Rolling Blackout Is Required for Synaptic Vesicle Exocytosis

Abstract

Rolling blackout (RBO) is a putative transmembrane lipase required for phospholipase C-dependent phosphatidylinositol 4,5-bisphosphate-diacylglycerol signaling in Drosophila neurons. Conditional temperature-sensitive (TS) rbo mutants display complete, reversible paralysis within minutes, demonstrating that RBO is acutely required for movement. RBO protein is localized predominantly in presynaptic boutons at neuromuscular junction (NMJ) synapses and throughout central synaptic neuropil, and rbo TS mutants display a complete, reversible block of both central and peripheral synaptic transmission within minutes. This phenotype appears limited to adults, because larval NMJs do not manifest the acute blockade. Electron microscopy of adult rbo TS mutant boutons reveals an increase in total synaptic vesicle (SV) content, with a concomitant shrinkage of presynaptic bouton size and an accumulation of docked SVs at presynaptic active zones within minutes. Genetic tests reveal a synergistic interaction between rbo and syntaxin1A TS mutants, suggesting that RBO is required in the mechanism of N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated SV exocytosis, or in a parallel pathway necessary for SV fusion. The rbo TS mutation does not detectably alter SNARE complex assembly, suggesting a downstream requirement in SV fusion. We conclude that RBO plays an essential role in neurotransmitter release, downstream of SV docking, likely mediating SV fusion.

Rolling blackout is required for bulk endocytosis in non-neuronal cells and neuronal synapses

“…synapses, similar to the results obtained in adult dorsal longitudinal muscle (DLM) synapse (Huang et al, 2006), posing us a conundrum. We therefore combined FM-dye-uptake experiments with photoconversion electron microscopy to reveal that loss of RBO function causes a specific blockade in the activity-dependent bulk-endocytosis generation of endosomes.…”

“…However, the exact RBO lipase substrate in the PtdIns(4,5)P 2 -DAG pathway has not been identified, despite exhaustive attempts (Vijayakrishnan and Broadie, 2006). Our previous data pointed to RBO function in SV exocytosis, with vesicles accumulating within minutes at the restrictive temperature, being arrested at the docking stage at presynaptic active zones (Huang et al, 2006). Crucial to this model, we showed that rbo ts displays a strong synergistic behavioral interaction with the syntaxin-1A TS allele syx 3-69 (T254I) (Littleton et al, 1998).…”

“…We previously reported that conditional temperature sensitive (TS) rbo ts mutants manifest complete blindness and paralysis within minutes upon shift to restrictive temperature (Huang et al, 2004;Huang et al, 2006). RBO is a well-conserved integral plasma-membrane protein and predicted lipase (Vijayakrishnan and Broadie, 2006).…”

Presynaptic Calcium Channel Localization and Calcium-Dependent Synaptic Vesicle Exocytosis Regulated by the Fuseless Protein

“…These phenotypes are consistent with direct disruption of Ca 2ϩ -triggered vesicle exocytosis. Similar vesicle accumulation characterizes syntaxin1A, dunc-13, comatose, and rolling blackout mutants, for example, each of which has a specific exocytosis deficit (Broadie et al, 1995;Kawasaki et al, 1998;Littleton et al, 1998;Aravamudan et al, 1999;Kidokoro, 2003;Huang et al, 2006). It has been shown that a specific block in SV exocytosis leads to a secondary accumulation of vesicles in pools upstream of the presynaptic membrane.…”

“…In controls, the number of docked vesicles was 2.0 Ϯ 0.2 (WT; N ϭ 21) and 2.0 Ϯ 0.1 (ϩ/Df; N ϭ 20), and it increased in mutants to 3.8 Ϯ 0.4 ( fusl 1 ; N ϭ 17) and 3.1 Ϯ 0.2 ( fusl 1 /Df; N ϭ 20). These defects are characteristic in known SV fusion mutants (Richmond and Broadie, 2002;Kidokoro, 2003;Huang et al 2006) and indicative of an impairment in SV exocytosis.…”