Biology subjects

Pathak, R.

Publications and source records attributed to Pathak, R..

9 recordsLinked to original sources

bioRxiv2026

Cryo-EM reveals a right-handed double-helix dimer architecture of PCDH15 critical for mechanotransduction

Tip links connect the stereocilia of mechanosensory hair cells in the inner ear and transmit force onto mechanotransduction (MET) channels. Tip links consist of protocadherin 15 (PCDH15) and cadherin 23 (CDH23), which assemble into an extracellular filament approximately 150 nm in length. Rare freeze-etched electron microscopy (EM) images have suggested that tip links could be right-handed double helices in vivo, but direct structural evidence has been lacking. Using cryo-EM we determined the structure of a large part of the extracellular PCDH15 domain. Two PCDH15 molecules form a parallel cis dimer stabilized by several dimerization interfaces, including two strand crossovers and two parallel contacts, yielding a right-handed double helix. Functional studies show that mutations in PCDH15 dimerization-domains impair MET. Our results establish the molecular foundation for how PCDH15 forms a right-handed double helix to enable mechanical sensing.

neuroscience↗

bioRxiv2026

Machine Learning Ensemble Reveals Distinct Molecular Pathways of Retinal Damage in Spaceflown Mice

BackgroundSpaceflight-associated neuro-ocular syndrome (SANS) poses significant risks to astronaut visual health during long-duration missions, yet its underlying molecular mechanisms remain incompletely understood. Oxidative stress and apoptosis are candidate molecular drivers, but their transcriptomic signatures in spaceflight-exposed retinal tissue have not been systematically characterized. MethodsWe applied a machine learning ensemble of linear regression models to predict two ocular phenotypes: 4-hydroxynonenal (4-HNE) immunostaining as a marker of lipid peroxidation-mediated oxidative damage; and TUNEL positivity as a marker of apoptotic cell death. In this observational study, we use bulk retinal gene expression data obtained from a controlled experiment with ground control and spaceflown mice to predict these phenotypes. Gene Ontology pathway enrichment was performed on the most predictive gene sets for each phenotype. ResultsThe 4-HNE phenotype was predicted by genes that converge on membrane-associated pathways, photoreceptor protein modification, synaptic dysfunction, and extracellular matrix dysregulation, including B2m, Tf, Cnga1, mt-Nd1, Snap25, and Efemp1. The genes predicting the TUNEL phenotype revealed a distinct signature emphasizing stress-induced apoptosis, rod photoreceptor degeneration, and endoplasmic reticulum dysfunction, with Ddit4, Nrl, Rom1, Reep6, and Gabarapl1 emerging as central regulators. ConclusionsOxidative lipid peroxidation and apoptotic cell death represent complementary and molecularly distinct pathological mechanisms in spaceflight-exposed murine retinal tissue. The gene signatures provide a putative molecular framework for developing noninvasive biomarkers and therapeutic targets to monitor and protect astronaut visual health during long-duration and deep-space missions.

bioinformatics↗

bioRxiv2025

Critical Evaluation of Sphingolipids Detection by MALDI-MSI

The increasing interest in the role of sphingolipids in (patho)physiology has led to the demand for visualization of these lipids within tissue samples (both from animal models and patient specimens) using techniques such as matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). While increasingly adopted, detection of sphingolipids with MALDI-MSI is challenging due to: i) the significant structural variations of sphingolipid molecules, ii) the potential breakdown of the more complex molecules into structurally simpler species which may confound the analysis, and iii) the great difference in levels among sphingolipid classes and subspecies, with the low-abundant ones often being close to the detection limit. In this study, we adopted a multi-pronged approach to establish a robust pipeline for the detection of sphingolipids by MALDI-MSI and to establish best practices and limitations of this technology. First, we evaluated the more commonly adopted methods [2,5-Dihydroxyacetophenon (DHA) or 2,5-Dihydroxybenzoic acid (DHB) matrix in positive ion mode and 1,5-Diaminonaphthalene (DAN) matrix in negative ion mode] using MALDI-MS on reference standards. These standards were used at ratios similar to their relative levels in biological samples to evaluate signal artifacts originating from fragmentation of more complex sphingolipids and impacting low level species. Next, by applying the most appropriate protocol for each sphingolipid class, MALDI-MSI signals were validated in cell culture by modulating specific sphingolipid species using sphingolipid enzymes and inhibitors. Finally, the optimized parameters were utilized on breast cancer tissue from the PyMT mouse model. We report the optimal signal for sphingomyelin (SM) and, for the first time, Sph in DHB positive ion mode (in cells and PyMT tissue), and the validated detection of ceramides and glycosphingolipids in DAN negative ion mode. We document the extensive fragmentation of SM into sphingosine-1-phosphate (S1P) and even more so into ceramide-1-phosphate (C1P) using DAN in negative ion mode and its effect in generating an artifactual C1P tissue signal; we also report the lack of detectable signal for S1P and C1P in biological samples (cells and tissue) using the more suitable DHB positive ion mode protocol.

cell biology↗

bioRxiv2024

The role of LEDGF in transcription is exploited by HIV-1 to position integration

HIV-1 integration occurs across actively transcribed genes due to the interaction of integrase with host chromatin factor LEDGF. Although LEDGF was originally isolated as a co-activator that stimulates promoter activity in purified systems, this role is inconsistent with LEDGF-mediated integration across gene bodies and with data indicating LEDGF is a histone chaperone that promotes transcriptional elongation. We found LEDGF is enriched in pronounced peaks that match the enrichments of H3K4me3 and RNA Pol II at transcription start sites (TSSs) of active promoters. Our genome-wide chromatin mapping revealed that MLL1 had a dominant role in recruiting LEDGF to promoters and the presence of LEDGF recruits RNA Pol II. Enrichment of LEDGF at TSSs correlates strongly with levels of integration across the transcribed sequences, indicating that LEDGF at TSSs contributed to integration across gene bodies. Although the N-terminal Pro-Trp-Trp-Pro (PWWP) domain of LEDGF interacts with nucleosomes containing H3K36me3, a modification thought to recruit LEDGF to chromatin, we found H3K36me3 does not contribute to gene specificity of integration. These data support a dual role model of LEDGF where it is tethered to promoters by MLL1 and recruits RNA Pol II. Subsequently, LEDGF travels across genes to effect HIV-1 integration. Our data also provides a mechanistic context for the contribution made by LEDGF to MLL1-based infant acute leukemia and acute myeloid leukemia in adults.

genomics↗

bioRxiv2022

Visualization of early RNA replication kinetics of SARS-CoV-2 by using single molecule RNA-FISH

SARS-CoV-2 infection has caused a major global burden. Despite intensive research, the mechanism and dynamics of early viral replication are not completely understood including the kinetics of formation of plus stranded genomic and subgenomic RNAs (gRNA and sgRNA) starting from the RNA from the first virus that enters the cell. We employed single-molecule RNA-fluorescence in situ hybridization (smRNA-FISH) to simultaneously detect viral gRNA and sgRNA in infected cells and carried out a time course analysis to determine the kinetics of their replication. We visualized the single molecules of gRNA within the cytoplasm of infected cells 30 minutes post-infection and detected the co-expression of gRNA and sgRNA within two hours post-infection. Furthermore, we observed the formation of a replication organelle (RO) from a single RNA, which led to the formation of multiple ROs within the same cells. Single molecule analysis indicated that while gRNA resided in the center of these ROs, the sgRNAs were found to radiate and migrate out of these structures. Our results also indicated that after the initial delay, there was a rapid but asynchronous replication, and the gRNA and sgRNAs dispersed throughout the cell within 4-5 hours post-infection forming multiple ROs that filled the entire cytoplasm. These results provide insight into the kinetics of early post-entry events of SARS-CoV-2 and the formation of RO, which will help to understand the molecular events associated with viral infection and facilitate the identification of new therapeutic targets that can curb the virus at a very early stage of replication to combat COVID-19. Author SummarySARS-CoV-2 infection continues to be a global burden. Soon after the entry, SARS-CoV-2 replicates by an elaborate process, producing genomic and subgenomic RNAs (gRNA and sgRNAs) within specialized structures called replication organelles (RO). Many questions including the timing of multiplication of gRNA and sgRNA, the generation, subcellular localization, and function of the ROs, and the mechanism of vRNA synthesis within ROs is not completely understood. Here, we have developed probes and methods to simultaneously detect the viral gRNA and a sgRNA at single cell single molecule resolution and have employed a method to scan thousands of cells to visualize the early kinetics of gRNA and sgRNA synthesis soon after the viral entry into the cell. Our results reveal that the replication is asynchronous and ROs are rapidly formed from a single RNA that enters the cell within 2 hours, which multiply to fill the entire cell cytoplasm within ~4 hours after infection. Furthermore, our studies provide a first glimpse of the gRNA and sgRNA synthesis within ROs at single molecule resolution. Our studies may facilitate the development of drugs that inhibit the virus at the earliest possible stages of replication to minimize the pathogenic impact of viral infection.

microbiology↗

bioRxiv2021

Mechanistic of Rac and Cdc42 synchronization at the cell edge by ARHGAP39-dependent signaling nodules and the impact on protrusion dynamics

Compartmentalization of GTPase regulators into signaling nodules dictates the GTPase pathways selected. Rac and Cdc42 are synchronized at the cell edge for effective protrusion in motile cells but how their activity is coordinated remains elusive. Here, we discovered that ARHGAP39, a Rac and Cdc42 GTPase-activating protein, sequentially interacts with WAVE and mDia2 to control Rac/lamellipodia and Cdc42/filopodia protrusions, respectively. Mechanistically, ARHGAP39 binds to WAVE and, upon phosphorylation by Src kinase, inactivates Rac to promote Cdc42-induced filopodia formation. With our optimized FRET biosensor, we detected active Cdc42 at the filopodia tips that controls filopodia extension. ARHGAP39 is transported to filopodia tips by Myosin-X where it binds mDia2 and inactivates Cdc42 leading to filopodia retraction. Failure in lamellipodia to filopodia switch by defective ARHGAP39 impairs cell invasion and metastasis. Our study reveals that compartmentalization of ARHGAP39 within Rac/Cdc42 signaling nodules orchestrates the synchronization of lamellipodia/filopodia crosstalk and highlights the intricate regulation of leading edge dynamics in migrating cells.

cell biology↗

bioRxiv2019

CTCF controls imprinted gene activity at the mouse Dlk1-Dio3 and Igf2- H19 domains by modulating allele-specific sub-TAD structure

Mammalian genomic imprinting is essential for development and provides a unique paradigm to explore intra-cellular differences in chromatin configuration. Here, we compared chromatin structure of the two conserved imprinted domains controlled by paternal DNA methylation imprints--the Igf2-H19 and the Dlk1-Dio3 domains--and assessed the involvement of the insulator protein CTCF. At both domains, CTCF binds the maternal allele of a differentially-methylated region (DMR), in addition to multiple instances of bi-allelic CTCF binding in their surrounding TAD (Topologically Associating Domain). On the paternal chromosome, bi-allelic CTCF binding alone is sufficient to structure a first level of sub-TAD organization. Maternal-specific CTCF binding at the DMRs adds a further layer of sub-TAD organization, which essentially hijacks the existing paternal sub-TAD organisation. Genome-editing experiments at the Dlk1-Dio3 locus confirm that the maternal sub-TADs are essential during development to maintain the imprinted Dlk1 gene in an inactive state on the maternal chromosome.

molecular biology↗

bioRxiv2018

Acetylation-Dependent Recruitment of the FACT Complex and Its Role in Regulating Pol II Occupancy Genome-Wide in Saccharomyces cerevisiae.

Histone chaperones, chromatin remodelers, and histone modifying complexes play a critical role in alleviating the nucleosomal barrier for DNA-dependent processes. Here, we have examined the role of two highly conserved yeast (Saccharomyces cerevisiae) histone chaperones, FACT and Spt6, in regulating transcription. We show that the H3 tail contributes to the recruitment of FACT to coding sequences in a manner dependent on acetylation. We found that deleting a H3 HAT Gcn5 or mutating lysines on the H3 tail impairs FACT recruitment at ADH1 and ARG1 genes. However, deleting the H4 tail or mutating the H4 lysines failed to dampen FACT occupancy in coding regions. Additionally, we show that FACT-depletion reduces Pol II occupancy in the 5 ends genome-wide. In contrast, Spt6-depletion leads to reduction in Pol II occupancy towards the 3 end, in a manner dependent on the gene-length. Severe transcription and histone eviction defects were also observed in a strain that was impaired for Spt6 recruitment (spt6{Delta}202) and depleted of FACT. Importantly, the severity of the defect strongly correlated with WT Pol II occupancies at these genes, indicating critical roles of Spt6 and Spt16 in promoting high-level transcription. Collectively, our results show that both FACT and Spt6 are important for transcription globally and may participate during different stages of transcription.

genetics↗

bioRxiv2018

Saccharomyces cerevisiae histone Chaperones FACT and Spt6 modulate Pol II and histone occupancy genome-wide

Histone chaperones, chromatin remodelers, and histone modifying complexes play a critical role in alleviating the nucleosomal barrier. Here, we have examined the role of two highly conserved yeast (Saccharomyces cerevisiae) histone chaperones, FACT and Spt6, in regulating transcription and histone occupancy. We show that the H3 tail contributes to the recruitment of FACT to coding sequences in a manner dependent on acetylation. We found that deleting a H3 HAT Gcn5 or mutating lysines on the H3 tail impairs FACT recruitment at ADH1 and ARG1 genes. However, deleting the H4 tail or mutating the H4 lysines failed to dampen FACT occupancy in coding regions. Additionally, we show that FACT-depletion greatly reduces Pol II occupancy in the 5 ends genome-wide. By contrast, Spt6-depletion led to reduction in Pol II occupancy towards the 3 end, in a manner dependent on the gene-length. Severe transcription and histone eviction defects were also observed in a strain that was impaired for Spt6 recruitment (spt6{Delta}202) and depleted of FACT. Importantly, the severity of the defect strongly correlated with WT Pol II occupancies at these genes, indicating critical roles of Spt6 and Spt16 in promoting high-level transcription. Collectively, our study shows cooperation, as well as redundancy between chaperones, FACT and Spt6, in regulating transcription and chromatin in coding regions of transcribed genes.

genetics↗