Biology subjects

Dhar, A.

Publications and source records attributed to Dhar, A..

16 recordsLinked to original sources

bioRxiv2026

Sustained presence of metabolically active mesophiles in the boiling vent-water of a low-salinity, slightly-alkaline, sulfate-rich geothermal spring characterized by an inequitable ecology of hyperthermophiles and thermophiles

Geothermal vent-heads exclusively support the proliferation of (hyper)thermophilic microorganisms. However, diverse mesophiles are stochastically thrust into these ecosystems by local geodynamic forces, and their population-level responses to the unfamiliar stressor hold critical implications for lifes intrinsic resilience to biophysical adversities. Here, we report a time-course exploration of the boiling vent-water of a Trans-Himalayan geothermal spring to determine the population dynamics, in situ functionalities, and potential ecological roles of mesophilic incomers in the context of indigenous (hyper)thermophiles. The temporally-consistent (hyper)thermophilic core-community, predominated in turn by a few Aquificia and Thermoproteota, was delineated as metagenome-assembled genomes. Mesophiles/moderate-thermophiles were identified as MAGs and/or pure-cultures, and whether temporally-consistent/inconsistent, had far lower prevalence. Overall, in situ transcriptional activity and prevalence had significant positive correlation; but many species exhibited high activity despite low prevalence and vice versa, implicating the decoupling of global metabolism from growth/proliferation. Bulked metatranscriptomic signatures identified the (hyper)thermophiles as primary producers of the ecosystem. Populations of mesophiles and moderate-thermophiles were apparently active, but not growing, in situ, with some being engaged in environment detoxification, and production of nutrients, metabolites and protective-biomolecules wanting in the (hyper)thermophiles. The findings engender an unprecedented portrayal of geothermal-vents as multi-community ecosystems where unfamiliar immigrants provide critical metabolic services.

microbiology↗

bioRxiv2026

A compact, portable degron tool derived from the 3'UTR of MTCH2 for tunable degradation of proteins

Targeted protein degradation relies on short sequence elements, termed degrons, that direct proteins to the cellular degradation machinery. Here, we report a nine-amino-acid degron (FYTVWRAFL) derived from the 3' untranslated region (UTR) of human MTCH2 and demonstrate its utility as a tuneable protein-degradation tool. When appended to the C-terminus of proteins, FYTVWRAFL induces rapid and robust degradation by the ubiquitin-proteasome system. Using pharmacological inhibition and genetic perturbation, we show that degradation mediated by this degron is dependent on the E3 ubiquitin ligase MDM2. Structural modeling revealed that FYTVWRAFL interacts with the hydrophobic cleft of MDM2 in a manner similar to the p53 degron. Three hydrophobic residues form the core interaction interface. By systematic mutagenesis of these residues, we generated a panel of degron variants that confer graded levels of protein stability. We demonstrate the versatility of this system by achieving tunable expression of the endogenous protein Elm1 in Saccharomyces cerevisiae. Collectively, our study establishes a compact, transportable, and tunable degron system as a robust toolkit for quantitative control of protein abundance across eukaryotic systems.

molecular biology↗

bioRxiv2025

In-vitro Knockdown and Pharmacological Inhibition of RIPK2 Attenuates Calcium Oxalate-Nephrocalcinosis Associated Inflammation and Oxidative Stress in NRK-52E and Primary Renal Epithelial Cells

Ectopic deposition of calcium oxalate in the parenchyma (nephrocalcinosis) or as stones in the collecting system (nephrolithiasis) causes inflammation and oxidative stress in renal tissue. Receptor interacting serine/threonine kinase 2 (RIPK2) is a well-known mediator of oxidative stress and inflammation. However, its role in nephrocalcinosis or nephrolithiasis remains unexplored. NRK-52E and primary renal cells were treated with calcium oxalate-monohydrate (COM) to induce nephrocalcinosis like pathological changes. siRNAs and commercially available inhibitor was used to block RIPK2s activity. Oxidative stress, inflammation, apoptosis, crystal adhesion, and changes in cell morphology were measured as endpoint markers. Exposure to COM but not to adenine, H2O2, and high-glucose significantly upregulated RIPK2 levels and induced oxidative stress, intracellular-calcium overload, and mitochondrial dysfunction. This was accompanied by the activation of NF-{kappa}B pathway, high levels of pro-inflammatory and low levels of anti-inflammatory cytokines, increased apoptosis, and epithelial-mesenchymal transition (EMT). RIPK2 silencing or pharmacological inhibition effectively mitigated these pathological changes and restored levels of antioxidant enzymes. Mechanistically, RIPK2 inhibition disrupted the NF-{kappa}B/TGF-{beta}1 signaling and reduced CaOx crystal adhesion. Preliminary data from our previously conducted in vivo CaOx mouse model study confirmed the CaOx-induced RIPK2 upregulation. Our data strongly supports the involvement of RIPK2 in CaOx-induced renal cell damage.

pharmacology and toxicology↗

bioRxiv2025

IntAct-U-ExM: Ultrastructure Expansion microscopy of actin networks via an internally-tagged actin

Expansion microscopy (ExM) has revolutionized super-resolution imaging in cell biology due to its simple and inexpensive workflow. The use of ExM has revealed several novel insights into the nanoscale architectures of cellular protein complexes, especially the microtubule cytoskeleton in model and non-model systems. Despite tremendous progress in expansion microscopy protocols that preserve cellular ultrastructure (U-ExM), compatible probes for imaging actin isoforms with U-ExM are still lacking and have hindered the study of diverse actin isoforms and networks across model systems. Here, we use IntAct, an internally tagged actin that incorporates into cellular actin networks, to develop and optimize U-ExM of diverse actin network types in both yeast and mammalian cells. Using expression of ALFA-tagged IntAct variants in yeast and mammalian cells, we show robust visualization of actin patches, cables, and rings in yeast and diverse actin networks such as actin cortex, stress fibers, filopodia, lamellipodium in mammalian cells at improved resolution. We also detect transient nuclear actin filaments using IntAct-U-ExM underscoring the advantages offered by our approach to image understudied actin structures. Overall, we demonstrate the effectiveness of IntAct-U-ExM for performing super-resolution imaging of various actin structures in an isoform-specific manner and highlight the potential of IntAct to study the nanoscale organization of diverse actin cytoskeletal networks across species. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/654030v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@169cd82org.highwire.dtl.DTLVardef@1334af3org.highwire.dtl.DTLVardef@7d9d43org.highwire.dtl.DTLVardef@dfc50b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Nim1-related kinases regulate septin organization and cytokinesis by modulating Hof1 at the cell division site

The septin scaffold recruits and organizes actomyosin ring (AMR) components, thus, ensuring faithful cytokinesis. The septin-associated kinases - Elm1, Gin4, Hsl1, and Kcc4 are thought to stabilize and regulate the septin architecture at the bud neck, but the underlying mechanisms remain largely unknown. Here, we present a comprehensive, quantitative analysis of these four septin-associated kinases and reveal major roles for Elm1 and Gin4 in septin stability and architectural transitions during the cell cycle. We find that Elm1 and Gin4 play a previously overlooked role in AMR organization and constriction during cytokinesis. We report that the Gin4 kinase interacts directly with the AMR component and F-BAR protein Hof1 via its C-terminal membrane-binding kinase associated-1 (KA1) domain, and is likely involved in the proper organization and anchoring of Hof1 at the bud neck, representing an unappreciated mode of regulation during cytokinesis. We further show that Gin4 controls septin organization and AMR constriction in a kinase-independent manner, similar to Elm1. Using an extensive GFP-GBP-based tethering assay in elm1{Delta} and gin4{Delta} cells, we identify an important role for Hsl1 in maintaining septin organization and cell shape in coordination with Elm1, Gin4, and Kcc4, independent of its role in the morphogenetic checkpoint. Furthermore, our data indicate that Hsl1 acts downstream of Elm1, with its membrane-binding KA1 domain being critical for its function. Together, these findings reveal new insights into the modes by which the kinases Gin4 and Elm1 regulate cytokinesis, highlight a redundant role for Hsl1 in controlling septin organization and cytokinesis, and uncover the inherent redundancy and adaptability of the septin kinase network in Saccharomyces cerevisiae.

cell biology↗

bioRxiv2025

Distinct filament morphology and membrane tethering features of the dual FtsZs in Odinarchaeota

The deep-branching Asgard archaea have emerged as a model to study Eukaryogenesis as they harbor homologs of many of the major eukaryotic protein families. In this study, we use Candidatus Odinarchaeota FtsZs as representatives to understand the probable origin, evolution, and assembly of the FtsZ-Tubulin protein superfamily in Asgard archaea. We have performed a comparative analysis of the biochemical properties and cytoskeletal assembly of FtsZ1 and FtsZ2, the two FtsZ isoforms in the Odinarchaeota metagenome. Our electron microscopy analysis reveals that OdinFtsZ1 assembles into curved single protofilaments, while OdinFtsZ2 forms stacked spiral ring-like structures. Upon sequence analysis, we identified an N-terminal amphipathic helix in OdinFtsZ1, which mediates direct membrane tethering. In contrast, OdinFtsZ2 is recruited to the membrane by the membrane-anchor OdinSepF via OdinFtsZ2s C-terminal tail. Overall, we report the presence of two distant evolutionary paralogs of FtsZ in Odinarchaeota, with distinct filament assemblies and differing modes of membrane targeting mechanisms, namely direct vs SepF-mediated membrane binding. Our findings highlight the diversity of FtsZ proteins in the transitional archaeal phylum Odinarchaeota, providing valuable insights into the evolution and differentiation of tubulin family proteins.

biochemistry↗

bioRxiv2025

Ensemble docking for intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) are implicated in many human diseases and are increasingly being pursued as drug targets. Conventional structure-based drug design methods that rely on well-defined binding sites are however, largely unsuitable for IDPs. Here, we present computationally efficient ensemble docking approaches to predict the relative affinities of small molecules to IDPs and characterize their dynamic, heterogenous binding mechanisms at atomic resolution. We demonstrate that these ensemble docking protocols accurately predict the relative binding affinities of small molecule -synuclein ligands measured by NMR spectroscopy and generate conformational ensembles of ligand binding modes in remarkable agreement with experimentally validated long-timescale molecular dynamics simulations. Our results display the potential of ensemble docking approaches for predicting small molecule binding to IDPs and suggest that these methods may be valuable tools for IDP drug discovery campaigns.

biophysics↗

bioRxiv2024

Functional redundancy and formin-independent localization of tropomyosin isoforms in Saccharomyces cerevisiae

Tropomyosin is an actin binding protein which protects actin filaments from cofilin-mediated disassembly. Distinct tropomyosin isoforms have long been hypothesized to differentially sort to subcellular actin networks and impart distinct functionalities. Nevertheless, a mechanistic understanding of the interplay between Tpm isoforms and their functional contributions to actin dynamics has been lacking. In this study, we present and charcaterize mNeonGreen-Tpm fusion proteins that exhibit good functionality in cells as a sole copy, surpassing limitations of existing probes and enabling real-time dynamic tracking of Tpm-actin filaments in vivo. Using these functional Tpm fusion proteins, we find that S. cerevisiae Tpm isoforms, Tpm1 and Tpm2, colocalize on actin cables and indiscriminately bind to actin filaments nucleated by either formin isoform-Bnr1 and Bni1 in vivo, in contrast to the long-held paradigm of Tpm-formin pairing. We show that cellular Tpm levels regulate endocytosis by affecting balance between linear and branched actin networks in yeast cells. Finally, we discover that Tpm2 can protect and organize functional actin cables in absence of Tpm1. Overall, our work supports a concentration-dependent and formin isoform independent model of Tpm isoform binding to F-actin and demonstrates for the first time, the functional redundancy of the paralog Tpm2 in actin cable maintenance in S. cerevisiae.

cell biology↗

bioRxiv2023

Injectable butyrate-prodrug micelles induce long-acting immune modulation and suppress autoimmune arthritis in mice

Dysbiosis is linked to autoimmune diseases such as rheumatoid arthritis (RA), where microbial metabolites, such as short chain fatty acids (SCFAs), mediate the so-called gut-joint axis. The therapeutic potential of SCFAs is limited due to the frequent and high oral dosage requirements. RA is characterized by aberrant activation of peripheral T cells and myeloid cells. We aim to deliver butyrate, an SCFA, directly to the lymphatics using a polymeric micelle as a butyrate prodrug, creating a depot for inducing long-lasting immunomodulatory effects. Notably, negatively charged micelles (Neg-ButM) demonstrate superior efficacy in targeting the lymphatics post-subcutaneous administration, and were retained in the draining lymph nodes, spleen, and liver for over a month. In a mouse RA model, we found that Neg-ButM substantially mitigated arthritis symptoms and promoted tolerogenic phenotypes in T cells and myeloid cells, both locally and systemically. These findings suggest potential applications of this approach in treating inflammatory autoimmune diseases.

bioengineering↗

bioRxiv2023

Seryl-butyrate: a prodrug that enhances butyrate's oral bioavailability and suppresses autoimmune arthritis and experimental autoimmune encephalomyelitis

Butyrate, a metabolite produced by commensal bacteria, has been intensively studied for its immunomodulatory effects on various immune cells, including T regulatory cells, macrophages, and dendritic cells. Butyrates development as a drug has been limited by its poor oral bioavailability due to its rapid metabolism in the gut, its low potency and thus high dosing, and its foul smell and taste. By simply esterifying butyrate to serine (O-butyryl-L-serine, SerBut), a design based on the concept of utilizing amino acid transporters to escape the gut and enhance systemic uptake thus increasing bioavailability, we developed an odorless and tasteless compound for oral administration. In the collagen antibody-induced arthritis (CAIA) and experimental autoimmune encephalomyelitis (EAE) murine models of rheumatoid arthritis and multiple sclerosis, we demonstrated that SerBut significantly ameliorated disease severity, modulated key immune cell populations both systemically and in disease-associated tissues, and reduced inflammatory responses without compromising global immune response to vaccination. Our findings highlight SerBut as a promising next-generation therapeutic agent for autoimmune and inflammatory diseases.

bioengineering↗

bioRxiv2022

ALIBY: ALFA Nanobody-Based Toolkit for Imaging and Biochemistry in Yeast

Specialized epitope tags continue to be an integral component in various biochemical and cell biological applications such as fluorescence microscopy, immunoblotting, immunoprecipitation, and protein purification. However, until recently, no single tag could offer this complete set of functionalities on its own. Here, we present a plasmid-based toolkit named ALIBY (ALFA Toolkit for Imaging and Biochemistry in Yeast) that provides a universal workflow to adopt the versatile ALFA tag/NbALFA system within the well-established model organism Saccharomyces cerevisiae. The kit comprises of tagging plasmids for labelling a protein-of-interest with the ALFA tag, and detection plasmids encoding a fluorescent protein-tagged NbALFA for live-cell imaging purposes. We demonstrate the suitability of ALIBY for visualizing the spatiotemporal localization of yeast proteins (i.e., cytoskeleton, nucleus, centrosome, divisome and exocyst) in live cells. Our approach has yielded an excellent signal-to-noise ratio without off-targeting or effect on cell growth. In summary, our yeast-specific toolkit aims to simplify and further advance the live-cell imaging of differentially abundant yeast proteins while also being suitable for biochemical applications. ImportanceIn yeast research, conventional fluorescent protein tags and small epitope tags are widely used to study the spatiotemporal dynamics and activity of proteins. Though proven to be efficient, these tags lack the versatility for usage across different cell biological and biochemical studies of a given protein-of-interest. Therefore, there is an urgent need for a unified platform for visualization, biochemical, and functional analyses of proteins-of-interest in yeast. Herein, we have engineered ALIBY, a plasmid-based toolkit which expands the benefits of the recently developed ALFA tag/NbALFA system to studies in the well-established model organism Saccharomyces cerevisiae. We demonstrate that ALIBY provides a simple and versatile strain construction workflow for long duration live-cell imaging and biochemical applications in yeast.

cell biology↗

bioRxiv2022

mNeonGreen-tagged fusion proteins and nanobodies reveal localization of tropomyosin to patches, cables, and contractile actomyosin rings in live yeast cells

Tropomyosins are structurally conserved -helical coiled-coil dimeric proteins that bind along the length of filamentous actin (F-actin) in fungi and animals. Tropomyosins play essential roles in the stability of actin filaments in non-muscle cells and are essential for calcium regulation of myosin II contractility in the muscle. Despite the crucial role of tropomyosin in actin cytoskeletal regulation, in vivo investigations of tropomyosin are limited, mainly due to the suboptimal live cell imaging tools currently available in many organisms. Here, we report mNeon-Green (mNG) tagged tropomyosin, with native promoter and linker length configuration, that clearly reports tropomyosin localization and dynamics in Schizosaccharomyces pombe (Cdc8), Schizosaccharomyces japonicus (Cdc8), and Saccharomyces cerevisiae (Tpm1 and Tpm2), in vivo and in isolated S. pombe cell division apparatuses. We extended this approach to also visualize the mammalian TPM2 isoform. Finally, we generated a camelid-nanobody against S. pombe Cdc8, which mimics the localization of mNG-Cdc8 in vivo without significantly influencing cell growth and dynamics of actin cytoskeleton. Using these tools, we report the presence of tropomyosin in previously unappreciated patch-like structures in fission and budding yeasts, show flow of tropomyosin (F-actin) cables to the cytokinetic actomyosin ring, and identify rearrangements of the actin cytoskeleton during mating. These powerful tools and strategies will aid better analyses of tropomyosin and actin cables in vivo.

cell biology↗

bioRxiv2021

Functional development of a V3-specific broadly neutralizing antibody isolated from a case of HIV superinfection

Stimulating broadly neutralizing antibodies (bnAbs) directly from germline remains a barrier for HIV vaccines. HIV superinfection elicits bnAbs more frequently than single infection, providing clues of how to elicit such responses. We used longitudinal antibody sequencing and structural studies to characterize bnAb development from a superinfection case. Mature bnAb QA013.2 bound both initial transmitted and superinfecting virus, but its inferred naive bound only the superinfecting strain and was not neutralizing. QA013.2 requires residues spanning FWRH1-CDRH1 to attain breadth, which is uncommon for V3-specific bnAbs. A 4.15 [A] cryo-EM structure of QA013.2 bound to heterologous native-like trimer showed recognition of V3 signatures (N301, N332, and GDIR). Antigenic profiling revealed that viral escape was achieved not only by changes in the structurally-defined epitope, but also by mutations in V1. These results highlight shared and distinct properties of QA013.2 relative to other V3-specific bnAbs in the setting of sequential, diverse antigenic variants.

microbiology↗

bioRxiv2020

Development of antibody-dependent cell cytotoxicity function in HIV-1 antibodies

A prerequisite for the design of an HIV vaccine that elicits protective antibodies is understanding the developmental pathways that result in desirable antibody features. The development of antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC) is particularly relevant because such antibodies have been associated with HIV protection in humans. We reconstructed the developmental pathways of six human HIV-specific ADCC antibodies using longitudinal antibody sequencing data. Most of the inferred naive antibodies did not mediate detectable ADCC. Gain of antigen binding and ADCC function typically required mutations in complementarity determining regions of one or both chains. Enhancement of ADCC potency often required additional mutations in framework regions. Antigen binding affinity and ADCC activity were correlated, but affinity alone was not sufficient to predict ADCC potency. Thus, elicitation of human ADCC antibodies may require mutations that first enable high affinity antigen recognition, followed by mutations that optimize factors contributing to functional ADCC activity.

immunology↗

bioRxiv2019

DNA clamp function of the mono-ubiquitinated Fanconi Anemia FANCI-FANCD2 complex

The FANCI-FANCD2 (ID) complex, mutated in the Fanconi Anemia (FA) cancer predisposition syndrome, is required for the repair of replication forks stalled at DNA interstrand crosslinks (ICL) and related lesions1. The FA pathway is activated when two replication forks converge onto an ICL2, triggering the mono-ubiquitination of the ID complex. ID mono-ubiquitination is essential for ICL repair by excision, translesion synthesis and homologous recombination, but its function was hitherto unknown1,3. Here, the 3.48 [A] cryo-EM structure of mono-ubiquitinated ID (IDUb) bound to DNA reveals that it forms a closed ring that encircles the DNA. Compared to the cryo-EM structure of the non-ubiquitinated ID complex bound to ICL DNA, described here as well, mono-ubiquitination triggers a complete re-arrangement of the open, trough-like ID structure through the ubiquitin of one protomer binding to the other protomer in a reciprocal fashion. The structures, in conjunction with biochemical data, indicate the mono-ubiquitinated ID complex looses its preference for ICL and related branched DNA structures, becoming a sliding DNA clamp that can coordinate the subsequent repair reactions. Our findings also reveal how mono-ubiquitination in general can induce an alternate structure with a new function.

molecular biology↗

bioRxiv2018

Rapid development of an infant-derived HIV-1 broadly neutralizing antibody lineage

HIV-infected infants develop broadly neutralizing plasma responses with more rapid kinetics than adults, suggesting the ontogeny of infant responses could better inform a path to achievable vaccine targets. We developed computational methods to reconstruct the developmental lineage of BF520.1, the first example of a HIV-specific broadly neutralizing antibody (bnAb) from an infant. The BF520.1 inferred naive precursor binds HIV Env and a bnAb evolved within six months of infection and required only 3% mutation. Mutagenesis and structural analyses revealed that for this infant bnAb, substitutions in the kappa chain were critical for activity, particularly in CDRL1. Overall, the developmental pathway of this infant antibody includes features distinct from adult antibodies, including several that may be amenable to better vaccine responses.

immunology↗