Synergistic roles of Synaptotagmin-1 and complexin in calcium-regulated neuronal exocytosis

Sathish Ramakrishnan; Manindra Bera; Jeff Coleman; James E Rothman; Shyam S Krishnakumar

doi:10.7554/eLife.54506

Figure 3.

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Syt1 and Cpx bind and clamp different pools of SNAREpins.

(A) In situ removal of Cpx from the Syt1/Cpx clamped state by extensive (40X) buffer wash triggers spontaneous fusion of the docked vesicles. This further confirms that both Syt1 and Cpx are required to produce a stable clamped state. (B) Inclusion of soluble cytoplasmic domain of t-SNAREs (CDT) blocked the spontaneous fusion events triggered by elimination of Cpx from the Syt1/Cpx clamped vesicles. The CDT-treated vesicles remain sensitive to Ca²⁺ influx and most of the vesicles fuse rapidly and synchronously following the addition of 1 mM Ca²⁺. This indicates that a sub-set of SNAREpins are protected against CDT even in the absence of Cpx, implying that Syt1 and Cpx likely engage and clamp different set of SNAREpins. It further shows that the Syt1-associated SNAREpins are sufficient to catalyze rapid Ca²⁺-triggered vesicle fusion. Data (average ± standard deviation) obtained from four to five independent experiments with at least 200 vesicles in total are shown. Note: Only single SNAREpins clamped by either Syt1 or Cpx is shown for illustrative purposes.

Figure 3—figure supplement 1.

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Representative fluorescence images showing that the extensive (40X) buffer wash results in complete washout of Cpx from clamped Syt1/Cpx vesicles.

This was assessed using fluorescently (Alexa488) labeled Cpx and fluorescence recorded prior to the addition of Cpx (left panel), upon addition of Cpx (middle) and after dilution by 40X buffer wash (right panel) are shown. Images corresponding to multiple 5 µm suspended bilayer is shown. Note: The vesicle and bilayer fluorescence are not shown for clarity.

Figure 3—figure supplement 2.

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Respresentative fluorescence images showing the effect of CDT wash and subsequent Ca²⁺ addition.

(A) Representative fluorescence (ATTO647N) image showing that clamped Syt1/Cpx vesicles (left panel) fuse spontaneously upon wash out of Cpx by dilution. Upon fusion, the ATTO647N dye on the vesicles mixes with suspended bilayer resulting in increase of the background ATTO647N fluorescence (right panel). (B) In the presence of excess inhibitory soluble cytoplasmic domain of the t-SNARE complex (CDT), the vesicles remain docked even after the removal of previously-bound Cpx by the buffer wash (middle panel). Subsequent addition of Ca²⁺ (1 mM) triggered rapid and synchronous fusion of these docked vesicles. Images corresponding to multiple 5 µm suspended bilayer is shown.