 also reduces spontaneous fusion in the same assay. Moreover, distinct effects of several complexin-1 truncation mutants on spontaneous and Ca2+-triggered fusion closely mimic those observed in neuronal cultures. The very N-terminal domain is essential for synchronization of Ca2+-triggered fusion, but not for suppression of spontaneous fusion, whereas the opposite is true for the C-terminal domain. By systematically varying the complexin-1 concentration, we observed differences in titration behavior for spontaneous and Ca2+-triggered fusion. Taken together, complexin-1 utilizes distinct mechanisms for synchronization of Ca2+-triggered fusion and inhibition of spontaneous fusion.


Author(s):  
Peter Mühlenbrock ◽  
Merve Sari ◽  
Claudia Steinem

AbstractNeuronal fusion mediated by soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) is a fundamental cellular process by which two initially distinct membranes merge resulting in one interconnected structure to release neurotransmitters into the presynaptic cleft. To get access to the different stages of the fusion process, several in vitro assays have been developed. In this review, we provide a short overview of the current in vitro single vesicle fusion assays. Among those assays, we developed a single vesicle assay based on pore-spanning membranes (PSMs) on micrometre-sized pores in silicon, which might overcome some of the drawbacks associated with the other membrane architectures used for investigating fusion processes. Prepared by spreading of giant unilamellar vesicles with reconstituted t-SNAREs, PSMs provide an alternative tool to supported lipid bilayers to measure single vesicle fusion events by means of fluorescence microscopy. Here, we discuss the diffusive behaviour of the reconstituted membrane components as well as that of the fusing synthetic vesicles with reconstituted synaptobrevin 2 (v-SNARE). We compare our results with those obtained if the synthetic vesicles are replaced by natural chromaffin granules under otherwise identical conditions. The fusion efficiency as well as the different fusion states observable in this assay by means of both lipid mixing and content release are illuminated.


eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Sathish Ramakrishnan ◽  
Manindra Bera ◽  
Jeff Coleman ◽  
James E Rothman ◽  
Shyam S Krishnakumar

Calcium (Ca2+)-evoked release of neurotransmitters from synaptic vesicles requires mechanisms both to prevent un-initiated fusion of vesicles (clamping) and to trigger fusion following Ca2+-influx. The principal components involved in these processes are the vesicular fusion machinery (SNARE proteins) and the regulatory proteins, Synaptotagmin-1 and Complexin. Here, we use a reconstituted single-vesicle fusion assay under physiologically-relevant conditions to delineate a novel mechanism by which Synaptotagmin-1 and Complexin act synergistically to establish Ca2+-regulated fusion. We find that under each vesicle, Synaptotagmin-1 oligomers bind and clamp a limited number of ‘central’ SNARE complexes via the primary interface and introduce a kinetic delay in vesicle fusion mediated by the excess of free SNAREpins. This in turn enables Complexin to arrest the remaining free ‘peripheral’ SNAREpins to produce a stably clamped vesicle. Activation of the central SNAREpins associated with Synaptotagmin-1 by Ca2+ is sufficient to trigger rapid (<100 msec) and synchronous fusion of the docked vesicles.


2012 ◽  
Vol 443 (1) ◽  
pp. 223-229 ◽  
Author(s):  
Ying Lai ◽  
Yeon-Kyun Shin

Syt1 (synaptotagmin 1) is a major Ca2+ sensor for synaptic vesicle fusion. Although Syt1 is known to bind to SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) complexes and to the membrane, the mechanism by which Syt1 regulates vesicle fusion is controversial. In the present study we used in vitro lipid-mixing assays to investigate the Ca2+-dependent Syt1 function in proteoliposome fusion. To study the role of acidic lipids, the concentration of negatively charged DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine) in the vesicle was varied. Syt1 stimulated lipid mixing by 3–10-fold without Ca2+. However, with Ca2+ there was an additional 2–5-fold enhancement. This Ca2+-dependent stimulation was observed only when there was excess PS (phosphatidylserine) on the t-SNARE (target SNARE) side. If there was equal or more PS on the v-SNARE (vesicule SNARE) side the Ca2+-dependent stimulation was not observed. We found that Ca2+ at a concentration between 10 and 50 μM was sufficient to give rise to the maximal enhancement. The single-vesicle-fusion assay indicates that the Ca2+-dependent enhancement was mainly on docking, whereas its effect on lipid mixing was small. Thus for Syt1 to function as a Ca2+ sensor, a charge asymmetry appears to be important and this may play a role in steering Syt1 to productively trans bind to the plasma membrane.


2012 ◽  
Vol 102 (3) ◽  
pp. 499a
Author(s):  
Jea-Yeol Kim ◽  
Bong-Kyu Choi ◽  
Mal-Gi Choi ◽  
Sun-Ae Kim ◽  
Yeon-Kyun Shin ◽  
...  

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Hanki Lee ◽  
Wook Jin ◽  
Byeong-Chul Jeong ◽  
Joo-Won Suh

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