Fig. 1.

Generation and localization of Syt 1 Ca²⁺-binding mutations. (A) Schematic diagrams of essential residues that coordinate Ca²⁺ binding to the Syt 1 C2A and C2B domain, rendered according to Ferandez et al. (18). The key Ca²⁺ binding D3 and D4 aspartate residues (highlighted in blue) were neutralized to asparagines in the C2A or C2B domain. (B) Immunostaining of hatching stage (21–24 h after fertilization) embryonic NMJs with anti-HRP (green), a neuronal membrane marker to highlight presynaptic terminals, and anti-Syt 1 (magenta). The merged signal is displayed in the bottom image. WT, as well as C2A and C2B mutant (indicated with D > N) proteins from transgenes, show similar levels and distribution of Syt 1 compared with endogenous protein in WT embryos (Left). Null mutants lack anti-Syt 1 immunoreactivity. (Scale bar, 5 μm.)