Temperature-sensitive paralytic mutants: insights into the synaptic vesicle cycle

Abstract

Forward genetic screens have identified numerous proteins with critical roles in neurotransmission. One particularly fruitful screening target in Drosophila has been TS (temperature-sensitive) paralytic mutants, which have revealed proteins acutely required in neuronal signalling. In the present paper, we review recent insights and current questions from one recently cloned TS paralytic mutant, rbo (rolling blackout). The rbo mutant identifies a putative integral lipase of the pre-synaptic plasma membrane that is required for the SV (synaptic vesicle) cycle. Identification of this mutant adds to a growing body of evidence that lipid-modifying enzymes locally control specialized lipid microenvironments and lipid signalling pathways with key functions regulating neurotransmission strength. The RBO protein is absolutely required for phospholipase C signalling in phototransduction. We posit that RBO might be required to regulate the availability of fusogenic lipids such as phosphatidylinositol 4,5-bisphosphate and diacylglycerol that may directly modify membrane properties and/or activate lipid-binding fusogenic proteins mediating SV exocytosis.

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

“…6A). The protein is primarily localized to the plasma membrane, as at the synapse (Vijayakrishnan and Broadie, 2006). In addition, RBO was also present in a small subset of internal organelles (Fig.…”

“…In rbo ts brain, phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P 2 ] accumulates and diacylglycerol (DAG) is concomitantly reduced within minutes upon shift to restrictive temperature. 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).…”

“…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). In rbo ts brain, phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P 2 ] accumulates and diacylglycerol (DAG) is concomitantly reduced within minutes upon shift to restrictive temperature.…”

Acquired temperature‐sensitive paralysis as a biomarker of declining neuronal function in aging Drosophila

“…Conversely, the ts paralytic phenotype has been associated with mutations that decrease membrane excitability and identify a number of loci encoding proteins necessary for membrane excitability, endocytosis and exocytosis (Vijayakrishnan & Broadie, 2006). These mutations both directly and indirectly affect excitability.…”

“…Forward genetic strategies have been extremely successful in identifying new genes involved in neuronal transmission. For instance, temperaturesensitive (ts) paralysis has been used as a powerful phenotypic paradigm to identify genes that encode key proteins for normal neuronal signaling or neurotransmission, such as ion channels, ion channel regulators, proteins necessary for synaptic transmission and mitochondrial function and others (Gnerer et al ., 2006;Vijayakrishnan & Broadie, 2006). Mutations in many of those genes cause some neural impairment even at permissive temperature, some shorten lifespan and about 10% cause some age-dependent neurodegeneration (Palladino et al ., 2002;Gnerer et al ., 2006).…”