Biology subjects

Shah, N.

Publications and source records attributed to Shah, N..

At least 19 recordsLinked to original sources

bioRxiv2026

TBX3 is required for the differentiation of a subset of retinal ganglion and cone photoreceptor cells

Retinogenesis is a key step driving the formation of the neurons that later coordinate angiogenesis in the retina. Understanding these complex interactions requires dissecting apart its cellular and molecular drivers. To tease apart the molecular links coupling retinogenesis to vascular development, we utilized conditional knockout mouse models to selectively delete the transcription factor T-box 3 (Tbx3) in 1) astrocytes, 2) postnatal retinal ganglion cells (RGCs), or 3) retinal progenitors. We demonstrate that the hypovascular retina phenotype observed upon Tbx3 loss most likely stems from a disruption of early retinogenesis. Embryonic analyses revealed that TBX3 is required during a critical retinogenic window coinciding with ATOH7 activity. While ATOH7+ progenitors are more abundant in Tbx3 mutants, the expression of key RGC and cone precursor factors (including TBX5, RXRgamma, ISL1/2, POU4F2, SHH, LHX4 and OTX2) is severely diminished, halting proper RGC and cone differentiation. Utilizing birthdating experiments and developmental immunostaining, we establish TBX3 as a novel upstream regulator of RGC and cone photoreceptor specification. Because the degeneration of RGCs is the definitive hallmark of diseases like glaucoma, identifying TBX3 as a driver of early RGC formation positions it as a promising new candidate biomarker for early-stage glaucoma diagnosis and potentially therapeutic targeting.

developmental biology↗

bioRxiv2026

Whole-organ surface mapping using multiview projection reconstruction

While light-sheet microscopy is emerging as a robust method for volumetric imaging with improved axial resolution, its capability regarding two-dimensional, surface-level mapping is often hindered by limitations in data redundancy and reconstruction efficiency stemming from volumetric registration methods. We demonstrate that a multiview imaging approach in an axially-swept, dithered light-sheet microscope paired with computational image reconstruction of view projections is able to address these trade-offs to enable large-scale mapping of surface structural features, leveraging the advantages of multiview light-sheet in scalable field of view, working distance, and near isotropic resolution across the entire imaging depth. To aid in the acquisition and analysis of two-dimensional surface structures, we present a tailored surface mapping workflow and a Fiji plugin for computational reconstruction, promoting robust and comprehensive visualization of surface features of uncleared volumetric samples. Our strategy, termed projection reconstruction for imaging surface morphology (PRISM), integrates axially swept dithered light-sheet microscopy and post-processing software for multiview imaging. The imaging hardware enables near-isotropic resolution across its entire field of view, while the software implementation leverages rigid and affine transformations to align two-dimensional projections of multiview samples. It is designed to work with the BigStitcher pipeline, leveraging its robust algorithm to provide support for two-dimensional image alignment and stitching. We demonstrate the capability of PRISM in studies of lymphatic network mapping in the epicardial layer of intact mouse hearts, as well as surface profiles of FaDu spheroids labeled with antibody-nanodiamond conjugates. This method allows us to quantify cardiac lymphatic branch numbers, diameters, and lengths of a Prox1-tdTomato mouse cardiac model, as well as cluster number and diameters of epidermal growth factor receptor within a FaDu spheroid labeled with a nanodiamond-antibody conjugate, with a significant reduction of post-processing data size. PRISM leverages multiview image projections to promote studies of cardiac lymphatics in mouse models and surface receptor distributions within spheroid models, enabling efficient surface mapping of large, intact, and uncleared biological samples across a variety of scales.

bioengineering↗

bioRxiv2026

Histological assessment of integrated human cortical organoid grafts after controlled cortical impact

Rodent models are a mainstay of traumatic brain injury (TBI) research, including investigations into the pathophysiology and treatment of this condition. However, there are fundamental molecular and cellular differences between rodent and human neurons, as well as other cells of the brain. Brain organoids derived from human pluripotent stem cells recapitulate key features of the human brain and have been used to model a variety of neurological disorders. Here, we developed a novel in vivo model of human TBI based on controlled cortical impact (CCI) injuries of human organoid grafts transplanted into the brains of young adult rats. Cortical organoids derived from human induced pluripotent stem cells (iPSCs) were grown for 50-60 days in vitro before transplantation into rat visual cortex. Injures were performed 2 months later, and histological outcomes were examined at 7 or 30 days after injury. Injury cavities in the integrated grafts were identified at both endpoints with a progression toward larger cavities sizes with time. The injured human tissue exhibited evidence of neuroinflammation with elevated numbers of IBA1+ cells and axonal injury with APP+ cells. There was evidence of increased cell proliferation in the injured grafts acutely after injury that decreased with time. The injured grafts also showed evidence of phosphorylated tau aggregates and accumulation of PNAG, a polysaccharide associated with microbial pathogens. These results support the feasibility of using human organoid grafts in rats as a model of TBI, potentially including the study of long-term neurodegeneration and microbial penetration of the brain after injury.

neuroscience↗

bioRxiv2026

Coral restoration alters reef soundscapes but machine learning and manual analyses suggest different recovery rates

Coral restoration is recognised as a critical tool to mitigate pantropical degradation of reef ecosystems. Robust monitoring of restoration progress is crucial for projects to evaluate their success, improve practice, and share knowledge. However, traditional visual surveys often fail to capture the full impact of coral restoration on reef function. Therefore, we employed Passive Acoustic Monitoring (PAM) to assess whether the soundscape of a coral restoration site in the Seychelles differs from adjacent healthy and degraded reference reefs. We applied two methods of soundscape analysis: manual detection of unidentified fish sounds; and machine learning-based Uniform Manifold Approximation and Projection analysis. Results were approach-specific: the manual approach highlighted similarities in fish calls between the restoration site and the healthy reference reef, while the machine learning approach extracted broader soundscape patterns, clustering the restoration site alongside the degraded reference reef. Although this is a single-site study, these findings suggest that a) coral restoration alters reef soundscapes, though recovery time may be taxon-specific, and b) multiple metrics are needed to bridge single-taxon and broad soundscape scales. This study contributes to the evolving field of soundscape ecology in coral reef ecosystems, highlighting the utility of PAM in monitoring changes to reef function through coral restoration.

ecology↗

bioRxiv2026

Brown and Lesser noddies as epidemiological reservoirs and sentinels of avian influenza virus in the South-western Indian Ocean

Avian influenza virus (AIV) epidemiology is well-documented in temperate regions but remains poorly understood in isolated ecosystems like tropical oceanic islands. On these islands, seabirds nest in dense interspecific colonies where the role of different species as reservoirs and dispersers of AIV may vary greatly. Here, we examine the role of noddies (Anous spp.) as potential reservoirs for low pathogenic AIV and evaluate their potential as sentinel species for highly pathogenic AIV introduction on tropical oceanic islands. We analyzed blood samples from 11 seabird species across eight islands in the southwestern Indian Ocean (2015-2020). Noddies exhibited high, stable seroprevalence (30-45%), comparable to reservoir host species in temperate regions. The detection of two N7-positive noddies, sampled the same year on two distinct islands, provided direct molecular evidence that AIV actively circulates on these island colonies. While most other species showed low exposure, Bridled Terns (Onychoprion anaethetus) had exceptionally high seroprevalence (80%), though their reservoir status requires further investigation due to limited sampling. Given noddies consistent exposure and regional distribution, we recommend prioritizing islands with large noddy populations for AIV surveillance. Continued investigation of viral dynamics within and among islands is now called for to elucidate the ecological drivers of AIV maintenance and transmission.

ecology↗

bioRxiv2026

Cross-Modal Training Using Xenium Spatial Transcriptomics Enables DINO-DETR Based Detection of Vascular Niches in H&E Whole-Slide Images

BackgroundTumor vasculature is a key driver of glioma progression, yet routine quantification depends on subjective histopathologic assessment or resource-intensive ancillary immunohistochemistry. A scalable, objective method for vascular phenotyping from routine histology remains an unmet need. MethodsWe leveraged 10x Genomics Xenium spatial transcriptomics data from a glioblastoma specimen to generate molecularly resolved annotations of GBM-associated endothelial cells and pericytes across 809,041 cells. These annotations were transferred to matched H&E-stained sections to train a DINO-DETR-based object detection model using a binary classification scheme (vascular vs. other). The model was validated on four independent Xenium patient slides and applied to a retrospective cohort of 119 diffuse gliomas spanning WHO grades 2-4 (oligodendroglioma, astrocytoma, and glioblastoma) with linked survival data. ResultsBinary vascular cell detection achieved a precision of 0.78, a recall of 0.63, and an F1 score of 0.70, with an overall accuracy of 98.6%. Orthogonal spatial validation confirmed that predicted vascular cells were preferentially localized within annotated blood vessel regions. In subtype-stratified survival analysis, high AI-derived vascular cell proportion was significantly associated with worse overall survival in astrocytoma patients (log-rank p < 0.019). ConclusionCross-modal AI training using spatial transcriptomics enables scalable, molecularly informed vascular quantification directly from routine H&E slides. Within the astrocytoma subtype, where tumor grade is most heterogeneous and vascular phenotype most variable, objective vascular quantification provides independent prognostic information demonstrating the potential of spatially supervised deep learning to extract clinically meaningful microenvironmental signals from universally available histologic material.

pathology↗

bioRxiv2025

scDNA: Single Cell DNA analysis software toolkit for subclonality discovery and assessment.

Advances in single cell multi-omics technologies have allowed for investigation into genotype-immunophenotype relationships and dynamic clonal changes in cancer patient samples. These technologies provide rich insights into genetic profiling, drivers of disease progression, and subclonal dynamics. Increased adoption and throughput of these technologies, necessitate accessible computational tools for processing and analysis. Toward developing easy to use computational tools, we introduce the Single Cell DNA (scDNA) package that allows for rapid analysis of single-cell molecular profiling. Our platform aims to provide a diagnostic summary tool for sample quality control, representative subclonal architecture within a sample, demultiplexing functionality for multi-sample processing, copy number variation, and mutation trajectory analysis to identify order of subclonal mutation acquisition. We showcase a series of vignettes on hematopoiesis datasets for each aim that reflects recently deployed uses of scDNA. Additionally, we show scDNA provides a modular, user-friendly framework that readily feeds into other standard software pipelines.

genomics↗

bioRxiv2025

Autofluorescence imaging reveals the impact of cryopreservation on T cell metabolism and activation response

Cryopreservation or the process of freezing cells is a cornerstone of most cell therapy protocols. Optimization of cryopreservation protocols and cryoprotectant agents to improve cell viability and functionality is under further investigation. However, the impact of cryopreservation on cellular metabolism and function immediately post-thaw is not fully understood. Here, we used label-free, non-invasive optical metabolic imaging (OMI) of NAD(P)H and FAD to characterize the activation response of frozen T cells from healthy donors and lymphoma patients post-thaw. Using OMI, we identified significant metabolic shift, along with delayed and diminished activation response in healthy donor T cells throughout the first 4.5 hours upon thawing. In cryopreserved peripheral T cells from lymphoma patients in our bispecific CD19/CD20 CAR T therapy clinical trial, OMI could identify early metabolic stress and allowed gating of metabolically-fit cells associated with post-thaw viability. Notably, in our pilot study, only metabolically-fit T cells from complete responders exhibited metabolic responses to activating stimuli within the first 4.5 hours post-thaw. Overall, our findings suggest that 4-5 hours post-thaw is a critical time window to assess the impact of cryopreservation and thawing, and support the potential of OMI to optimize cryopreservation protocols and evaluate patient T cell quality for cell therapy.

immunology↗

bioRxiv2025

Red Blood Cell Transfusion is a Non-Canonical Immune Stimulus Characterized by the Suboptimal Induction of CD4+ T Cell Help

Red blood cell (RBC) alloimmunization to non-ABO antigens is a major clinical complication for chronically transfused patients. When exposed to transfused RBCs carrying foreign antigens, some patients generate IgG antibodies that target these antigens, creating potential barriers to future transfusions. Interestingly, other patients produce only IgM antibodies against the same non-ABO antigens, which generally have fewer clinical consequences. Despite the stark differences in their impact, the factors regulating IgM versus IgG production in response to transfused RBCs remain poorly understood. This study explores the balance between IgM and IgG production following transfusion, comparing it to the well-characterized antibody response induced by vaccination in mouse models. By directly assessing antibody levels following RBC transfusion versus Alum-adjuvanted vaccination, we demonstrate that transfusion of RBCs expressing a model antigen is a relatively weak inducer of IgG class switching. Additionally, loss-of-function experiments using CD40L blockade and CD4 depletion confirmed that T cell help is essential for class switching after transfusion but has no effect on IgM production. Most notably, providing supra-physiological levels of T cell help enhanced class switching in a dose-dependent manner after transfusion, whereas vaccination-induced class switching remained unaffected. These findings support a model in which the limited IgG class switching following transfusion stems from suboptimal T cell help compared to vaccination. Furthermore, they suggest that transfusion activates T cells through a non-canonical pathway, distinct from the mechanisms driving immune responses to standard Alum vaccination.

immunology↗

bioRxiv2025

A scalable framework for current steering at single-neuron resolution

Electrical stimulation at cellular resolution to restore the function of neural circuits is limited by the density of available electrode arrays. Although current steering with multi-electrode stimulation can be used to target cells between electrodes, it has not been proven for systematically targeting individual cells. We develop a framework for cellular-resolution current steering, leveraging the biophysics of electrically-evoked spike generation, and test its efficacy in isolated macaque and human retina. Currents were passed through three electrodes simultaneously using large-scale high-density microelectrode arrays, directly evoking single spikes in retinal ganglion cells. The currents combined either linearly or nonlinearly to drive spiking, depending on the geometry of the electrodes relative to the cell. These findings were captured by a biophysical model and by a simpler parametric model in which spikes can initiate at several sites on the cell membrane, and were leveraged to efficiently identify multi-electrode stimulation patterns that optimized cellular selectivity.

bioengineering↗

bioRxiv2025

Transfusion of allogenic murine HOD red blood cells preferentially induces low-affinity, short-lived IgG antibodies that are germinal center independent

Transfusion-induced anti-red blood cell (RBC) alloantibodies pose a significant risk to patients who require chronic transfusions. Anti-RBC alloantibodies can be remarkably short-lived (i.e. evanescent), leading to clinically relevant alloantibodies that are not detected in later pre-transfusion antibody screens. Subsequent transfusion of alloantigen-positive RBCs stimulates a rapid memory antibody response that may induce a delayed hemolytic transfusion reaction (DHTR), causing morbidity and occasional mortality in chronically transfused patients. It is unclear why transfusions favor evanescent antibody responses over long-lived antibodies typically observed upon infections and vaccinations. We therefore turned to the HOD mouse model of RBC alloimmunization to elucidate regulators of antibody persistence in response to allogenic transfusions. By following antibody responses over time in transfused mice, we found that HOD-specific alloantibodies rapidly decay within three months while vaccination-induced antibodies remain constant. Thus, the HOD model recapitulates RBC antibody evanescence. The rapid antibody evanescence suggests that transfusion is a poor inducer of germinal centers (GCs), specialized immunological structures where B cells differentiate into germinal center B (GC B) cells and undergo iterative rounds of affinity maturation, ultimately differentiating into long-lived plasma cells that can produce antibodies for decades. Consistent with this hypothesis, we failed to observe an increase in GC B cell formation in response to transfusion, and the majority of anti-RBC alloantibodies were low affinity when compared to vaccination. To formally test the functional requirement for GCs in anti-RBC alloantibody production, we employed two orthogonal approaches to disrupt GC formation: i) day 4 CD40L blockade and ii) genetic disruption of the GC-transcription factor BCL6 selectively in B cells. Both approaches fully blocked GC formation, yet anti-RBC alloantibody production was unchanged. Collectively, our data demonstrate that anti-HOD RBC alloantibodies are GC-independent, low affinity and short-lived. The GC-independence of HOD RBC IgG responses has important implications for understanding the cellular and molecular pathways that regulate the humoral immune response to transfused RBCs, potentially explaining anti-RBC alloantibody evanescence patterns in patients.

immunology↗

bioRxiv2024

The two crystal structures of unloaded and cargo-loaded Vacuolar Sorting Receptor 1 lumenal domain (VSR1-Nt) reveal two cargo binding sites and a mechanism of transport.

Vacuolar sorting receptors (VSRs) are type I membrane proteins crucial for seed germination and plant development. While VSR trafficking has been extensively studied, the mechanism of cargo binding and release by the N-terminal lumenal region is less understood. We have elucidated the crystal structures of unloaded and cargo-loaded forms of the lumenal region of VSR1 containing the protease-associated domain, the Central domain and epidermal growth factor-like repeats. Calcium coordination induces remodeling of the linkers between domains, triggering large conformational changes that expose two binding sites, one across the PA and Central domain and a second site in the Central domain. Our findings provide a mechanistic model for cargo binding in a calcium rich environment, where VSR is locked in a conformation exposing the cargo binding sites, while cargo release is favoured by lower calcium concentrations and trimer formation. These results advance our current knowledge on VSRs and will inform future studies on vacuolar trafficking and cargo binding/release.

plant biology↗

bioRxiv2024

Single-Mitochondrion Sequencing Uncovers Distinct Mutational Patterns and Heteroplasmy Landscape in Mouse Astrocytes and Neurons.

BackgroundMitochondrial (mt) heteroplasmy can cause adverse biological consequences when deleterious mtDNA mutations accumulate disrupting normal mt-driven processes and cellular functions. To investigate the heteroplasmy of such mtDNA changes we developed a moderate throughput mt isolation procedure to quantify the mt single-nucleotide variant (SNV) landscape in individual mouse neurons and astrocytes In this study we amplified mt-genomes from 1,645 single mitochondria (mts) isolated from mouse single astrocytes and neurons to 1. determine the distribution and proportion of mt-SNVs as well as mutation pattern in specific target regions across the mt-genome, 2. assess differences in mtDNA SNVs between neurons and astrocytes, and 3. Study cosegregation of variants in the mouse mtDNA. Results1. The data show that specific sites of the mt-genome are permissive to SNV presentation while others appear to be under stringent purifying selection. Nested hierarchical analysis at the levels of mitochondrion, cell, and mouse reveals distinct patterns of inter- and intra-cellular variation for mt-SNVs at different sites. 2. Further, differences in the SNV incidence were observed between mouse neurons and astrocytes for two mt-SNV 9027:G>A and 9419:C>T showing variation in the mutational propensity between these cell types. Purifying selection was observed in neurons as shown by the Ka/Ks statistic, suggesting that neurons are under stronger evolutionary constraint as compared to astrocytes. 3. Intriguingly, these data show strong linkage between the SNV sites at nucleotide positions 9027 and 9461. f ConclusionThis study suggests that segregation as well as clonal expansion of mt-SNVs is specific to individual genomic loci, which is important foundational data in understanding of heteroplasmy and disease thresholds for mutation of pathogenic variants.

neuroscience↗

bioRxiv2024

Extracellular Matrix-Induced Genes May Reduce Response to Rapamycin in LAM

RationaleLymphangioleiomyomatosis (LAM) is a rare cystic lung disease driven by nodules containing TSC2-/- LAM cells and recruited LAM associated fibroblasts (LAFs). Although rapamycin reduces lung function loss, some patients continue to decline meaning additional therapies are needed. ObjectivesTo investigate how the LAM nodule environment affects LAM cell proliferation and the response to rapamycin. MethodsChanges in advanced LAM were identified using shotgun proteomics and immunohistochemistry in tissue from carefully phenotyped patients. Genes potentially associated with rapamycin insensitivity of cells grown on LAF-derived extracellular matrix were identified by RNA sequencing and validated using repurposed pharmacologic inhibitors. Main ResultsMore advanced disease was associated with increasing nodules adjacent to lung cysts and greater decline in forced expiratory volume in 1 sec (FEV1) when treated with rapamycin (p=0.005). In late-stage LAM, proteomics identified upregulation of pathways associated with accumulation of activated fibroblasts, including extracellular matrix deposition, glucose metabolism and the actin cytoskeleton. Picrosirius red staining and immunohistochemistry confirmed deposition of extracellular matrix within LAM nodules. The growth of TSC2-/- model LAM cells was increased on LAF-derived extracellular matrix (LAF ECM), and incompletely supressed by rapamycin (p<0.0001). RNA sequencing of cells grown on LAF ECM identified upregulation of pathways driving cell cycle control, transcription and metabolism in cells. Tractable, pro-proliferative, rapamycin insensitive genes included CDK7, GAS6 and PLAU. Repurposed inhibitors of these pathways inhibited LAM cell proliferation and enhanced the anti-proliferative effect of rapamycin. ConclusionsExtracellular matrix deposited by LAM associated fibroblasts upregulates expression of genes which potentially blunt the response to rapamycin, but offer additional therapeutic opportunities for patients with established LAM.

cell biology↗

bioRxiv2024

Comparative transcriptome reprogramming in oak galls containing asexual or sexual generations of gall wasps

O_LIOak gall wasps have evolved strategies to manipulate the developmental pathways of their host to induce gall formation. This provides shelter and nutrients for the developing larva. Galls are entirely host tissue; however, the initiation, development, and physical appearance are controlled by the inducer. The underlying molecular mechanisms of gall formation, by which one or a small number of cells are reprogrammed and commit to a novel developmental path, are poorly understood. In this study, we sought a deeper insight into the molecular underpinnings of this process. C_LIO_LIOak gall wasps have two generations each year, one sexual, and one asexual. Galls formed by these two generations exhibit a markedly different appearance. We sequenced transcriptomes of both the asexual and sexual generations of Neuroterus quercusbaccarum and Neuroterus numismalis. We then deployed Nanopore sequencing to generate long-read sequences to test the hypothesis that gall wasps introduce DNA insertions to determine gall development. C_LIO_LIWe detected potential genome rearrangements, but did not uncover any non-host DNA insertions. Transcriptome analysis revealed that the transcriptomes of the sexual generations of distinct species of wasp are more similar than inter-generational comparisons from the same species of wasp. C_LIO_LIOur results highlight the intricate interplay between the host leaves and gall development, suggesting that season and requirements of the gall structure play a larger role than species in controlling gall development and structure. C_LI Summary StatementOak gall wasps, Neuroterus quercusbaccarum and Neuroterus numismalis, induce species-specific galls on Quercus robur leaves. We demonstrate that the sexual generation of distinct species of wasps induce more similar changes in the host than different generation galls from the same species.

plant biology↗

bioRxiv2023

Reactive Oxygen Species Generation by Reverse Electron Transfer at Mitochondrial Complex I Under Simulated Early Reperfusion Conditions.

Ischemic tissues accumulate succinate, which is rapidly oxidized upon reperfusion, driving a burst of mitochondrial reactive oxygen species (ROS) generation that triggers cell death. In isolated mitochondria with succinate as the sole metabolic substrate under non-phosphorylating conditions, 90% of ROS generation is from reverse electron transfer (RET) at the Q site of respiratory complex I (Cx-I). Together, these observations suggest Cx-I RET is the source of pathologic ROS in reperfusion injury. However, numerous factors present in early reperfusion may impact Cx-I RET, including: (i) High [NADH]; (ii) High [lactate]; (iii) Mildly acidic pH; (iv) Defined ATP/ADP ratios; (v) Presence of the nucleosides adenosine and inosine; and (vi) Defined free [Ca2+]. Herein, experiments with mouse cardiac mitochondria revealed that under simulated early reperfusion conditions including these factors, overall mitochondrial ROS generation was only 56% of that seen with succinate alone, and only 52% of this ROS was assignable to Cx-I RET. The residual non-RET ROS could be partially assigned to complex III (Cx-III) with the remainder likely originating from other ROS sources upstream of the Cx-I Q site. Together, these data suggest the relative contribution of Cx-I RET ROS to reperfusion injury may be overestimated, and other ROS sources may contribute a significant fraction of ROS in early reperfusion.

biochemistry↗

bioRxiv2023

De novo distillation of thermodynamic affinity from deep learning regulatory sequence models of in vivo protein-DNA binding

Transcription factors (TF) are proteins that bind DNA in a sequence-specific manner to regulate gene transcription. Despite their unique intrinsic sequence preferences, in vivo genomic occupancy profiles of TFs differ across cellular contexts. Hence, deciphering the sequence determinants of TF binding, both intrinsic and context-specific, is essential to understand gene regulation and the impact of regulatory, non-coding genetic variation. Biophysical models trained on in vitro TF binding assays can estimate intrinsic affinity landscapes and predict occupancy based on TF concentration and affinity. However, these models cannot adequately explain context-specific, in vivo binding profiles. Conversely, deep learning models, trained on in vivo TF binding assays, effectively predict and explain genomic occupancy profiles as a function of complex regulatory sequence syntax, albeit without a clear biophysical interpretation. To reconcile these complementary models of in vitro and in vivo TF binding, we developed Affinity Distillation (AD), a method that extracts thermodynamic affinities de-novo from deep learning models of TF chromatin immunoprecipitation (ChIP) experiments by marginalizing away the influence of genomic sequence context. Applied to neural networks modeling diverse classes of yeast and mammalian TFs, AD predicts energetic impacts of sequence variation within and surrounding motifs on TF binding as measured by diverse in vitro assays with superior dynamic range and accuracy compared to motif-based methods. Furthermore, AD can accurately discern affinities of TF paralogs. Our results highlight thermodynamic affinity as a key determinant of in vivo binding, suggest that deep learning models of in vivo binding implicitly learn high-resolution affinity landscapes, and show that these affinities can be successfully distilled using AD. This new biophysical interpretation of deep learning models enables high-throughput in silico experiments to explore the influence of sequence context and variation on both intrinsic affinity and in vivo occupancy.

genomics↗

bioRxiv2022

Golgi localized Arl15 regulates cargo transport, cell adhesion and motility

Arf-like GTPases (Arls) regulate membrane trafficking and cytoskeletal organization. Genetic studies predicted a role for Arl15 in type-2 diabetes, insulin resistance, adiposity, and rheumatoid arthritis. Recent studies indicate a possible role for Arl15 in multiple physiological processes, including magnesium homeostasis. However, the molecular function of Arl15 is poorly defined. We evaluated the role of Arl15 in vesicular transport using techniques to quantify cargo trafficking, to mechanobiology. Fluorescence microscopy of stably expressing Arl15-GFP HeLa cells showed its localization to the Golgi and cell surface, including filopodia, and a cohort to recycling endosomes. The dissociation of Golgi, using small molecular inhibitors or the expression of Arf1 dominant-negative mutant, completely mislocalized Arl15 to the cytosol. Interestingly, site-directed mutagenesis analysis identified a novel V80A mutation in the GTP-binding domain that turns Arl15 into a dominant-negative form with reduced number of filopodia. Depletion of Arl15 in HeLa cells caused mislocalization of cargo, such as caveolin-2 and STX6, from the Golgi. Arl15 knockdown cells displayed reduced filopodial number, altered focal adhesion kinase organization, and enhanced soluble and receptor-mediated cargo uptake without affecting the TfR recycling. Arl15 knockdown decreased cell migration and enhanced cell spreading and adhesion strength. Traction force microscopy experiments revealed that Arl15 depleted cells exert higher tractions and generate multiple focal adhesion points during the initial phase of cell adhesion as compared to control cells. Collectively, these studies demonstrated a functional role for Arl15 in the Golgi, which includes regulating cargo transport to organize membrane domains at the cell surface. Key pointsO_LIArl15 primarily localizes to Golgi and plasma membrane, including filopodia C_LIO_LIMembrane localization of Arl15 is dependent on Golgi integrity or Arf1 activation C_LIO_LIArl15 knockdown mislocalizes STX6-dependent Golgi localized cargo required for cell surface organization and reduces the filopodial number C_LIO_LIArl15 is involved in cell spreading, adhesion, and migration C_LI

cell biology↗