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.
Highlights
Neurotransmitter or hormone release requires a basal membrane fusion machinery, and one or more Ca2+-sensors to link fusion to the electrical activity of the cell
Since chromaffin cells do not have a limited number of release sites, Ca2+-dependent priming leads to a large and readily measurable increase in the size of the pool of primed large dense-core vesicles (LDCV) when prestimulation [Ca2+] exceeds 100–200 nM
By expressing either Syt-1 or Syt-7 in Syt-1/Syt-7 double KO (DKO) cells, we showed that both synaptotagmins are able to act as stand-alone calcium sensors, with Syt-7 being a slower sensor than Syt-1 both on the population level, and at the level of single LDCV fusion (Figure 1)
Summary
Neurotransmitter or hormone release requires a basal membrane fusion machinery, and one or more Ca2+-sensors to link fusion to the electrical activity of the cell. The machinery driving vesicle-to-membrane fusion consists of the SNAREs (Fang and Lindau, 2014; Jahn and Fasshauer, 2012), associated proteins Munc and Munc necessary for SNARE-complex assembly (Rizo and Xu, 2015), whereas the Ca2+-sensors are proteins of the synaptotagmin (Syt) family (Pinheiro et al, 2016), which act together with complexins (Makke et al, 2018; Trimbuch and Rosenmund, 2016). Syts harbor two C2-domains, which can bind to Ca2+ and phospholipids (in 8 of the 17 Syt isoforms present in the mammalian genome), and to SNAREs (Sudhof, 2002). The co-existence of two Syts with very different kinetics and Ca2+-affinities in the same cell raises the question whether they
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