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Compromised endocytosis in neurons leads to synapse overgrowth and altered organization of synaptic proteins. However, the molecular players and the signaling pathways which regulate the process remains poorly understood. Here we show that σ2-adaptin, one of the subunits of the AP2-complex, genetically interacts with BMP type I receptor, Thickveins (Tkv), and Daughter against decapentaplegic (Dad), two of the components of BMP signaling. We found that mutations in σ2-adaptin lead to an accumulation of Tkv receptors at the NMJ and results in a significant reduction in Tkv-positive early endosomes in the presynaptic terminals. Interestingly, the level of small GTPase Rab11 was significantly reduced in the σ2-adaptin mutant synapses. Consistent with the role of σ2-adaptin and Rab11 in the regulation of the same signaling pathway, a mutation in Rab11 or overexpression of a GDP-locked form of Rab11 (Rab11S25N) phenocopies the morphological and signaling defects of the σ2-adaptin mutants. Finally, we demonstrate that σ2-adaptin mutants show an accumulation of large vesicles and massive membranous structures, akin to endosomes at the synapse. Thus, we propose a model in which AP2 regulates Tkv internalization and recycling through a process that requires Rab11 activity to control the synaptic growth.

Polarized hyphal growth of filamentous pathogenic fungi is an essential event for host penetration and colonization. The long-range early endosomal trafficking during the hyphal growth is crucial for nutrient uptake, sensing of host-specific cues, and regulation of effector production. Bin1/Amphiphysin/Rvs167 (BAR) domain-containing proteins mediate fundamental cellular processes, including membrane remodeling and endocytosis. Here, we identified an F-BAR domain protein (ArF-BAR) in the necrotrophic fungusAscochyta rabieiand demonstrate its involvement in endosome-dependent fungal virulence on the host plant,Cicer arietinum. We show that ArF-BAR regulates endocytosis at the hyphal tip, localizes to the early endosomes, and is involved in actin dynamics. Functional studies involving gene knockout and complementation experiments reveal that ArF-BAR is essential for virulence. The loss-of-function of ArF-BAR results in delayed formation of first septa from the hyphal tip, crucial for host penetration and proliferation. ArF-BAR was induced in response to oxidative stress and infection and localized to endocytic vesicles within the fungal hyphae. We also show that ArF-BAR is able to tubulate synthetic liposomes, suggesting the functional role of F-BAR domain in membrane tubule formationin vivo. Further, our studies identified a stress-induced transcription factor, ArCRZ1 (Calcineurin-responsive zinc finger 1) as key regulator for transcriptional reprogramming of ArF-BAR. We propose a model in which ArCRZ1 functions upstream of ArF-BAR to regulate fungal pathogenesis through a mechanism that involves membrane remodeling and actin cytoskeleton regulation.Author summaryBAR-domain superfamily is known to mold amorphous lipid bilayer into defined tubular shapes and critical for endosome formation and trafficking. Although these processes are studied earlier in the context of their structural and biochemical properties, there is limited evidence on the direct role of F-BAR domain proteins in the pathophysiological development of other economically important fungi. Our study assumes functional significance for plant infection as we identified an F-BAR domain-containing protein that is regulated by a distinct transcriptional network. We characterized F-BAR in a necrotrophic fungal pathogen,Ascochyta rabieithat causes the Ascochyta blight (AB) disease in chickpea plants. Additionally, we have also identified a calcium-regulated CRZ1 transcription factor that regulates the transcription ofArF-BAR. Our study will help to understand the complex interplay underlying the endosome formation required for fungal virulence.

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 sensitive protein 1 (DMon1) in regulating glutamate receptor (GluRIIA) levels at the nmj. Dmon1 is an evolutionarily conserved protein which, in complex with 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 a defects in synaptic morphology and a strong increase in glutamate receptor GluRIIA levels. The mechanism by which Dmon1 regulates GluRIIA is unclear.In this study, we have described the characterization the mutation in an EMS mutant referred to as pog1 and demonstrate this mutant to be an allele of Dmon1. Further, we have examined the role of rab7 in regulation the of GluRIIA. We show that similar to Dmon1, knock-down of rab7 using RNAi in neurons, and not muscles, leads to an increase in GluRIIA. Loss of one copy each of Dmon1 with rab7 leads to a synergistic increase in receptor expression. Further, overexpression of an activated Rab7 can rescue the GluRIIA phenotype observed in Dmon1Δ181 mutants. Together, these results highlight a neuronal role for Rab7 in GluRIIA regulation and underscores the important of the endo-lysosomal pathway in this process.

Summary StatementELYS, a nucleoporin spatiotemporally regulates NF-κB pathway dynamics during development inDrosophilaand its misregulation in post-embryonic stages leads to apoptosis mediated abnormalities.AbstractNuclear pores are the exclusive conduit to facilitate the nucleocytoplasmic transport in a precisely regulated manner. ELYS, a constituent protein of nuclear pores, initiates assembly of nuclear pore complexes (NPCs) into functional nuclear pores towards the end of mitosis. Using cellular, molecular and genetic tools, here, we report that ELYS orthologue (dElys) plays critical roles duringDrosophiladevelopment. Throughin silicoanalyses, we find all conserved structural features indElys except for the presence of non-canonical AT-hook motif strongly binding with DNA.dElys localized to nuclear rim in interphase cells, but during mitosis, it was present on chromatin. RNAi mediated depletion ofdElys leads to aberrant development and defects in the nuclear lamina and NPCs assembly at the cellular level. Furthermore, we demonstrate that indElys depletion NF-κB is activated and accumulates inside the nucleus which results in illimed expression of critical molecules.dElys depletion sustains NF-κB into the nucleus in post-embryonic stages. Prolonged NF-κB inside nucleus induces apoptosis in response to hitherto unknown quality check mechanism and highlights on the under-appreciated apoptotic paradigm of NF-κB pathway.

Insect mushroom bodies (MB) have an ensemble of synaptic connections well-studied for their role in experience-dependent learning and several higher cognitive functions. MB requires neurotransmission for an efficient flow of information across synapses with the different flexibility to meet the demand of the dynamically changing environment of an insect. Neurotransmitter transporters coordinate appropriate changes for an efficient neurotransmission at the synapse. Till date, there is no transporter reported for any of the previously known neurotransmitters in the intrinsic neurons of MB. In this study, we report a highly enriched expression of Choline Transporter (ChT) in Drosophila MB. We demonstrate that knockdown of ChT in a sub-type of MB neurons called α/β core (α/βc) neurons leads to eclosion failure, peristaltic defect in larvae, and altered NMJ phenotype. These defects were neither observed on knockdown of proteins of the cholinergic locus in α/βc neurons nor by knockdown of ChT in cholinergic neurons. Thus, our study provides insights into non-canonical roles of ChT in MB.