Biology subjects

Varshney, N.

Publications and source records attributed to Varshney, N..

9 recordsLinked to original sources

bioRxiv2026

A tissue-targeted auxin-inducible degradation toolkit for C. elegans embryogenesis

Many proteins required for embryogenesis are expressed in multiple tissues and across developmental time, often with tissue- and stage-specific roles. To resolve where and when proteins function, we developed a tissue-targeted auxin-inducible degradation toolkit for C. elegans embryogenesis. We generated a plasmid set compatible with single-copy insertion or high-copy extrachromosomal array generation that employs tissue-specific promoters (active in the germline, intestine, muscle, pharynx, neuroblasts, neurons, sensory neurons, epidermis, and pan soma) to drive an operon expressing an optimized auxin-dependent plant F-box factor TIR1 and, in most cases, a fluorescent histone. Using a ubiquitous nuclear envelope marker as a test substrate, we identified single-copy insertions and arrays that exhibit efficient auxin-dependent, tissue-specific target degradation. We also establish a workflow that ensures all functionally important isoforms of a target are tagged and that degradation reproduces the loss-of-function phenotype prior to tissue-specific analysis. Applying the toolkit to the broadly expressed Rho kinase LET-502, we find that degradation in the epidermis, but not in other embryonic tissues, disrupts elongation. Unexpectedly, temporal analysis revealed that LET-502 is required for late as well as early elongation.

developmental biology↗

bioRxiv2026

Inhibitor-2 directs formation of PP1 holoenzymes through a docking motif-dependent transfer of catalytic subunits to adapters

The catalytic subunits of protein phosphatase 1 (PP1) achieve spatiotemporal substrate specificity by assembling with diverse regulatory adapters to form holoenzymes. Three conserved proteins--Sds22, Inhibitor-2 and Inhibitor-3--facilitate loading of PP1 catalytic subunits (PP1cs) onto adapters. We show here that Inhibitor-2 is central to a dynamic cycle that directs formation of adapter-bound PP1 holoenzymes. Inhibitor-2 engages PP1cs via two adapter-like docking motifs (RVxF and SILK) and an active site-binding inhibitory region. While Inhibitor-2 depletion produced moderate phenotypes, mutation of its RVxF docking motif caused severe defects resembling global PP1c inhibition. The RVxF mutant did not prevent PP1c binding or reduce PP1c stability but inhibited formation of adapter-bound holoenzymes. The severe effects of the RVxF mutation were suppressed by linked mutation of the inhibitory active site-binding motif. These results suggest that Inhibitor-2 is integral to a dynamic cycle that delivers PP1cs to adapters, with its RVxF motif being critical for coupling relief of active site inhibition to adapter handoff.

cell biology↗

bioRxiv2026

Unique pore architecture underlies constitutive gating of human retinalTRPM1

Transient receptor potential melastatin 1 (TRPM1), a Ca{superscript 2}-permeable nonselective cation channel essential for retinal ON bipolar cell signaling and night vision, and implicated in congenital night blindness, has remained structurally and functionally poorly characterized. Here we report the first cryo-electron microscopy structure of human TRPM1 in conducting state. Although the channel assembles as a tetramer, it adopts an unexpected clockwise domain-swapped pore module with rotational geometry inverse to that observed in previously characterized 6-TM tetrameric channels. This inverted topology is accompanied by extensive remodeling of the S5-P-S6 module, dilation of the selectivity filter, expansion of the central cavity, and splaying of S6 to form a wide intracellular gate. Single-channel recordings reveal constitutive activity consistent with the conductive state captured. Together, these findings uncover a new 6-TM fold in tetrameric channel and provide a framework for understanding TRPM1 gating and disease-associated dysfunction.

biochemistry↗

bioRxiv2025

Calcium Dependent Protein Kinase 3 is not essential for the asexual replication of Plasmodium falciparum

Calcium dependent protein kinases are essential at various stages of malaria parasite development within the vertebrate host and the mosquitoes. In the rodent malaria parasite, Plasmodium berghei, CDPK3 is essential for the sexual stage development of the parasite within mosquitoes. However, the function of CDPK3 in the most lethal human malaria parasite, P. falciparum remains unexplored. Moreover, the calcium dependent kinase activity of CDPK3 has not been experimentally tested. With the aim to biochemically characterize CDPK3 and perform its functional evaluation in the asexual replication of P. falciparum, here we have expressed the full-length CDPK3 in E. coli and demonstrated that it is a bona fide calcium dependent kinase using a luminescence-based assay and a semi-synthetic epitope approach. Complete knock-out of cdpk3 using CRISPR/Cas9 did not show any defect in the asexual replication of the parasite and the knockout parasites look morphologically similar to the wild type. Our study suggests that cdpk3 is not essential for the asexual replication of the parasite under in vitro culture conditions. Perhaps CDPK3 is required for the sexual development of P. falciparum like its ortholog in the rodent malaria parasite. CDPK3 may be a good target for the development of transmission blocking drugs.

microbiology↗

bioRxiv2023

Cyclin B3 is a dominant fast-acting cyclin that drives rapid early embryonic mitoses

In many species, early embryonic mitoses proceed at a very rapid pace, but how this pace is achieved is not understood. Here we show that in the early C. elegans embryo, cyclin B3 is the dominant driver of rapid embryonic mitoses. Metazoans typically have three cyclin B isoforms that associate with and activate Cdk1 kinase to orchestrate mitotic events: the related cyclins B1 and B2 and the more divergent cyclin B3. We show that whereas embryos expressing cyclins B1 and B2 support slow mitosis (NEBD to Anaphase [~] 600s), the presence of cyclin B3 dominantly drives the [~]3-fold faster mitosis observed in wildtype embryos. CYB-1/2-driven mitosis is longer than CYB-3-driven mitosis primarily because the progression of mitotic events itself is slower, rather than delayed anaphase onset due to activation of the spindle checkpoint or inhibitory phosphorylation of the anaphase activator CDC-20. Addition of cyclin B1 to cyclin B3-only mitosis introduces an [~]60s delay between the completion of chromosome alignment and anaphase onset, which likely ensures segregation fidelity; this delay is mediated by inhibitory phosphorylation on CDC-20. Thus, the dominance of cyclin B3 in driving mitotic events, coupled to introduction of a short cyclin B1-dependent delay in anaphase onset, sets the rapid pace and ensures fidelity of mitoses in the early C. elegans embryo.

cell biology↗

bioRxiv2023

Bacterial deamidases modulate ubiquitin structure and dynamics to dysregulate ubiquitin signaling

The deamidases secreted by Burkholderia pseudomallei and Enteropathogenic Ecoli modify the residue 40 in ubiquitin from a Glutamine (Q) to Glutamate (E), triggering several downstream processes to cause cell cycle arrest and activate immune responses. Deamidation hampers the activity of ubiquitin and its interaction with ubiquitin chain receptors by an unknown mechanism. Here, we study the effect of deamidation on ubiquitin structure and dynamics. We report the crystal structure of the deamidated ubiquitin, supported by NMR and molecular dynamics simulations. The structure reveals a new intra-molecular salt bridge between the deamidated region and the C-terminal tail of ubiquitin. The salt bridge perturbs the dynamics of the ubiquitin tail to reduce affinity for ubiquitin receptors like the p62 ubiquitin-associated domain. The salt bridge disrupts the transition to catalytically active E2~Ub closed conformation. Consequently, RING E3s fail to interact with E2~Ub, reducing ubiquitination activity. Our findings reveal that deamidation-induced intramolecular salt bridges in ubiquitin modulate conformational ensembles to inactivate ubiquitination.

biochemistry↗

bioRxiv2020

Helicobacter pylori and Epstein-Barr virus coinfection stimulates the aggressiveness in gastric cancer through the regulation of gankyrin

Persistent coinfection of Helicobacter pylori (H. pylori) and Epstein-Barr virus (EBV) promotes aggressive gastric carcinoma. The molecular mechanisms underlying the aggressiveness in H. pylori and EBV coinfected gastric cancer is not well characterized. In the current study, we investigated the molecular mechanism involved in the cooperation of H. pylori and EBV-driven proliferation of gastric epithelial cells. Results showed that the coinfections are significantly more advantageous to the pathogens to create a microenvironment that favors the higher pathogen-associated gene expression. The EBV latent genes EBNA1 and EBNA3C are highly overexpressed in the coinfections compared to individual EBV infection at different time points (12 and 24 hrs). The H. pylori-associated genes 16s rRNA, CagA, and BabA has also been highly overexpressed in coinfections compared to H. pylori alone. Gankyrin is a small protein of 25 KDa involved in multiple biological and physiological processes. The upregulation of gankyrin modulates the various cell signaling pathways, leading to oncogenesis. The gankyrin shows a similar expression pattern as EBNA3C at both transcript and protein levels, suggesting a possible correlation. Further EBV and H. pylori create microenvironments that induce cell transformation and oncogenesis by dysregulation of the cell-cycle regulator, GC marker, cell migration, DNA response, and antiapoptotic genes in infected gastric epithelial cells by enhancing the expression of gankyrin. Our study provides new insights into the molecular mechanism where the interplay between two oncogenic agents (H. pylori and EBV) leads to the enhanced carcinogenic activity of gastric epithelial cells through overexpression of oncoprotein gankyrin. ImportanceIn the present study, we have evaluated the synergistic effect of EBV and H. pylori infection on gastric epithelial cells in various coinfection models. These coinfection models depict the first exposures of gastric epithelial cells with EBV and then the H. pylori. While other coinfection models narrated the first exposures of H. pylori followed by the infection of EBV. This led to an enhanced oncogenic phenotype in gastric epithelial cells. We determined the coinfection of EBV and H. pylori enhanced the expression of oncogenic protein gankyrin. The interplay between EBV and H. pylori promotes the oncogenic properties of AGS cells through the newly discovered oncoprotein gankyrin. EBV and H. pylori mediated upregulation of gankyrin further dysregulates various cancer-associated hallmarks of genes such as cell-migratory, gastric cancer marker, tumor suppressor, DNA damage response, and proapoptotic genes.

microbiology↗

bioRxiv2019

MTOC clustering and nuclear division through proper spindle positioning in yeast: a microtubule grow-and-catch model

The dynamic process of mitotic spindle assembly depends on multitudes of inter-dependent interactions involving kinetochores (KTs), microtubules (MTs), spindle pole bodies (SPBs), and molecular motors. Before forming the mitotic spindle, multiple visible microtubule organizing centers (MTOCs) coalesce into a single focus to serve as a SPB in the pathogenic budding yeast, Cryptococcus neoformans. To explain this unusual phenomenon in the fungal kingdom, we propose a search and capture model, in which cytoplasmic MTs (cMTs) nucleated by MTOCs grow and capture each other to promote MTOC clustering. Our quantitative modeling identifies multiple redundant mechanisms mediated by a combination of cMT-cell cortex interactions and inter-cMT coupling to facilitate MTOC clustering within the physiological time limit as determined by time-lapse live-cell microscopy. Besides, we screen various possible mechanisms by computational modeling and propose optimal conditions that favor proper spindle positioning - a critical determinant for timely chromosome segregation. These analyses also reveal that a combined effect of MT buckling, dynein pull, and cortical push maintains spatiotemporal spindle localization.

biophysics↗

bioRxiv2019

Aurora kinase Ipl1 facilitates bilobed distribution of clustered kinetochores to ensure error-free chromosome segregation in Candida albicans

Candida albicans, an ascomycete, has an ability to switch to diverse morphological forms. While C. albicans is predominatly diploid, it can tolerate aneuploidy as a survival strategy under stress. Aurora kinase B homolog Ipl1 is a critical ploidy regulator that controls microtubule dynamics and chromosome segregation in Saccharomyces cerevisiae. In this study, we show that Ipl1 in C. albicans has a longer activation loop than that of the well-studied ascomycete S. cerevisiae. Ipl1 localizes to the kinetochores during the G1/S phase and associates with the spindle during mitosis. Ipl1 regulates cell morphogenesis and is required for cell viability. Ipl1 monitors microtubule dynamics which is mediated by separation of spindle pole bodies. While Ipl1 is dispensable for maintaining structural integrity and clustering of kinetochores in C. albicans, it is required for the maintenance of kinetochore geometry to form bilobed structures along the mitotic spindle, a feature of Ipl1 that was not observed in other yeasts. Depletion of Ipl1 results in erroneous kinetochore-microtubule attachments leading to aneuploidy-associated resistance to fluconazole, the most common anti-fungal drug used to treat Candida infections. Taking together, we suggest that Ipl1 spatiotemporally ensures kinetochore geometry to facilitate bipolar spindle assembly crucial for ploidy maintenance in C. albicans.

cell biology↗