Synaptotagmin-7 places dense-core vesicles at the cell membrane to promote Munc13-2- and Ca2+-dependent priming

Abstract

Synaptotagmins confer calcium-dependence to the exocytosis of secretory vesicles, but how coexpressed synaptotagmins interact remains unclear. We find that synaptotagmin-1 and synaptotagmin-7 when present alone act as standalone fast and slow Ca2+-sensors for vesicle fusion in mouse chromaffin cells. When present together, synaptotagmin-1 and synaptotagmin-7 are found in largely non-overlapping clusters on dense-core vesicles. Synaptotagmin-7 stimulates Ca2+-dependent vesicle priming and inhibits depriming, and it promotes ubMunc13-2- and phorbolester-dependent priming, especially at low resting calcium concentrations. The priming effect of synaptotagmin-7 increases the number of vesicles fusing via synaptotagmin-1, while negatively affecting their fusion speed, indicating both synergistic and competitive interactions between synaptotagmins. Synaptotagmin-7 places vesicles in close membrane apposition (<6 nm); without it, vesicles accumulate out of reach of the fusion complex (20-40 nm). We suggest that a synaptotagmin-7-dependent movement toward the membrane is involved in Munc13-2/phorbolester/Ca2+-dependent priming as a prelude to fast and slow exocytosis triggering.

Phorbolester-activated Munc13-1 and ubMunc13-2 exert opposing effects on dense-core vesicle secretion

“…1A; traces are the mean of all measured cells). The pre-stimulation calcium concentration was ∼0.9 µM, which is near the optimal concentration for calcium-dependent vesicle priming in these cells (Pinheiro et al, 2013; Tawfik et al, 2021). We performed kinetic analysis of the capacitance traces by fitting them with a sum of two exponential functions and a straight line, using an automatic fitting routine (Tawfik et al, 2021).…”

“…Synaptotagmin-1 binds to Ca 2+ and triggers release in an interplay with complexin and the SNAREs themselves (Mohrmann, Dhara, & Bruns, 2015; Sudhof, 2013). Synaptotagmin-7 (Syt7) is a slower Ca 2+ -sensor, which is able to both trigger slow release downstream of SNARE-complex formation (Bacaj et al, 2013; Schonn, Maximov, Lao, Sudhof, & Sorensen, 2008), but also to act upstream of complex formation in a role leading to Ca 2+ -dependent vesicle priming (H. Liu et al, 2014; Tawfik et al, 2021). The exocytotic cascade culminates in the formation of a fusion pore through which water-soluble signaling molecules can escape (Alvarez de Toledo, Montes, Montenegro, & Borges, 2018; Chang, Chiang, & Jackson, 2017; Sharma & Lindau, 2018).…”

Implication of synaptotagmins 4 and 7 in activity-dependent somatodendritic dopamine release in the ventral midbrain

“…A role for Syt7 in asynchronous release was previously demonstrated [ 50 ]. This isoform has also been shown to synergize with Syt1 [ 51 ]. The function of Syt4 is more controversial because it has been proposed to act either as a negative regulator of vesicular exocytosis [ 52 – 54 ] or to promote some forms of neurotransmitter release [ 3 , 55 , 56 ], depending on the system investigated.…”

Allosteric stabilization of calcium and phosphoinositide dual binding engages three synaptotagmins in fast exocytosis

“…Besides syt1/2, Munc13 and syt7 have been proposed to function by bridging the vesicular and plasma membranes (Liu et al, 2016; Quade et al, 2019; Tawfik et al, 2021; Voleti et al, 2017). Differences in the distance these bridges may span, the binding rates and affinities for Ca 2+ and membranes, which determine the Ca 2+ sensitivity, duration, speed and sequence of molecular interaction, might explain their diverse roles in SV docking, priming and fusion and versatile effects on spontaneous-, synchronous- and delayed neurotransmission (Augustin et al, 1999; Bacaj et al ., 2013; Basu et al, 2007; Liu et al, 2014; Luo and Sudhof, 2017; Quade et al ., 2019; Varoqueaux et al, 2002; Vevea et al, 2021; Wen et al, 2010; Xu et al, 2017).…”

“…This is a simplification, as we did not take the individual SV/syt distances to the plasma membrane into account. This distance is affected by several synaptic proteins, including syt1, Munc13 and Syt7 (Chen et al ., 2021; Imig et al, 2014; Liu et al, 2016; Quade et al, 2019; Tawfik et al, 2021; Voleti et al, 2017). A role of these proteins in short term plasticity is firmly established, yet precise mechanistic details are still lacking (Jackman et al, 2016; Rosenmund et al, 2002; Shin et al, 2010).…”