Abstract
The assembly of SNARE complexes between syntaxin, SNAP-25 and synaptobrevin is required to prime synaptic vesicles for fusion. Since Munc18 and tomosyn compete for syntaxin interactions, the interplay between these proteins is predicted to be important in regulating synaptic transmission. We explored this possibility, by examining genetic interactions between C. elegans unc-18(Munc18), unc-64(syntaxin) and tom-1(tomosyn). We have previously demonstrated that unc-18 mutants have reduced synaptic transmission, whereas tom-1 mutants exhibit enhanced release. Here we show that the unc-18 mutant release defect is associated with loss of two morphologically distinct vesicle pools; those tethered within 25 nm of the plasma membrane and those docked with the plasma membrane. In contrast, priming defective unc-13 mutants accumulate tethered vesicles, while docked vesicles are greatly reduced, indicating tethering is UNC-18-dependent and occurs in the absence of priming. C. elegans unc-64 mutants phenocopy unc-18 mutants, losing both tethered and docked vesicles, whereas overexpression of open syntaxin preferentially increases vesicle docking, suggesting UNC-18/closed syntaxin interactions are responsible for vesicle tethering. Given the competition between vertebrate tomosyn and Munc18, for syntaxin binding, we hypothesized that C. elegans TOM-1 may inhibit both UNC-18-dependent vesicle targeting steps. Consistent with this hypothesis, tom-1 mutants exhibit enhanced UNC-18 plasma membrane localization and a concomitant increase in both tethered and docked synaptic vesicles. Furthermore, in tom-1;unc-18 double mutants the docked, primed vesicle pool is preferentially rescued relative to unc-18 single mutants. Together these data provide evidence for the differential regulation of two vesicle targeting steps by UNC-18 and TOM-1 through competitive interactions with syntaxin.
Highlights
Rapid, calcium-regulated release of neurotransmitter is dependent on the availability of a readily releasable, primed synaptic vesicle pool (Sudhof, 2004)
UNC-18-dependent synaptic vesicle tethering occurs in the absence of docking C. elegans unc-18 mutants exhibit defects in evoked and spontaneous synaptic transmission and a concomitant reduction in the size of the readily releasable synaptic vesicle pool (Weimer et al, 2003)
Morphometric analyses of unc-18 mutant synaptic profiles by EM revealed a reduction in two vesicle pools; those docked at the plasma membrane and those tethered within one vesicle diameter (>0 nm up to 25 nm) of the plasma membrane (Figure 1A)
Summary
Calcium-regulated release of neurotransmitter is dependent on the availability of a readily releasable, primed synaptic vesicle pool (Sudhof, 2004). Current models suggest that Munc13s promote SNARE complex assembly and stability, through interactions with syntaxin (Betz et al, 1997; Richmond et al, 2001; Basu et al, 2005; Madison et al, 2005; Stevens et al, 2005) This function of Munc was initially attributed to a post vesicle docking stage, on the basis of ultrastructural data from aldehyde fixed tissues which showed normal docking in null mutants (Aravamudan et al, 1999; Richmond et al, 1999; Varoqueaux et al, 2002). That this recently revealed docking defect reflects the loss of assembled SNARE complexes in Munc13/unc-13 mutants is supported by the observation that C. elegans syntaxin null mutants prepared by HPF fixation exhibit a similar docking defect (Hammarlund et al, 2007)
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