CG17192 is a Phospholipase That Regulates Signaling Lipids in the Drosophila Gut upon Infection.

The chemoproteomics technique, activity-based protein profiling (ABPP), has proven to be an invaluable tool in assigning functions to enzymes. The serine hydrolase (SH) enzyme superfamily, in particular, has served as an excellent example in displaying the versatility of various ABPP platforms and has resulted in a comprehensive cataloging of the biochemical activities associated within this superfamily. Besides SHs, in mammals, several other enzyme classes have been thoroughly investigated using ABPP platforms. However, the utility of ABPP platforms in fly models remains underexplored. Realizing this knowledge gap, leveraging complementary ABPP platforms, we reported the full array of SH activities during various developmental stages and adult tissues in the fruit fly (Drosophila melanogaster). Following up on this study, using ABPP, we mapped SH activities in adult fruit flies in an infection model and found that a gut-resident lipase CG17192 showed increased activity during infection. To assign a biological function to this uncharacterized lipase, we performed an untargeted lipidomics analysis and found that phosphatidylinositols were significantly elevated when CG17192 was depleted in the adult fruit fly gut. Next, we overexpressed this lipase in insect cells, and using biochemical assays, we show that CG17192 is a secreted enzyme that has phospholipase C (PLC) type activity, with phosphatidylinositol being a preferred substrate. Finally, we show during infection that heightened CG17192 regulates phosphatidylinositol levels and, by doing so, likely modulates signaling pathways in the adult fruit fly gut that might be involved in the resolution of this pathophysiological condition.

Age-dependent dynamics of neuronal VAPBALS inclusions in the adult brain.

Amyotrophic Lateral Sclerosis (ALS) is a relentlessly progressive and fatal disease, caused by the degeneration of upper and lower motor neurons within the brain and spinal cord in the ageing human. The dying neurons contain cytoplasmic inclusions linked to the onset and progression of the disease. Here, we use a Drosophila model of ALS8 (VAPP58S) to understand the modulation of these inclusions in the ageing adult brain. The adult VAPP58S fly shows progressive deterioration in motor function till its demise 25 days post-eclosion. The density of VAPP58S-positive brain inclusions is stable for 5-15 days of age. In contrast, adding a single copy of VAPWT to the VAPP58S animal leads to a large decrease in inclusion density with concomitant rescue of motor function and lifespan. ER stress, a contributing factor in disease, shows reduction with ageing for the disease model. Autophagy, rather than the Ubiquitin Proteasome system, is the dominant mechanism for aggregate clearance. We explored the ability of Drosophila Valosin-containing protein (VCP/TER94), the ALS14 locus, which is involved in cellular protein clearance, to regulate age-dependent aggregation. Contrary to expectation, TER94 overexpression increased VAPP58S punctae density, while its knockdown led to enhanced clearance. Expression of a dominant positive allele, TER94R152H, further stabilised VAPP58S puncta, cementing roles for an ALS8-ALS14 axis. Our results are explained by a mechanism where autophagy is modulated by TER94 knockdown. Our study sheds light on the complex regulatory events involved in the neuronal maintenance of ALS8 aggregates, suggesting a context-dependent switch between proteasomal and autophagy-based mechanisms as the larvae develop into an adult. A deeper understanding of the nucleation and clearance of the inclusions, which affect cellular stress and function, is essential for understanding the initiation and progression of ALS.

Caspar, an adapter for VAPB and TER94, modulates the progression of ALS8 by regulating IMD/NFκB-mediated glial inflammation in a Drosophila model of human disease.

Amyotrophic lateral sclerosis (ALS) is a fatal, late-onset, progressive motor neurodegenerative disorder. A key pathological feature of the disease is the presence of heavily ubiquitinated protein inclusions. Both the unfolded protein response and the ubiquitin-proteasome system appear significantly impaired in patients and animal models of ALS. We have studied cellular and molecular mechanisms involved in ALS using a vesicle-associated membrane protein-associated protein B (VAPB/ALS8) Drosophila model [Moustaqim-Barrette, A., Lin, Y.Q., Pradhan, S., Neely, G.G., Bellen, H.J. and Tsuda, H. (2014) The ALS 8 protein, VAP, is required for ER protein quality control. Hum. Mol. Genet., 23, 1975-1989], which mimics many systemic aspects of the human disease. Here, we show that VAPB, located on the cytoplasmic face of the endoplasmic reticulum membrane, interacts with Caspar, an orthologue of human fas associated factor 1 (FAF1). Caspar, in turn, interacts with transitional endoplasmic reticulum ATPase (TER94), a fly orthologue of ALS14 (VCP/p97, valosin-containing protein). Caspar overexpression in the glia extends lifespan and also slows the progression of motor dysfunction in the ALS8 disease model, a phenomenon that we ascribe to its ability to restrain age-dependent inflammation, which is modulated by Relish/NFκB signalling. Caspar binds to VAPB via an FFAT motif, and we find that Caspar's ability to negatively regulate NFκB signalling is not dependent on the VAPB:Caspar interaction. We hypothesize that Caspar is a key molecule in the pathogenesis of ALS. The VAPB:Caspar:TER94 complex appears to be a candidate for regulating both protein homeostasis and NFκB signalling, with our study highlighting a role for Caspar in glial inflammation. We project human FAF1 as an important protein target to alleviate the progression of motor neuron disease.

Bidirectional regulation between AP-1 and SUMOylation pathway genes modulates inflammatory signaling during Salmonella infection.

Post-translational modifications (PTMs), such as SUMOylation, are known to modulate fundamental processes of a cell. Infectious agents such as Salmonella Typhimurium (STm), which causes gastroenteritis, utilize the PTM mechanism SUMOylation to hijack the host cell. STm suppresses host SUMO pathway genes UBC9 (also known as UBE2I) and PIAS1 to perturb SUMOylation for an efficient infection. In the present study, the regulation of SUMO pathway genes during STm infection was investigated. A direct binding of c-Fos (encoded by FOS), a component of activator protein-1 (AP-1), to promoters of both UBC9 and PIAS1 was observed. Experimental perturbation of c-Fos led to changes in the expression of both UBC9 and PIAS1. STm infection of fibroblasts with SUMOylation-deficient c-Fos (c-FOS-KOSUMO-def-FOS) resulted in uncontrolled activation of target genes, leading to massive immune activation. Infection of c-FOS-KOSUMO-def-FOS cells favored STm replication, indicating misdirected immune mechanisms. Finally, chromatin immunoprecipitation assays confirmed a context-dependent differential binding and release of AP-1 to and from target genes due to its phosphorylation and SUMOylation, respectively. Overall, our data point towards the existence of a bidirectional cross-talk between c-Fos and the SUMO pathway and highlight their importance in AP-1 function in STm infection and beyond. This article has an associated First Person interview with the first author of the paper.

SUMOylation of Jun fine-tunes the Drosophila gut immune response.

Post-translational modification by the small ubiquitin-like modifier, SUMO can modulate the activity of its conjugated proteins in a plethora of cellular contexts. The effect of SUMO conjugation of proteins during an immune response is poorly understood in Drosophila. We have previously identified that the transcription factor Jra, the Drosophila Jun ortholog and a member of the AP-1 complex is one such SUMO target. Here, we find that Jra is a regulator of the Pseudomonas entomophila induced gut immune gene regulatory network, modulating the expression of a few thousand genes, as measured by quantitative RNA sequencing. Decrease in Jra in gut enterocytes is protective, suggesting that reduction of Jra signaling favors the host over the pathogen. In Jra, lysines 29 and 190 are SUMO conjugation targets, with the JraK29R+K190R double mutant being SUMO conjugation resistant (SCR). Interestingly, a JraSCR fly line, generated by CRISPR/Cas9 based genome editing, is more sensitive to infection, with adults showing a weakened host response and increased proliferation of Pseudomonas. Transcriptome analysis of the guts of JraSCR and JraWT flies suggests that lack of SUMOylation of Jra significantly changes core elements of the immune gene regulatory network, which include antimicrobial agents, secreted ligands, feedback regulators, and transcription factors. Mechanistically, SUMOylation attenuates Jra activity, with the TFs, forkhead, anterior open, activating transcription factor 3 and the master immune regulator Relish being important transcriptional targets. Our study implicates Jra as a major immune regulator, with dynamic SUMO conjugation/deconjugation of Jra modulating the kinetics of the gut immune response.

Drosophila Mon1 constitutes a novel node in the brain-gonad axis that is essential for female germline maturation.

Monensin-sensitive 1 (Mon1) is an endocytic regulator that participates in the conversion of Rab5-positive early endosomes to Rab7-positive late endosomes. In Drosophila, loss of mon1 leads to sterility as the mon1 mutant females have extremely small ovaries with complete absence of late stage egg chambers - a phenotype reminiscent of mutations in the insulin pathway genes. Here, we show that expression of many Drosophila insulin-like peptides (ILPs) is reduced in mon1 mutants and feeding mon1 adults an insulin-rich diet can rescue the ovarian defects. Surprisingly, however, mon1 functions in the tyramine/octopaminergic neurons (OPNs) and not in the ovaries or the insulin-producing cells (IPCs). Consistently, knockdown of mon1 in only the OPNs is sufficient to mimic the ovarian phenotype, while expression of the gene in the OPNs alone can 'rescue' the mutant defect. Last, we have identified ilp3 and ilp5 as critical targets of mon1. This study thus identifies mon1 as a novel molecular player in the brain-gonad axis and underscores the significance of inter-organ systemic communication during development.

A Superfamily-wide Activity Atlas of Serine Hydrolases in Drosophila melanogaster.

The serine hydrolase (SH) superfamily is, perhaps, one of the largest functional enzyme classes in all forms of life and consists of proteases, peptidases, lipases, and carboxylesterases as representative members. Consistent with the name of this superfamily, all members, without any exception to date, use a nucleophilic serine residue in the enzyme active site to perform hydrolytic-type reactions via a two-step ping-pong mechanism involving a covalent enzyme intermediate. Given the highly conserved catalytic mechanism, this superfamily has served as a classical prototype in the development of several platforms of chemical proteomics techniques, activity-based protein profiling (ABPP), to globally interrogate the functions of its different members in various native, yet complex, biological settings. While ABPP-based proteome-wide activity atlases for SH activities are available in numerous organisms, including humans, to the best of our knowledge, such an analysis for this superfamily is lacking in any insect model. To address this, we initially report a bioinformatics analysis toward the identification and categorization of nonredundant SHs in Drosophila melanogaster. Following up on this in silico analysis, leveraging discovery chemoproteomics, we identify and globally map the full complement of SH activities during various developmental stages and in different adult tissues of Drosophila. Finally, as a proof of concept of the utility of this activity atlas, we highlight sexual dimorphism in SH activities across different tissues in adult D. melanogaster, and we propose new research directions, resources, and tools that this study can provide to the fly community.

Drosophila DNA/RNA methyltransferase contributes to robust host defense in aging animals by regulating sphingolipid metabolism.

Drosophila methyltransferase (Mt2) has been implicated in the methylation of both DNA and tRNA. In this study, we demonstrate that loss of Mt2 activity leads to an age-dependent decline of immune function in the adult fly. A newly eclosed adult has mild immune defects that are exacerbated in a 15 day old Mt2-/- fly. The age-dependent effects appear to be systemic, including disturbances in lipid metabolism, changes in cell shape of hemocytes and significant fold-changes in levels of transcripts related to host defense. Lipid imbalance, as measured by quantitative lipidomics, correlates with immune dysfunction, with high levels of immunomodulatory lipids, sphingosine-1-phosphate (S1P) and ceramides, along with low levels of storage lipids. Activity assays on fly lysates confirm the age-dependent increase in S1P and concomitant reduction of S1P lyase activity. We hypothesize that Mt2 functions to regulate genetic loci such as S1P lyase and this regulation is essential for robust host defense as the animal ages. Our study uncovers novel links between age--dependent Mt2 function, innate immune response and lipid homeostasis.

A Simple Immunofluorescence Method to Characterize Neurodegeneration and Tyrosine Hydroxylase Reduction in Whole Brain of a Drosophila Model of Parkinson's Disease.

Dopaminergic (DAergic) neurodegeneration in the substantia nigra pars compacta of the human brain is the pathological feature associated with Parkinson's disease (PD). Drosophila also exhibits mobility defects and diminished levels of brain dopamine on exposure to neurotoxicants mimicking PD. Our laboratory demonstrated in a Drosophila model of sporadic PD that there is no decrease in DAergic neuronal number; instead, there is a significant reduction in tyrosine hydroxylase (TH) fluorescence intensity (FI). Here, we present a sensitive assay based on the quantification of FI of the secondary antibody (ab). As the FI is directly proportional to the amount of TH synthesis, its reduction under PD conditions denotes the decrease in the TH synthesis, suggesting DAergic neuronal dysfunction. Therefore, FI quantification is a refined and sensitive method to understand the early stages of DAergic neurodegeneration. FI quantification is performed using the ZEN 2012 SP2 single-user software; a license must be acquired to utilize the imaging system to interactively control image acquisition, image processing, and analysis. This method will be of good use to biologists, as it can also be used with little modification to characterize the extent of degeneration and changes in the level of degeneration in response to drugs in different cell types. Unlike the expensive and cumbersome confocal microscopy, the present method will be an affordable option for fund-constrained neurobiology laboratories. Key features • Allows characterizing the incipient DAergic and other catecholaminergic neurodegeneration, even in the absence of loss of neuronal cell body. • Great alternative for the fund-constrained neurobiology laboratories in developing countries to utilize this method in different cell types and their response to drugs/nutraceuticals.

The Hydra small ubiquitin-like modifier.

SUMO is a protein posttranslational modifier. SUMO cycle components are believed to be conserved in all eukaryotes. Proteomic analyses have lead to the identification a wealth of SUMO targets that are involved in almost every cellular function in eukaryotes. In this article, we describe the characterization of SUMO Cycle components in Hydra, a Cnidarian with an ability to regenerate body parts. In cells, the translated SUMO polypeptide cannot conjugate to a substrate protein unless the C-terminal tail is cleaved, exposing the di-Glycine motif. This critical task is done by SUMO proteases that in addition to SUMO maturation are also involved in deconjugating SUMO from its substrate. We describe the identification, bioinformatics analysis, cloning, and biochemical characterization of Hydra SUMO cycle components, with a focus on SUMO and SUMO proteases. We demonstrate that the ability of SUMO proteases to process immature SUMO is conserved from Hydra to flies. A transgenic Hydra, expressing a SUMO-GFP fusion protein under a constitutive actin promoter, is generated in an attempt to monitor the SUMO Cycle in vivo as also to purify and identify SUMO targets in Hydra.

SUMOylation of Dorsal attenuates Toll/NF-κB signaling.

In Drosophila, Toll/NF-κB signaling plays key roles in both animal development and in host defense. The activation, intensity, and kinetics of Toll signaling are regulated by posttranslational modifications such as phosphorylation, SUMOylation, or ubiquitination that target multiple proteins in the Toll/NF-κB cascade. Here, we have generated a CRISPR-Cas9 edited Dorsal (DL) variant that is SUMO conjugation resistant. Intriguingly, embryos laid by dlSCR mothers overcome dl haploinsufficiency and complete the developmental program. This ability appears to be a result of higher transcriptional activation by DLSCR. In contrast, SUMOylation dampens DL transcriptional activation, ultimately conferring robustness to the dorso-ventral program. In the larval immune response, dlSCR animals show an increase in crystal cell numbers, stronger activation of humoral defense genes, and high cactus levels. A mathematical model that evaluates the contribution of the small fraction of SUMOylated DL (1-5%) suggests that it acts to block transcriptional activation, which is driven primarily by DL that is not SUMO conjugated. Our findings define SUMO conjugation as an important regulator of the Toll signaling cascade, in both development and host defense. Our results broadly suggest that SUMO attenuates DL at the level of transcriptional activation. Furthermore, we hypothesize that SUMO conjugation of DL may be part of a Ubc9-dependent mechanism that restrains Toll/NF-κB signaling.

SUMOylation of Arginyl tRNA Synthetase Modulates the Drosophila Innate Immune Response.

SUMO conjugation of a substrate protein can modify its activity, localization, interaction or function. A large number of SUMO targets in cells have been identified by Proteomics, but biological roles for SUMO conjugation for most targets remains elusive. The multi-aminoacyl tRNA synthetase complex (MARS) is a sensor and regulator of immune signaling. The proteins of this 1.2 MDa complex are targets of SUMO conjugation, in response to infection. Arginyl tRNA Synthetase (RRS), a member of the sub-complex II of MARS, is one such SUMO conjugation target. The sites for SUMO conjugation are Lys 147 and 383. Replacement of these residues by Arg (RRS K147R,K383R ), creates a SUMO conjugation resistant variant (RRS SCR ). Transgenic Drosophila lines for RRS WT and RRS SCR were generated by expressing these variants in a RRS loss of function (lof) animal, using the UAS-Gal4 system. The RRS-lof line was itself generated using CRISPR/Cas9 genome editing. Expression of both RRS WT and RRS SCR rescue the RRS-lof lethality. Adult animals expressing RRS WT and RRS SCR are compared and contrasted for their response to bacterial infection by gram positive M. luteus and gram negative Ecc15. We find that RRS SCR , when compared to RRS WT , shows modulation of the transcriptional response, as measured by quantitative 3' mRNA sequencing. Our study uncovers a possible non-canonical role for SUMOylation of RRS, a member of the MARS complex, in host-defense.

SUMO conjugation regulates immune signalling.

Post-translational modifications (PTMs) are critical drivers and attenuators for proteins that regulate immune signalling cascades in host defence. In this review, we explore functional roles for one such PTM, the small ubiquitin-like modifier (SUMO). Very few of the SUMO conjugation targets identified by proteomic studies have been validated in terms of their roles in host defence. Here, we compare and contrast potential SUMO substrate proteins in immune signalling for flies and mammals, with an emphasis on NFκB pathways. We discuss, using the few mechanistic studies that exist for validated targets, the effect of SUMO conjugation on signalling and also explore current molecular models that explain regulation by SUMO. We also discuss in detail roles of evolutionary conservation of mechanisms, SUMO interaction motifs, crosstalk of SUMO with other PTMs, emerging concepts such as group SUMOylation and finally, the potentially transforming roles for genome-editing technologies in studying the effect of PTMs.

Drosophila Mon1 and Rab7 interact to regulate glutamate receptor levels at the neuromuscular junction.

Regulation of post-synaptic receptors plays an important role in determining synaptic strength and plasticity. The Drosophila larval neuromuscular junction (nmj) has been used extensively as a model to understand some of these processes. In this context, we are interested in the role of Drosophila Monensin sensitivity protein 1 (DMon1) in regulating glutamate receptor (GluRIIA) levels at the nmj. DMon1 is an evolutionarily conserved protein which, in complex with calcium caffeine zinc sensitivity1 (CCZ1), regulates the conversion of early endosomes to late endosomes through recruitment of Rab7. C-terminal deletion mutants of Dmon1 (Dmon1Δ181) exhibit lethality. The escapers have a short life span and exhibit severe motor defects. At the nmj, these mutants show defects in synaptic morphology and a strong increase in GluRIIA levels. The mechanism by which Dmon1 regulates GluRIIA is unclear. In this study, we have characterized an EMS mutant referred to as pog1 and demonstrate it to be an allele of Dmon1. Further, we have examined the role of rab7 in regulating GluRIIA. We show that similar to Dmon1, knock-down of rab7 using RNAi in neurons, but not muscles, leads to an increase in GluRIIA. Loss of one copy each of Dmon1 and rab7 leads to a synergistic increase in receptor expression. Further, overexpression of an activated Rab7 can rescue the GluRIIA phenotype observed in Dmon1 Δ181 mutants. Together, these results highlight a neuronal role for Rab7 in GluRIIA regulation and underscore the importance of the endo-lysosomal pathway in this process.

SOD1 activity threshold and TOR signalling modulate VAP(P58S) aggregation via reactive oxygen species-induced proteasomal degradation in a Drosophila model of amyotrophic lateral sclerosis.

Familial amyotrophic lateral sclerosis (ALS) is an incurable, late-onset motor neuron disease, linked strongly to various causative genetic loci. ALS8 codes for a missense mutation, P56S, in VAMP-associated protein B (VAPB) that causes the protein to misfold and form cellular aggregates. Uncovering genes and mechanisms that affect aggregation dynamics would greatly help increase our understanding of the disease and lead to potential therapeutics. We developed a quantitative high-throughput Drosophila S2R+ cell-based kinetic assay coupled with fluorescent microscopy to score for genes involved in the modulation of aggregates of the fly orthologue, VAP(P58S), fused with GFP. A targeted RNA interference screen against 900 genes identified 150 hits that modify aggregation, including the ALS loci Sod1 and TDP43 (also known as TBPH), as well as genes belonging to the mTOR pathway. Further, a system to measure the extent of VAP(P58S) aggregation in the Drosophila larval brain was developed in order to validate the hits from the cell-based screen. In the larval brain, we find that reduction of SOD1 levels or decreased mTOR signalling reduces aggregation, presumably by increasing the levels of cellular reactive oxygen species (ROS). The mechanism of aggregate clearance is, primarily, proteasomal degradation, which appears to be triggered by an increase in ROS. We have thus uncovered an interesting interplay between SOD1, ROS and mTOR signalling that regulates the dynamics of VAP aggregation. Mechanistic processes underlying such cellular regulatory networks will lead to better understanding of the initiation and progression of ALS.This article has an associated First Person interview with the first author of the paper.

Monensin Sensitive 1 Regulates Dendritic Arborization in Drosophila by Modulating Endocytic Flux.

Monensin Sensitive 1 (Mon1) is a component of the Mon1:Ccz1 complex that mediates Rab5 to Rab7 conversion in eukaryotic cells by serving as a guanine nucleotide exchange factor for Rab7 during vesicular trafficking. We find that Mon1 activity modulates the complexity of Class IV dendritic arborization (da) neurons during larval development. Loss of Mon1 function leads to an increase in arborization and complexity, while increased expression, leads to reduced arborization. The ability of Mon1 to influence dendritic development is possibly a function of its interactions with Rab family GTPases that are central players in vesicular trafficking. Earlier, these GTPases, specifically Rab1, Rab5, Rab10, and Rab11 have been shown to regulate dendritic arborization. We have conducted genetic epistasis experiments, by modulating the activity of Rab5, Rab7, and Rab11 in da neurons, in Mon1 mutants, and demonstrate that the ability of Mon1 to regulate arborization is possibly due to its effect on the recycling pathway. Dendritic branching is critical for proper connectivity and physiological function of the neuron. An understanding of regulatory elements, such as Mon1, as demonstrated in our study, is essential to understand neuronal function.

Dorsal interacting protein 3 potentiates activation by Drosophila Rel homology domain proteins.

Dorsal interacting protein 3 (Dip3) contains a MADF DNA-binding domain and a BESS protein interaction domain. The Dip3 BESS domain was previously shown to bind to the Dorsal Rel homology domain. We show here that Dip3 also binds to the Relish Rel homology domain and enhances Rel family transcription factor function in both dorsoventral patterning and the immune response. While Dip3 is not essential, Dip3 mutations enhance the embryonic patterning defects that result from dorsal haplo-insufficiency, indicating that Dip3 may render dorsoventral patterning more robust. Dip3 is also required for optimal resistance to immune challenge since Dip3 mutant adults and larvae infected with bacteria have shortened lifetimes relative to infected wild-type flies. Furthermore, the mutant larvae exhibit significantly reduced expression of antimicrobial defense genes. Chromatin immunoprecipitation experiments in S2 cells indicate the presence of Dip3 at the promoters of these genes, and this binding requires the presence of Rel proteins at these promoters.

Stonewall and Brickwall: Two Partially Redundant Determinants Required for the Maintenance of Female Germline in Drosophila.

Proper specification of germline stem cells (GSCs) in Drosophila ovaries depends on niche derived non-autonomous signaling and cell autonomous components of transcriptional machinery. Stonewall (Stwl), a MADF-BESS family protein, is one of the cell intrinsic transcriptional regulators involved in the establishment and/or maintenance of GSC fate in Drosophila ovaries. Here we report identification and functional characterization of another member of the same protein family, CG3838/ Brickwall (Brwl) with analogous functions. Loss of function alleles of brwl exhibit age dependent progressive degeneration of the developing ovarioles and loss of GSCs. Supporting the conclusion that the structural deterioration of mutant egg chambers is a result of apoptotic cell death, activated caspase levels are considerably elevated in brwl- ovaries. Moreover, as in the case of stwl mutants, on several instances, loss of brwl activity results in fusion of egg chambers and misspecification of the oocyte. Importantly, brwl phenotypes can be partially rescued by germline specific over-expression of stwl arguing for overlapping yet distinct functional capabilities of the two proteins. Taken together with our phylogenetic analysis, these data suggest that brwl and stwl likely share a common MADF-BESS ancestor and they are expressed in overlapping spatiotemporal domains to ensure robust development of the female germline.

A Presynaptic Regulatory System Acts Transsynaptically via Mon1 to Regulate Glutamate Receptor Levels in Drosophila.

Mon1 is an evolutionarily conserved protein involved in the conversion of Rab5 positive early endosomes to late endosomes through the recruitment of Rab7. We have identified a role for Drosophila Mon1 in regulating glutamate receptor levels at the larval neuromuscular junction. We generated mutants in Dmon1 through P-element excision. These mutants are short-lived with strong motor defects. At the synapse, the mutants show altered bouton morphology with several small supernumerary or satellite boutons surrounding a mature bouton; a significant increase in expression of GluRIIA and reduced expression of Bruchpilot. Neuronal knockdown of Dmon1 is sufficient to increase GluRIIA levels, suggesting its involvement in a presynaptic mechanism that regulates postsynaptic receptor levels. Ultrastructural analysis of mutant synapses reveals significantly smaller synaptic vesicles. Overexpression of vglut suppresses the defects in synaptic morphology and also downregulates GluRIIA levels in Dmon1 mutants, suggesting that homeostatic mechanisms are not affected in these mutants. We propose that DMon1 is part of a presynaptically regulated transsynaptic mechanism that regulates GluRIIA levels at the larval neuromuscular junction.