Drosophila synaptotagmin I null mutants survive to early adulthood

Abstract

Synaptotagmin is a synaptic vesicle protein required for efficient neurotransmitter release, yet its exact role in the synaptic vesicle cycle is unclear. Drosophila presents an ideal organism for studies aimed at determining the in vivo functions of proteins. However, synaptotagmin studies have been limited by the early (embryonic or first instar) lethality previously reported for Drosophila synaptotagmin I null (syt(null)) mutants. Here we report a new culturing technique that enhances survival of severely uncoordinated mutants thereby permitting Drosophila syt(null) mutants to survive through early adulthood. We examined synapses in syt(null) third instar larvae by electrophysiology and found that they exhibit severely decreased and asynchronous evoked neurotransmitter release, as well as an increased rate of spontaneous neurotransmitter release, as previously seen in first instar syt(null) larvae. The ability to examine severe synaptotagmin mutants as third instar larvae, a stage where electrophysiological and morphological analyses are more easily accomplished, will facilitate structure/function studies.

Mutations in aDrosophilaα2δ Voltage-Gated Calcium Channel Subunit Reveal a Crucial Synaptic Function

“…10 -20 larvae were raised at 25°C, separated from heterozygous siblings as second-instar larvae and grown on agar apple plates with yeast coating (Loewen et al, 2001). Third-instar larvae were dissected in saline then bathed in modified HL-3 saline with the following ionic concentrations (in mM): NaCl 70, KCl 5, MgCl 2 10, NaHCO 3 10, sucrose 115, Trehalose 5, HEPES 5, pH7.2, and CaCl 2 as indicated.…”

“…Despite the variety of molecular lesions in the d␣ 2 ␦-3 gene, all 5 of the d␣ 2 ␦-3 mutants (DD106, DD131, DD173, DD174, and DD196 ) die as well formed, late-stage embryos when homozygous or when in combination with each other or either of two different deficiencies, Df(2)CX1 or Df(2)Ex7128. However, the k10814 insertion, when homozygous or in combination with either deficiency, survives to third-instar stages in standard conditions or to adulthood when raised apart from heterozygous siblings (Loewen et al, 2001); these adults are ataxic, unable to fly, and die a few days after eclosion. This allele is therefore likely to be a hypomorphic mutation, perhaps resulting in a reduction in wild-type levels of the protein.…”

Drosophila NAB (dNAB) is an orphan transcriptional co‐repressor required for correct CNS and eye development

“…dnab e310 /dnab e310 mutant larvae are less active than wild-type larvae and appear uncoordinated (Table 1). To determine the lethal phase for this and all other mutants in this study, we have used the larval culturing technique of Loewen et al, in which larvae are reared on dilute yeast solutions in high humidity chambers (see the Experimental Procedures section; Loewen et al, 2001). This technique has been shown previously to extend the life span of synaptotagmin I mutant larvae, which exhibit severe defects in locomotion.…”

“…Again, for these analyses, larvae were cultured on dilute solutions of yeast in high humidity chambers (Loewen et al, 2001; see Experimental Procedures section). dnab G26 /dnab G26 flies died as first to second instar larvae; larvae appeared uncoordinated and initiated fewer movements than wild-type flies.…”

“…When larvae are cultured under more permissive conditions, however, syt mutants can survive to adulthood, where they again exhibit severe defects in locomotion. Synaptotagmin 1 encodes a synaptic vesicle protein required for efficient neurotransmitter release (Loewen et al, 2001). Similarly, endophilin A (D-endoA) mutants die during early larval development due to severe locomotion defects.…”