Biology subjects

Adhikary, B.

Publications and source records attributed to Adhikary, B..

6 recordsLinked to original sources

bioRxiv2026

Calcium and actomyosin pulses drive tricellular zipping to remodel epithelial cell-cell junctions and facilitate cell packing

Epithelial morphogenesis requires coordinated changes in cell geometry, neighbor cell arrangement, and cell packing to generate functional tissue architectures. However, the spatiotemporal regulation of upstream signals that drive local junctional remodeling in highly proliferative developing epithelial tissues remains unclear. Here, we characterize a new junction remodeling process, which we call "tricellular zipping". It resolves long, thin cell extensions generated by cell-division forces that terminate at transient higher-order vertices. We show that tricellular zipping facilitates epithelial cell packing in blastula-stage Xenopus laevis embryos through asymmetric displacement of a tricellular vertex and formation of a de novo bicellular interface. Similarly, we observe tricellular zipping in developing mouse embryos. Combining modeling and live imaging approaches, we find that displacement of the remodeling vertex depends on actomyosin-dependent asymmetric force generation and on enrichment of adaptor proteins coupling actomyosin to junctions, which enables effective force transmission. In contrast, the stationary vertex remains close to mechanical equilibrium and therefore undergoes no net displacement. Furthermore, we find that calcium flashes drive the localized, pulsatile actomyosin contractility required for efficient tricellular zipping. Extending our observations, we show that disrupting transient calcium signaling also affects cell packing in Drosophila embryos, thereby impairing mesoderm internalization. Overall, by combining multidisciplinary and multisystem approaches, we uncover a new mode of junctional remodeling and highlight the importance of spatiotemporally regulated calcium signaling in driving local junctional rearrangements to facilitate morphogenesis.

cell biology↗

bioRxiv2026

Zyxin and LPP differ in their sensing of strained actin filaments at tricellular junctions

The actomyosin cytoskeleton plays important roles in cell-cell adhesion by generating and responding to forces. Actin-binding proteins support actomyosin networks by reinforcing actin filaments, promoting actin remodeling, and transmitting forces to transmembrane adhesion proteins. One family of actin-binding proteins, LIM domain-containing proteins, is recruited to strained actin filaments. Here, we investigate two members of the LIM domain family, Zyxin and Lipoma-Preferred Partner (LPP), using the embryonic epithelium of Xenopus laevis, where actin-associated cell-cell junctions connect cells to promote tissue integrity and barrier function. Specifically, we compare Zyxin and LPP's response to increased tension at tricellular junctions (TCJs), sites of heightened mechanical strain within the tissue. Upon increased tension, Zyxin and LPP mechanoaccumulate to different extents relative to F-actin, suggesting distinct mechanisms. Our results demonstrate that Zyxin's and LPP's LIM domain-containing regions (LCRs) are sufficient for mechanoaccumulation as well as responsible for the difference in mechanoaccumulation, while their N-termini play a regulatory role in their mechanosensitive responses. Docking simulations show that a hydrophobic interaction between the LCR and the "crack site" that forms in F-actin filaments under high force may underlie the LCR's sensing of strained F-actin. Additionally, the docking simulations reveal that previously-identified conserved residues are present at the binding interface. Mutations of conserved residues in Zyxin or LPP reduce the extent of mechanoaccumulation at TCJs, supporting the docking prediction. Together, this study advances our understanding of Zyxin and LPP's sensing of strained actin at TCJs under mechanical challenge.

cell biology↗

bioRxiv2026

Cryoelectron tomography of HIV-1 cell-cell transmission conjugates reveals a secluded environment for viral assembly and transfer

Cell-cell transmission of HIV-1 (CCT) is a highly efficient mode of intrahost transmission that provides viral particles broad protection from antiretroviral agents including antiretroviral drugs, host restriction factors, and broadly neutralizing antibodies. Interestingly, the exact factors that grant viral particles increased protection and efficiency during intrahost spread remain unclear. Using a T cell co-culturing system and correlative cryoelectron tomography (cryoET) workflow, we investigated the architecture of HIV-1 CCT sites in a near-native, in situ context to address why CCT is efficient and grants this resistance. Contrary to previous models which suggest large, virus-packed sites, our 3D reconstructions reveal that CCT occurs within small intercellular spaces containing on average 1-5 viral particles. Surrounding these spaces are long, tight membrane interfaces that seclude the viral particles from the remaining extracellular environment. These observations suggest a model where spatial isolation may limit accessibility of antiretroviral agents to the virus-containing spaces thereby conferring protection to viral particles during transmission. To functionally validate these structural insights, we developed a quantitative flow cytometry strategy that decouples CCT conjugate formation from successful CCT infection through the analysis of multicell events. This methodology allows us to systematically compare the impact different molecules such as Env and CD4 have on HIV-1 CCT in a quantitative and population-level manner. Using this approach, we determined that initial HIV-1 CCT conjugate formation is partially dependent on CD4, but the Env involved in conjugate formation may differ from Env used for viral entry. Overall, these findings may prompt a rethinking of intervention strategies for HIV-1 CCT and have further implications with cytoplasmic trafficking, nuclear import, and host restriction factors.

microbiology↗

bioRxiv2025

PAK4 promotes vertex remodeling to maintain epithelial integrity and barrier function

Cell-cell junctions are essential for epithelial integrity and barrier function, but the mechanisms regulating their remodeling remain unclear. Here, we investigated the role of the junctional kinase PAK4 in vertex remodeling. PAK4 localized to apical junctions and accumulated at multicellular vertices in MCDK cells and Xenopus embryos. Inhibition or knockout of PAK4 increased higher-order vertices, caused junctional discontinuities, and impaired barrier function in MDCK cells. PAK4 recruitment required the scaffolding protein Afadin. Afdn-KO cells exhibited severe junctional defects and reduced barrier function. Artificial targeting of PAK4 in Afdn-KO cells partially restored junctional continuity and barrier function. In Xenopus embryos, live imaging showed dynamic PAK4 accumulation at remodeling vertices, and PAK4 inhibition hindered resolution of multicellular vertices. Expression of an amino-terminal fragment (PAK4-NT) impaired remodeling and induced cytokinetic failure. Live imaging in Xenopus revealed barrier leakage at multicellular vertices upon PAK4 inhibition. These findings indicate that PAK4 and Afadin cooperate to maintain epithelial integrity and barrier function by promoting vertex remodeling. SummaryAdhikary et al. show that PAK4, recruited to epithelial junctions by Afadin, drives dynamic vertex remodeling in cells and embryos. Inhibiting PAK4 disrupts tissue packing and compromises junctional continuity and barrier function, implicating PAK4 and Afadin as critical partners in vertex remodeling to preserve tissue integrity.

cell biology↗

bioRxiv2024

Enhanced Yield and Gentle Purification of HIV for Cryo-Electron Tomography Analysis of Virion Maturation.

HIV is a lentivirus characterized by the formation of its mature core. Visualization and structural examination of HIV requires purification of virions to high concentrations. The yield and integrity of these virions are crucial for ensuring a uniform representation of all viral particles in subsequent analyses. In this study, we present a method for purification of HIV virions which minimizes forces applied to virions while maximizing the efficiency of collection. This method allows us to capture between 1,000 and 5,000 HIV virions released from individual HEK293 cells after transfection with the NL4.3 HIV backbone, a 10 fold advantage over other methods. We utilized this approach to investigate HIV core formation from several constructs: pNL4-3(RT:D185A&D186A) with an inactive reverse transcriptase, NL4.3(IN: V165A&R166A) with a type-II integrase mutation, and NL4.3(: {Delta}(105-278)&{Delta}(301-332)) featuring an edited packaging signal. Notably, virions from NL4.3(: {Delta}(105-278)&{Delta}(301-332)) displayed a mixed population, comprising immature virions, empty cores, and cores with detectable internal density. Conversely, virions derived from NL4.3(IN: V165A&R166A) exhibited a type II integrase mutant phenotype characterized by empty cores and RNP density localized around the cores, consistent with previous studies. In contrast, virions released from pNL4-3(RT:D185A&D186A) displayed mature cores containing detectable RNP density. We suggest that the purification methods developed in this study can significantly facilitate the characterization of enveloped viruses.

microbiology↗

bioRxiv2022

Human-sloth bear conflict in Nepal - case study from an unprotected Churia forest

Human-sloth bear conflict, generally in the form of human attacks, is common throughout most areas where sloth bears co-occur with humans. The situation is more prevalent in multi-use forest landscapes outside protected areas. Although sloth bears are a conflict-prone species in Nepal, there is a clear lack of systematic studies that can inform human safety and conflict mitigation. We used data from questionnaire-based interviews with conflict-affected people and witnesses to provide detailed information about human-sloth bear conflict (1990- 2021) in the Trijuga forest, an important sloth bear stronghold outside protected areas in Nepal. The data were analyzed using descriptive statistics, chi-square tests, and regression analysis. For the time period, 66 conflict incidents involving 69 human individuals were recorded, with an annual average of 2.06 (SD = 1.48) incidents and 1.75 (SD = 1.34) attacks. Conflicts primarily impacted working-age group (25-55 years old) men, whose primary occupation was farming and who frequented the forest regularly. They typically occurred between 0900 and 1500, inside forests, and in habitats associated with poor land cover visibility. Poor visibility was also a significant positive determinant of bear attacks on humans. Fifty-six conflict incidents resulted in attacks that injured 59 people, with a fatality rate of 8.47%. Victims of bear attacks frequently had serious injuries, especially to the head and neck areas of the body. Serious injuries were more likely to occur to lone individuals than to people who were in groups of two or more. We suggest identification of conflict-risk habitats through a participatory mapping approach and education programs for the local people for effective human-sloth bear conflict management in Nepals unprotected forests.

ecology↗