Systematic Discovery of Rab GTPases with Synaptic Functions in Drosophila

Department of Physiology, UT Southwestern Medical Center, Dallas, TX 75390, USA.
Current biology: CB (Impact Factor: 9.92). 10/2011; 21(20):1704-15. DOI: 10.1016/j.cub.2011.08.058
Source: PubMed

ABSTRACT Neurons require highly specialized intracellular membrane trafficking, especially at synapses. Rab GTPases are considered master regulators of membrane trafficking in all cells, and only very few Rabs have known neuron-specific functions. Here, we present the first systematic characterization of neuronal expression, subcellular localization, and function of Rab GTPases in an organism with a brain.
We report the surprising discovery that half of all Drosophila Rabs function specifically or predominantly in distinct subsets of neurons in the brain. Furthermore, functional profiling of the GTP/GDP-bound states reveals that these neuronal Rabs are almost exclusively active at synapses and the majority of these synaptic Rabs specifically mark synaptic recycling endosomal compartments. Our profiling strategy is based on Gal4 knockins in large genomic fragments that are additionally designed to generate mutants by ends-out homologous recombination. We generated 36 large genomic targeting vectors and transgenic rab-Gal4 fly strains for 25 rab genes. Proof-of-principle knockout of the synaptic rab27 reveals a sleep phenotype that matches its cell-specific expression.
Our findings suggest that up to half of all Drosophila Rabs exert specialized synaptic functions. The tools presented here allow systematic functional studies of these Rabs and provide a method that is applicable to any large gene family in Drosophila.

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    • "Although the tagged Rab26 protein variants were expressed throughout development, no lethality or delayed development was observed (data not shown). Consistent with previous observations, analysis of third instar larvae nerve-muscle preparations revealed an exclusive localization of Rab26 to presynaptic compartments of the neuromuscular junction without staining of axons and cell bodies (Figure 3—figure supplement 2; see also [Chan et al., 2011]). "
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    eLife Sciences 02/2015; 4. DOI:10.7554/eLife.05597 · 8.52 Impact Factor
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    • "These stocks facilitate rapidly testing any gene's involvement in a given process. In addition, because RNA interference does not generally lead to complete loss-of-function mutants, homologous recombination has also been used to engineer knockout mutants, including for whole-gene families (e.g., Chan et al., 2011). Finally, the Drosophila genome has been fully sequenced and annotated and shows extensive gene conservation with humans, though with less genetic redundancy (Adams et al., 2000). "
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