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. 2019 Jan;593(2):154-162.
doi: 10.1002/1873-3468.13317. Epub 2019 Jan 18.

Synaptotagmin oligomers are necessary and can be sufficient to form a Ca2+ -sensitive fusion clamp

Sathish Ramakrishnan  1 , Manindra Bera  1 , Jeff Coleman  1 , Shyam S Krishnakumar  1   2 , Frederic Pincet  1   3 , James E Rothman  1   2
Affiliations

Synaptotagmin oligomers are necessary and can be sufficient to form a Ca2+ -sensitive fusion clamp

Sathish Ramakrishnan et al. FEBS Lett. 2019 Jan.

Abstract

The buttressed-ring hypothesis, supported by recent cryo-electron tomography analysis of docked synaptic-like vesicles in neuroendocrine cells, postulates that prefusion SNAREpins are stabilized and organized by Synaptotagmin (Syt) ring-like oligomers. Here, we use a reconstituted single-vesicle fusion analysis to test the prediction that destabilizing the Syt1 oligomers destabilizes the clamp and results in spontaneous fusion in the absence of Ca2+ . Vesicles in which Syt oligomerization is compromised by a ring-destabilizing mutation dock and diffuse freely on the bilayer until they fuse spontaneously, similar to vesicles containing only v-SNAREs. In contrast, vesicles containing wild-type Syt are immobile as soon as they attach to the bilayer and remain frozen in place, up to at least 1 h until fusion is triggered by Ca2+ .

Keywords: SNARE proteins; Synaptotagmin; calcium; fusion clamp; single-vesicle analysis.

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Figures

Figure 1
Figure 1
Overview of the single‐vesicle docking and fusion analysis with a pore‐spanning bilayer (A) VAMP2 (± Syt1)‐containing SUVs are added to the t‐PSM from the top and monitored, using a confocal microscope from the bottom. (B) The docking, mobility and the fusion of the SUVs with the t‐PSM is tracked, using the fluorescence marker (ATTO647N‐PE) included in the SUVs. A representative fluorescence trace showing a behavior of a typical vesicle is shown. The time between docking and fusion (τ) is measured for each docked vesicle and the results for the whole population are presented as a survival curve (C). (D) To monitor the fate of substantial numbers of individual vesicles from each experimental trial, the SUVs were allowed to interact with t‐PSM for 10 min and critical parameters, including vesicle docking, mobility of the docked vesicles and Ca2+‐independent spontaneous fusion were acquired. The chamber was subsequently washed with buffer and then Ca2+ (1 mm) was added from the top to record (~ 5 min) Ca2+‐dependent fusion events.
Figure 2
Figure 2
The mobility of docked vesicles containing VAMP2 with or without Syt. (A) Representative time‐lapse fluorescence (ATTO647N) images of a single docked SUVs show that all vSUVs are diffusively mobile upon docking and fuse spontaneous with a half‐time of ~ 1 s (Fig. 3). In contrast, the vSUVs with Syt1WT (Syt1WT‐vSUV) are stably docked in‐place and largely immobile and do not fuse, until triggered by Ca2+. When Syt1 oligomerization is compromised with a targeted mutation (F349A), a large majority (85%) of these SUVs (Syt1349‐vSUV) are mobile and spontaneous fuse similar to vSUVs, while a minority (15%) are immobile upon docking and never fuse. Representative video files corresponding to these images are included as Videos [Link], [Link], [Link], [Link]. (B) The immobile fractions of Syt1WT‐vSUV (100%) and Syt1349‐vSUV (~ 15%) remain stably clamped and Ca2+‐sensitive for at least 1 h, the observation period limited by photo‐bleaching and bilayer stability. In such experiments, designed to test the stability of docked vesicles, it was necessary to minimize fluorescence bleaching by only imaging every 10 min prior to Ca2+‐addition, and every 1 min post‐Ca2+‐addition. The NBD‐fluorescence included on the pore‐spanning bilayer was recorded at each time point to verify the continued integrity of the pore‐spanning suspended bilayer.
Figure 3
Figure 3
Syt1 oligomers form a Ca2+‐sensitive fusion clamp. (A) The cumulative docking‐to‐fusion delays represented as the survival percentage shows that vSUVs spontaneous fuse with a half‐time of ~ 1 s, but Syt1WT‐vSUV remain stably docked. Destabilizing the Syt1 oligomers (Syt1349) destabilizes the fusion ‘clamp’ with the majority of vesicles proceeding to fuse spontaneously. (B) Clamped SUVs containing Syt1WT or Syt1349 are triggered to fuse by Ca2+ (1 mm). End‐point analysis at 5 min post‐Ca2+‐addition shows that > 90% of all clamped vesicles fuse following Ca2+ addition. Representative video file corresponding to fluorescence change associated with Ca2+‐triggered exocytosis is shown in Videos [Link], [Link]. The average values and standard deviations at each time point from three independent experiments are shown for each condition. In total, > 500 vesicles were analyzed for each condition.
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References

    1. Li X, Radhakrishnan A, Grushin K, Kasula R, Chaudhuri A, Gomathinayagam S, Krishnakumar SS, Liu J and Rothman JE (2018) Symmetrical organization of proteins under docked vesicles. FEBS Lett, 10.1002/1873-3468.13316. - DOI - PMC - PubMed
    1. Wang J, Bello O, Auclair SM, Wang J, Coleman J, Pincet F, Krishnakumar SS, Sindelar CV and Rothman JE (2014) Calcium sensitive ring‐like oligomers formed by Synaptotagmin. Proc Natl Acad Sci USA 111, 13966–13971. - PMC - PubMed
    1. Wang J, Li F, Bello OD, Sindelar CV, Pincet F, Krishnakumar SS and Rothman JE (2017) Circular oligomerization is an intrinsic property of Synaptotagmin. Elife 6, e27441. - PMC - PubMed
    1. Zanetti MN, Bello OD, Wang J, Coleman J, Cai Y, Sindelar CV, Rothman JE and Krishnakumar SS (2016) Ring‐like oligomers of SYNAPTOTAGMINs and related C2 domain proteins. ELife 5, 1–15. - PMC - PubMed
    1. Bello OD, Jouannot O, Chaudhuri A, Stroeva E, Coleman J, Volynski KE, Rothman JE and Krishnakumar SS (2018) Synaptotagmin oligomerization is essential for calcium control of regulated exocytosis. Proc Natl Acad Sci USA 115, E7624–E7631. - PMC - PubMed

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