Fig 1.
Synaptotagmin structure and C2A mutations.
A, Protein alignment of loops 1 and 3 of the C2A domain of synaptotagmin 1 from Human, Mouse, Rat, and Drosophila (* = Ca2+ binding aspartates, boxes = loop 1 and loop 3 hydrophobic tip residues) B, Crystal structure of synaptotagmin and the SNARE complex showing a postulated role of the C2 domains in triggering fusion, adapted from [19]. Negatively charged residues of the Ca2+ binding pockets are shown as sticks in red, the hydrophobic residues at the tips of these pockets are shown as sticks in gray, and Ca2+ ions are shown as green spheres. VM = vesicle membrane and PM = presynaptic membrane. C, A cartoon depiction of the C2A domain. Colors as in panel B. D, Hydrophilic glutamic acid substitutions are indicated in white. Sequential mutation of C2A hydrophobic tip residues to hydrophilic residues is predicted to increasingly disrupt synaptotagmin’s ability to penetrate, warp and disorder lipids of the presynaptic membrane.
Fig 2.
Mutation of the hydrophobic tip residues disrupts evoked transmitter release.
The single hydrophobic mutation decreased neurotransmitter release by 50% while the double mutation inhibited release to a greater extent than that seen in sytnull mutants. A, Representative traces of EJPs for P[sytWT], P[sytA-ME], and P[sytA-ME,FE]. B, Mean EJP amplitude ± SEM for P[sytWT], P[sytA-ME], and P[sytA-ME,FE] (Tukey multiple comparisons, p < 0.0001 = ****). C, Representative traces of EJPs for P[sytA-ME,FE] and sytnull. D, Mean EJP amplitude ± SEM for P[sytA-ME,FE] and sytnull (Tukey multiple comparisons, p < 0.01 = **).
Fig 3.
Expression and localization of synaptotagmin are unaffected by hydrophobic mutations.
A, Representative western blots of transgenic synaptotagmin expression levels with actin as a loading control. B, Mean protein expression levels ± SEM, normalized to actin (One-way ANOVA, no significant differences). C, Representative confocal images of larval neuromuscular junctions labeled with anti-synaptotagmin antibodies (scale bar = 20 μm). Synaptotagmin is appropriately concentrated at synaptic sites in all three genotypes.
Fig 4.
Spontaneous events are unaffected by hydrophobic mutations.
A, Representative traces of mEJPs for P[sytWT], P[sytA-ME], and P[sytA-ME,FE]. B, Mean mEJP amplitudes ± SEM (Kruskal Wallis test with Dunn’s multiple comparison test, no significance = ns). C, Mean mEJP frequency ± SEM (Kruskal Wallis test with Dunn’s multiple comparison test, no significance = ns).
Fig 5.
Probability of release is decreased by hydrophobic mutations.
Probability of release was determined using a paired pulse protocol with interpulse intervals of 10, 20, 50, and 100ms. A, Representative traces of paired EJPs for P[sytWT], P[sytA-ME], and P[sytA-ME,FE] with a 20ms interpulse interval. B, Mean paired pulse ratios ± SEM for P[sytWT], P[sytA-ME], and P[sytA-ME,FE] (Two-way Repeated Measures ANOVA with Tukey post-hoc, p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***, p < 0.0001 = ****). Indicated differences are between mutants and P[sytWT], though the paired pulse ratio was significantly different (p < 0.05) between P[sytA-ME], and P[sytA-ME,FE], for all interpulse intervals except 100ms.
Fig 6.
The apparent Ca2+ affinity of release is decreased by hydrophobic mutations.
A, Mean EJP amplitude ± SEM across a range of Ca2+ concentrations fit with a nonlinear regression. B, Ca2+ curves normalized to maximum EJP amplitude predicted by the nonlinear regression. The significant rightward shift in the curve (EC50, non overlapping confidence intervals) indicates a decrease in the apparent Ca2+ affinity of neurotransmitter release.