Syt linker mutants are targeted to synapses. (a) The linker between the C2A and C2B domains of syt (residues 264–272, SAEKEEQEK) was modified as indicated. TMD stands for Transmembrane Domain. (b) Syt KO hippocampal neurons were infected with lentivirus expressing each mutant, and immunostained for syt (red) and synapsin (green). WT littermates, that harbor native endogenous syt, were used as controls (WT). (c) Colocalization of WT and linker mutant forms of syt with synapsin; in all cases the mutant forms of syt were well targeted to synaptic sites. For each condition, two distinct random regions were imaged from each of three coverslips that were derived from three independent litters of mice, yielding an N of six images for each construct. The total number of puncta was 1,180–1,320 per condition. The mean values ± SEM are indicated. Statistical significance was determined using a bootstrap approach (see Online Methods); no significant differences were observed (Supplementary Statistics).