Biology subjects

Bisht, A.

Publications and source records attributed to Bisht, A..

8 recordsLinked to original sources

bioRxiv2024

Endocytosis restricts synapse growth by attenuating Highwire/PHR1-dependent JNK signaling in a pathway parallel to the BMP signaling

Synaptic growth and organization are orchestrated by pre - and post-synaptic signaling, neuronal activity, and environmental cues. Although endocytosis is known to attenuate synaptic growth, the underlying signaling mechanisms have remained elusive. Here, we uncover a previously unrecognized mechanism by which endocytosis constrains synaptic growth through regulation of the neuronal E3 ubiquitin ligase Highwire (Hiw/Phr1). We show that loss of endocytosis causes Hiw to accumulate in neuronal cell bodies, leading to elevated MAP3K Wallenda (Wnd)/DLK levels and hyperactivation of JNK signaling. The accumulated Hiw assembles into dynamic liquid-liquid phase-separated condensates, as revealed by their rapid and reversible dissolution with 1,6-hexanediol. Acute blockade of endocytosis using a temperature-sensitive dynamin mutant Shibiretssimilarly triggered robust Hiw phase separation. We further demonstrate that Rab11-positive recycling endosomes are essential for proper Hiw localization and turnover, directly linking endosomal trafficking to the control of JNK signaling. Finally, we show that both BMP and JNK signaling are necessary and sufficient to guide synaptic morphogenesis at the Drosophila NMJ, thereby integrating endocytic trafficking with synaptic growth signaling. Our findings establish endocytosis as a critical regulator of Hiw/Phr1-dependent JNK signaling via liquid-liquid phase separation, with implications that extend beyond synaptic morphogenesis to axon injury and degeneration pathways.

neuroscience↗

bioRxiv2024

Prefoldin 5 is a microtubule-associated protein that suppresses Tau-aggregation and neurotoxicity

Tauopathies represent a major class of neurodegenerative disorders associated with intracellular aggregates of the microtubule-associated protein Tau. To identify molecular modulators of Tau toxicity, we used a genetic screen to identify protein chaperones whose RNAi-mediated knockdown could modulate hTauV337M-induced eye-ommatidial degeneration in Drosophila. This screen identified the Prefoldins Pfdn5 and Pfdn6 as strong modifiers of hTauV337M cytotoxicity. Consistent with the known function of Pfdn as a cotranslational chaperone for tubulin, Pfdn5 mutants showed substantially reduced levels of tubulin monomer. However, additional microtubule-related functions were indicated by the robust unexpected association of Pfdn5 with axonal microtubules in vivo, as well as binding with stabilized microtubules in biochemical assays. Loss of Pfdn5 resulted in neuromuscular junctions (NMJ) defects similar to those previously described in hTau-expressing flies: namely, increased supernumerary boutons and fewer microtubule loops within mature presynaptic boutons. Significantly, synaptic phenotypes caused by hTauV337M overexpression were also strongly enhanced in a Pfdn5 mutant background. Consistent with a role in modulating Tau toxicity, not only did loss of Pfdn5 result in increased accumulations of Tau-aggregates in hTauV337M expressing neurons, but also neuronal overexpression of Prefoldin strikingly ameliorated age-dependent neurodegeneration and memory deficits induced by pathological hTau. Together, these and other observations described herein: (a) provide new insight into Prefoldin-microtubule interactions; (b) point to essential posttranslational roles for Pfdn5 in controlling Tau-toxicity in vivo; and (c) demonstrate that Pfdn5 overexpression is sufficient to restrict Tau-induced neurodegeneration.

neuroscience↗

bioRxiv2024

Drosophila Asap regulates cellular protrusions via dArf6-dependent actin regulatory pathway

Membrane protrusions are fundamental to cellular functions like migration, adhesion, and communication and depend upon the dynamic reorganization of the cytoskeleton. The GAP-dependent GTP hydrolysis of Arf proteins regulates actin-dependent membrane remodeling. Here, we show that the dAsap regulates membrane protrusions in S2R+ cells by a mechanism that critically relies on its ArfGAP domain and re-localization of actin regulators, SCAR, and Ena. While our data reinforce the preference of dAsap for Arf1 GTP hydrolysis in vitro, we demonstrate that induction of membrane protrusions in S2R+ cells depends on Arf6 inactivation. This study furthers our understanding of how dAsap-dependent GTP hydrolysis maintains a balance between active and inactive states of dArf6 to regulate cell shape.

cell biology↗

bioRxiv2023

A conserved graft formation process in Picea abies and Arabidopsis thaliana identifies the PAT gene family as central regulators of wound healing

The widespread use of plant grafting has enabled eudicots and gymnosperms to join with closely related species and grow as one. Gymnosperms have dominated forests for over 200 million years and despite their economic and ecological relevance, we know little about how they graft. Here, we developed a micrografting method in conifers using young tissues that allowed efficient grafting between closely related species and distantly related genera. Conifer graft junctions rapidly connected vasculature and differentially expressed thousands of genes including auxin and cell wall-related genes. By comparing these genes to those induced during Arabidopsis thaliana graft formation, we found a common activation of cambium, cell division, phloem and xylem-related genes. A gene regulatory network analysis in Picea abies (Norway spruce) predicted that PHYTOCHROME A SIGNAL TRANSDUCTION 1 (PAT1) acted as a core regulator of graft healing. This gene was strongly upregulation during both P. abies and Arabidopsis grafting, and Arabidopsis mutants lacking PAT-genes failed to attach tissues or successfully graft. Complementing Arabidopsis PAT mutants with the P. abies PAT1 homolog rescued tissue attachment and enhance callus formation. Together, our data demonstrate an ability for young tissues to facilitate grafting with distantly related species and identifies the PAT gene family as conserved regulators of graft healing and tissue regeneration.

plant biology↗

bioRxiv2023

Arf GAP containing dAsap regulates NMJ organization and synaptic calcium through Arf6-dependent signaling in Drosophila

Synaptic morphogenesis involves an interplay of multiple signaling pathways and requires membrane remodeling and cytoskeleton dynamics. We identified the BAR-domain protein dAsap (Arf GAP, SH3, Ankyrin repeat, and PH domain) as one of the regulators of synaptic morphogenesis. Loss of dAsap results in decreased bouton numbers, increased inter-bouton diameter, and disrupted microtubule organization at the nerve terminals. Electrophysiological analysis of the mutants revealed a gain in neurotransmission compared to control neuromuscular junctions (NMJs). dAsap mutant NMJs have increased evoked amplitude, increased spontaneous miniature frequency, and significantly fewer synaptic failures in low calcium. Consistent with these observations, dAsap mutants have increased active zone number. Additional pharmacological and genetic manipulations that are known to impair calcium release from stores suppress the dAsap phenotypes. Finally, we show that expressing a GDP-locked form of Arf6 in dAsap mutants restored the NMJ morphological defects, disrupted cytoskeleton, and aberrant neurotransmission. Thus, we propose a model in which dAsap regulates NMJ morphogenesis and synaptic calcium homeostasis through Arf6-dependent neuronal signaling.

neuroscience↗

bioRxiv2022

Enhanced epithelial to mesenchymal transition and chemoresistance in advanced Retinoblastoma tumors is driven by miR-181a

Advanced retinoblastoma (Rb) tumors can infiltrate distant tissues and cause a potent threat to vision and life. Through transcriptomic profiling, we discovered key epithelial to mesenchymal transition (EMT) and chemotherapy resistance genes at higher expression levels in advanced Rb tumors. Rb-/- tumor cells acquire metastasis-like phenotype through the EMT program that critically contributes to chemoresistance. We demonstrate that prolonged chemo-drug exposure in Rb cells elicits an EMT program through ZEB1 and SNAI2 that further acquires therapeutic resistance through cathepsin L and MDR1 mediated drug efflux mechanisms. Further, 16 significantly differentially expressed miRNAs were identified in patient tumors, of which miR-181a-5p was significantly reduced in advanced Rb tumors and associated with altered EMT and drug resistance genes. Enhancing miR-181a-5p levels in Rb-/- cells and Rb-/- chemo-resistant sublines controls EMT transcription factors ZEB1 and SNAI2 and halts the transition switch, thereby reversing drug resistance. We thus identify miR-181a-5p as a potential therapeutic target for EMT triggered drug-resistant cancers that can halt their invasion and sensitize them to low dose chemotherapy drugs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=171 SRC="FIGDIR/small/501381v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@114edeorg.highwire.dtl.DTLVardef@50da16org.highwire.dtl.DTLVardef@1df21borg.highwire.dtl.DTLVardef@1ea79_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2021

The regeneration factors ERF114 and ERF115 act as transducers of mechanical cues to developmental pathways

Plants show an unparalleled regenerative capacity, allowing them to survive severe stress conditions, such as injury, herbivory attack and harsh weather conditions. This potential not only replenishes tissues and restores damaged organs, but can also give rise to whole plant bodies, highlighting the intertwined nature of development and regeneration. It suggests that regeneration and developmental processes respond to the same upstream signals, but how a cell knows which of the two processes to engage is currently unknown. Here, we demonstrate that next to being regulators of regeneration, ETHYENE RESPONSE FACTOR 114 (ERF114) and ERF115 govern developmental growth in the absence of wounding or injury. Increased ERF114 and ERF115 activity is correlated with enhanced xylem maturation and lateral root formation, whereas their knockout results in a decrease in lateral roots and xylem connectivity following grafting. Moreover, we provide evidence that mechanical cues contribute to ERF114 and ERF115 expression in correlation with BZR1 mediated brassinosteroid signaling under both regenerative and developmental conditions. Antagonistically, negative regulation of cell wall extensibility via cell wall-associated mechanosensory FERONIA signaling suppresses their expression under both conditions. Our data suggest a molecular framework in which mechanical perturbations too great to be compensated by adaptive cell wall remodeling results in strong ERF114 and ERF115 expression, switching their role from developmental to regenerative regulators. One-Sentence SummaryMechanical cues drive ERF114/ERF115 expression during lateral root development as well as regeneration.

plant biology↗

bioRxiv2018

Cholesterol bound Plasmodium falciparum co-chaperone ‘PFA0660w’ complexes with major virulence factor ‘PfEMP1’ via chaperone ‘PfHsp70-x’

Lethality of Plasmodium falciparum (Pf) caused malaria results from cytoadherence, which is effected by exported Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family. Several exported Pf proteins (exportome) including chaperones alongside cholesterol rich microdomains are crucial for PfEMP1 translocation to infected erythrocyte surface. An exported Hsp40 (heat shock protein 40) PFA0660w functions as a co-chaperone of PfHsp70-x, and these co-localize to specialized intracellular mobile structures termed J-dots. Our studies attempt to understand the function of PFA0660w-PfHsp70-x chaperone pair using recombinant proteins. Biochemical assays reveal that N and C-terminal domains of PFA0660w and PfHsp70-x respectively are critical for their activity. We show the novel direct interaction of PfHsp70-x with the cytoplasmic tail of PfEMP1, and binding of PFA0660w with cholesterol. PFA0660w operates both as a chaperone and lipid binding molecule via its separate substrate and cholesterol binding sites. PfHsp70-x binds cholesterol linked PFA0660w and PfEMP1 simultaneously in vitro to form a complex. Collectively, our results and the past literature support the hypothesis that PFA0660w-PfHsp70-x chaperone pair assists PfEMP1 transport across the host erythrocyte through cholesterol containing J-dots. Since PFA0660w seems essential for parasite survival, characterization of its interaction with PfHsp70-x and J-dots may form the basis for development of future antimalarials.

molecular biology↗