Figure 1

Plasma membrane targeted cytoplasmic domain of syt1 rescues SV exocytosis in syt1 KO neurons. (a) Diagram of the GAP43-C2AB chimera. The cytoplasmic domain of syt1 (C2AB) was targeted to the presynaptic plasma membrane by fusing it to the first 20 residues of GAP-43. (b) Immunocytochemistry, using an anti-C2A domain antibody, indicated that GAP43-C2AB was localized to the plasma membrane while soluble GFP was cytosolic. Scale bar, 10 μm. (c) Immunostaining with an antibody specific for the C2A domain of syt1 showing that GAP43-C2AB was localized to synapsin I labeled presynaptic boutons. Scale bar, 4 μm. (d) Immunoblot analysis revealed that syt1^FL was expressed in syt1 KO neurons at levels similar to endogenous syt1 in WTcells; GAP43-C2AB was expressed at somewhat lower levels (57 %). (e) Distribution of expressed syt1 constructs analyzed via subcellular fractionation of WT neurons, syt1 KO neurons, and KO neurons expressing syt1^FL or GAP43-C2AB. Antibodies directed against the NMDA receptor (NMDAR) or synaptophysin were used to distinguish plasma membrane from SV fractions, respectively. Vertical lines denote different blots. (f) Typical traces of evoked EPSCs recorded from syt1 KO neurons (KO) and lentivirus-infected KO neurons expressing syt1^FL or GAP43-C2AB. (g) Bar graph showing the peak amplitude of EPSCs recorded from syt1 KO neurons (n = 24), KO neurons expressing syt1^FL (n = 21) or GAP43-C2AB (n = 20). (h) Average normalized cumulative EPSC charge transfer over 0.5 s for KO neurons expressing the indicated constructs. ** p<0.001. Error bars represent SEM.