Official websites use. Share sensitive information only on official, secure websites. Correspondence should be addressed to Dr. Noreen E. Email: Noreen. Reist Colostate. Here we report the most severe disruption of function by any synaptotagmin mutation identified to date.
A , Alignment of synaptotagmin I from rat and Drosophila. Within the alignment, conserved residues are shown in gray and identical residues are boxed. Phenylalanine Fig. Drosophila embryos were transfected with the mutant pUAST plasmids as described previously Paddock et al. Because no gender selection was used, a mix of male and female animals were used for all experiments. Standard genetic techniques were used to cross the transgenes into the syt null background; selected larvae were green fluorescent protein GFP negative and y negative without regard to gender.
Unless specifically noted, experiments were performed in the syt null background to express the transgene in the absence of endogenous synaptotagmin I. The effect of the syt B-IE mutant protein was examined both in the absence and presence of expression from the wild type synaptotagmin gene. Parental lines contain the native syt gene on the CyO balancer chromosome to maintain healthy stocks, and transgene expression is under the control of a UAS promoter that requires Gal4 to promote expression.
Saline used was HL3 [in m m : 5 KCl, 1. Immunolabeling and immunoblotting of third-instar larvae were performed by standard techniques Paddock et al. The cuticle was perforated and opened along the dorsal midline with a glass needle, and the gut was removed to expose the CNS. After changing to HL3 containing 1. Evoked EJPs were generated by stimulating segmental nerves with 1 ms pulses of 30β50 nA at 0.
Intracellular recordings of spontaneous mEJPs were collected and analyzed as described previously Mackler and Reist, To record synaptic currents, Drosophila embryos 22β24 h after egg laying were dechorionated with bleach and manually dissected as described previously Featherstone et al. Evoked synaptic currents were triggered by 5β10 V electrical stimulation of the appropriate segmental nerve using a suction electrode filled with bath solution.
