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: 9.57). 10/2011; 21(20):1704-15. DOI: 10.1016/j.cub.2011.08.058
Source: PubMed


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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    • "The Drosophila genome predicts 33 Rab proteins based on sequence similarity, and there is evidence that 27 are expressed (Chan et al., 2011; Zhang et al., 2007). Fourteen of these Rabs were already present in the LECA (between 2–3 billion years ago), and the other 13 first arose by duplication and divergence at the root of the metazoan lineage (500 million years ago). "
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    ABSTRACT: Membrane trafficking is key to the cell biological mechanisms underlying development. Rab GTPases control specific membrane compartments, from core secretory and endocytic machinery to less-well-understood compartments. We tagged all 27 Drosophila Rabs with YFP(MYC) at their endogenous chromosomal loci, determined their expression and subcellular localization in six tissues comprising 23 cell types, and provide this data in an annotated, searchable image database. We demonstrate the utility of these lines for controlled knockdown and show that similar subcellular localization can predict redundant functions. We exploit this comprehensive resource to ask whether a common Rab compartment architecture underlies epithelial polarity. Strikingly, no single arrangement of Rabs characterizes the five epithelia we examine. Rather, epithelia flexibly polarize Rab distribution, producing membrane trafficking architectures that are tissue- and stage-specific. Thus, the core machinery responsible for epithelial polarization is unlikely to rely on polarized positioning of specific Rab compartments. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Developmental Cell 05/2015; 33(3):351-365. DOI:10.1016/j.devcel.2015.03.022 · 9.71 Impact Factor
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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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    ABSTRACT: eLife digest Our brain contains billions of cells called neurons that form an extensive network through which information is readily exchanged. These cells connect to each other via junctions called synapses. Our developing brain starts off with far more synapses than it needs, but the excess synapses are destroyed as the brain matures. Even in adults, synapses are continuously made and destroyed in response to experiences and learning. Inside neurons there are tiny bubble-like compartments called vesicles that supply the synapses with many of the proteins and other components that they need. There is a growing body of evidence that suggests these vesicles are rapidly destroyed once a synapse is earmarked for destruction, but it is not clear how this may occur. Here, Binotti, Pavlos et al. found that a protein called Rab26 sits on the surface of the vesicles near synapses. This protein promotes the formation of clusters of vesicles, and a membrane sometimes surrounds these clusters. Further experiments indicate that several proteins involved in a process called autophagy—where unwanted proteins and debris are destroyed—may also be found around the clusters of vesicles. Autophagy starts with the formation of a membrane around the material that needs to be destroyed. This seals the material off from rest of the cell so that enzymes can safely break it down. Binotti, Pavlos et al. found that one of the autophagy proteins—called Atg16L—can bind directly to Rab26, but only when Rab26 is in an ‘active’ state. These findings suggest that excess vesicles at synapses may be destroyed by autophagy. Further work will be required to establish how this process is controlled and how it is involved in the loss of synapses. DOI:
    eLife Sciences 02/2015; 4(4). DOI:10.7554/eLife.05597 · 9.32 Impact Factor
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    • "Expressing Rab26-DN in IPCs had a weaker effect on growth inhibition (7% reduction in size), compared with a 20% reduction in size achieved with Rab26 RNAi, suggesting that the DN construct is less effective than the RNAi construct (Figure S1). Rab1 is expressed ubiquitously in the brain including in all 14 IPCs (Figure 6C) (Chan et al. 2011). Producing Rab1-DN in IPCs dramatically reduced pupal size to only 61% of the controls, in keeping with the RNAi data (Figure 6B and Figure S3A). "
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    ABSTRACT: Insulin-producing cells (IPCs) in the Drosophila brain produce and release insulin-like peptides (ILPs) to hemolymph. ILPs are crucial for growth and regulation of metabolic activity in flies, functions analogous to those of mammalian insulin and insulin-like growth factors (IGFs). To identify components functioning in IPCs to control ILP production, we employed genomic and candidate gene approaches. We used laser microdissection and mRNA sequencing to characterize the transcriptome of larval IPCs. IPCs highly express many genes homologous to genes active in insulin-producing beta cells of the mammalian pancreas. The genes in common encode insulin-like peptides and proteins that control insulin metabolism, storage, secretion, and beta cell proliferation, and some not previously linked to insulin production or beta cell function. Among these novelties is unc-104, a Kinesin 3 family gene, which is more highly expressed in IPCs compared to most other neurons. Knockdown of unc-104 in IPCs impaired ILP secretion and reduced peripheral insulin signaling. Unc-104 appears to transport ILPs along axons. As a complementary approach, we tested dominant-negative Rab genes to find Rab proteins required in IPCs for ILP production or secretion. Rab1 was identified as crucial for ILP trafficking in IPCs. Inhibition of Rab1 in IPCs increased circulating sugar levels, delayed development, and lowered weight and body size. Immunofluorescence labeling of Rab1 showed its tight association with ILP2 in the Golgi of IPCs. Unc-104 and Rab1 join other proteins required for ILP transport in IPCs.
    Genetics 02/2014; 197(1). DOI:10.1534/genetics.113.160663 · 5.96 Impact Factor
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