Biology subjects

Johnson, K. R.

Publications and source records attributed to Johnson, K. R..

18 recordsLinked to original sources

bioRxiv2026

Solving Paleobotany's 200-year problem with fluorescence and artificial intelligence

Paleobotany has a 200-year problem: leaf fossils are often too poorly preserved to make proper taxonomic identification. Though they are the most abundant, and well-studied macroscopic fossil plant organ, many fossil leaves-- including specimens held in museum and university collections-- appear to lack the detailed features needed to take crucial next steps, like placement into phylogenetic context, generation of ecological interpretations, or reconstruction of paleoclimate. However, we have discovered that under UV and laser light [~]50% of leaves across a diverse survey of fossil deposits fluoresce in a way that reveals spectacularly detailed leaf venation and gross morphology that is invisible under white light. These 'Fossil Ciphers' bring new life into each step of the paleobotanical method, unlocking previously unattainable taxonomic classifications, quantitative morphometrics, and promising new avenues for the incorporation of AI into fossil leaf analyses. With the aid of fluorescence techniques, we can help solve paleobotany's 200-year problem.

paleontology↗

bioRxiv2026

AAV-Delivered RNAi Targeting Mutant LDB3 Prevents and Reverses Myofibrillar Myopathy through Mechanosignaling Restoration

The autosomal dominant p.Ala165Val mutation in LIM Domain Binding Protein 3 (LDB3) causes myofibrillar myopathy marked by Z-disc disruption, accumulation of filamin-C (FLNc) and chaperone proteins, and progressive muscle weakness. We previously showed that this mutation interferes with the LDB3-protein kinase C alpha (PKC)-FLNc mechanosensing axis and impairs chaperone-assisted selective autophagy (CASA), establishing a gain-of-function mechanism. In this study, we examined whether mutant allele-specific knockdown could reverse the disease or mitigate disease progression in-vivo. A single intramuscular-injection of an AAV9-delivered microRNA-based shRNA produced substantial knockdown of mutant Ldb3 transcripts and protein in Ldb3Ala165Val/+ knock-in mice treated either before or after the onset of pathology. Treatment after disease onset reduced filamin-C and CASA protein aggregates and improved muscle strength, whereas early intervention prevented development of molecular and histological features of myopathy. Phosphoproteomic profiling further showed broad remodeling of dysregulated phosphorylation networks, including restoration of PKC-responsive sites and normalization of altered sarcomeric and cytoskeletal signaling observed in Ldb3Ala165Val/+ mice. These findings identify disruption of the LDB3-PKC-FLNc mechanosensing pathway as a central disease driver and suggest that restoring this signaling axis may complement mutant allelespecific RNA interference (RNAi). Overall, our results support RNAi as a promising therapeutic strategy for dominant LDB3-related myofibrillar myopathy.

neuroscience↗

bioRxiv2026

Proximity-Informed Graph Learning Defines Spatial Protein Communities for Tumor-Associated Proximity Antigen Discovery

The spatial organization of membrane proteins is an underexplored dimension of cell-surface biology. Spatial proximity shapes cellular function and therapeutic targetability, yet efforts to identify tumor-associated antigens (TAAs) have largely focused on expression alone. Here, we developed an industrialized surface-protein proximity mapping workflow to interrogate TAAs within their membrane microenvironments. In the process, we generated 248 proximity maps across 12 receptor tyrosine kinases (RTKs) and 28 tumor cell systems. This proximity atlas enabled two advances: first, MetaMap, a correlation-based analytical framework that defines spatial protein communities and infers non-targeted proximal proteins from reproducible proximity signatures; and second, tumor-associated proximity antigens (TAPAs), a conceptual class of co-targets defined by disease-specific spatial proximity to TAAs rather than expression alone. Applying these proximity-derived relationships within a multimodal prioritization framework, we identified and validated an EGFRxCDCP1 TAA-TAPA pair that enhanced tumor cell killing across therapeutic modalities. By integrating spatial organization with multimodal data, this work expands the design space for precision-guided therapeutic strategies.

cancer biology↗

bioRxiv2025

Maintenance of chronic neuroinflammation in multiple sclerosis via interferon signaling and CD8 T cell-mediated cytotoxicity

Chronic neuroinflammation and neurodegeneration are critical but unresolved drivers of disability accumulation in progressive multiple sclerosis (MS). Chronic active white matter lesions (CAL), identifiable radiologically as paramagnetic rim lesions (PRL), indicate progression-relevant chronic neuroinflammation. Using single-cell transcriptomics (scRNAseq) and T-cell receptor sequencing (scTCR-seq), we profiled cerebrospinal fluid (CSF) and blood immune cells of 34 radiologically characterized adults with MS (17 untreated, 6 treated with B-cell-depletion) and 5 healthy controls. Coupled with proteomics, we found PRL-associated enrichment of interferon (IFN) signaling and upregulation of TCR signaling in CSF and blood. This was accompanied by clonal expansion of CD8+ T effector memory (TEM) cells, with the highly expanded clonal cells exhibiting T helper type 1 (TH1) and cytotoxic profiles. Validating the cytotoxic immune profile in blood using flow cytometry, we identified a cellular correlate of PRL exhibiting features of CD8+ TEMRA cells. Despite B-cell depletion, PRL-associated neuroinflammation, driven by myeloid activation and CD8+ T-cell cytotoxicity, persisted. Serum and CSF proteomic networks showed PRL-pertinent signatures, including networks unaffected by B-cell depletion. Using in silico perturbation, we nominated therapeutic targets, including MYD88, TNF, MYC, TYK2, JAK2, and BTK, for alleviating chronic neuroinflammation in MS. Our findings highlight mechanisms of chronic neuroinflammation in MS and point to potential biomarkers for monitoring disease progression.

immunology↗

bioRxiv2024

Diversification of Dentate Gyrus Granule Cell Subtypes is Regulated by Neuregulin1 Nuclear Back Signaling.

Neuronal heterogeneity is a defining feature of the developing mammalian brain, but the mechanisms regulating the diversification of closely related cell types remain elusive. In this study, we investigated the heterogeneity of dentate gyrus (DG) granule cells (GCs) and the influence of a psychosis associated V321L mutation in Neuregulin1 (Nrg1) on GC subtype composition. Using morpho-electric characterization, single-nucleus gene expression, and chromatin accessibility profiling, we identified distinct morphological and molecular features of typical GCs and a rare subtype known as semilunar granule cells (SGCs). The V321L mutation disrupts Nrg1 nuclear back-signaling, resulting in an overabundance of SGC-like cells. We discovered pseudotime gene expression trajectories suggesting the potential for GC-to-SGC transitions, supported by the accessibility of SGC-specific genes in other GCs. Intriguingly, we found an increase in SGC-marker expression over the adolescence to adulthood transition window in wild-type mice, coinciding with a decline in Nrg1 nuclear back-signaling capacity. This suggests that intact Nrg1 signaling suppresses SGC-like fate acquisition, and that its natural downregulation may underlie the emergence of SGC-like cells during postnatal development. Similarly, a pathological block of nuclear back signaling by the V321L mutation in Nrg1, may result in acquisition of the SGC-like fate due to loss of the repressive mechanisms maintained by intact nuclear back signaling. Our findings reveal a novel role for Nrg1 in maintaining DG cell-type composition and suggest that disrupted subtype regulation may contribute to disease-associated changes in DG GC morphology and function. Understanding these mechanisms provides new insights into mechanisms of cell-type diversity and its potential role in psychiatric pathology.

neuroscience↗

bioRxiv2024

Maintenance of neuronal TDP-43 expression requires axonal lysosome transport

TDP-43 mislocalization and pathology occurs across a range of neurodegenerative diseases, but the pathways that modulate TDP-43 in neurons are not well understood. We generated a Halo-TDP-43 knock-in iPSC line and performed a genome-wide CRISPR interference FACS-based screen to identify modifiers of TDP-43 levels in neurons. A meta-analysis of our screen and publicly available screens identified both specific hits and pathways present across multiple screens, the latter likely responsible for generic protein level maintenance. We identified BORC, a complex required for anterograde lysosome transport, as a specific modifier of TDP-43 protein, but not mRNA, levels in neurons. BORC loss led to longer half-life of TDP-43 and other proteins, suggesting lysosome location is required for proper protein turnover. As such, lysosome location and function are crucial for maintaining TDP-43 protein levels in neurons.

neuroscience↗

bioRxiv2024

Emergence and interstate spread of highly pathogenic avian influenza A(H5N1) in dairy cattle

Highly pathogenic avian influenza (HPAI) viruses cross species barriers and have the potential to cause pandemics. In North America, HPAI A(H5N1) viruses related to the goose/Guangdong 2.3.4.4b hemagglutinin phylogenetic clade have infected wild birds, poultry, and mammals. Our genomic analysis and epidemiological investigation showed that a reassortment event in wild bird populations preceded a single wild bird-to-cattle transmission episode. The movement of asymptomatic cattle has likely played a role in the spread of HPAI within the United States dairy herd. Some molecular markers in virus populations were detected at low frequency that may lead to changes in transmission efficiency and phenotype after evolution in dairy cattle. Continued transmission of H5N1 HPAI within dairy cattle increases the risk for infection and subsequent spread of the virus to human populations.

microbiology↗

bioRxiv2024

Triglyceride metabolism controls inflammation and APOE4-associated disease states in microglia

Microglia modulate their cell state in response to various stimuli. Changes to cellular lipids often accompany shifts in microglial cell state, but the functional significance of these metabolic changes remains poorly understood. In human induced pluripotent stem cell-derived microglia, we observed that both extrinsic activation (by lipopolysaccharide treatment) and intrinsic triggers (the Alzheimers disease-associated APOE4 genotype) result in accumulation of triglyceride-rich lipid droplets. We demonstrate that lipid droplet accumulation is not simply concomitant with changes in cell state but rather necessary for microglial activation. We discovered that both triglyceride biosynthesis and catabolism are needed for the transcription and secretion of proinflammatory cytokines and chemokines in response to extrinsic stimuli. Additionally, we reveal that triglyceride biosynthesis and catabolism are necessary for the activation-associated phagocytosis of multiple substrates including the disease-associated amyloid-beta peptide. In microglia harboring the Alzheimers disease risk APOE4 genotype, triglyceride-rich lipid droplets accumulate even in the absence of any external stimuli. Inhibiting triglyceride biosynthesis in APOE4 microglia not only modifies the transcription of immune response genes but also attenuates disease-associated transcriptional states. This work establishes that triglyceride metabolism is necessary for microglia to respond to extrinsic activation. In APOE4 microglia, this metabolic process modulates both immune signaling and a disease-associated transcriptional state. Importantly, our work identifies metabolic pathways that can be used to tune microglial immunometabolism in APOE4-associated disease.

cell biology↗

bioRxiv2024

Metabolic Quadrivalency in RSeT Human Embryonic Stem Cells

One of the most important properties of human embryonic stem cells (hESCs) is related to their pluripotent states. In our recent study, we identified a previously unrecognized pluripotent state induced by RSeT medium. This state makes primed hESCs resistant to conversion to naive pluripotent state. In this study, we have further characterized the metabolic features in these RSeT hESCs, including metabolic gene expression, metabolomic analysis, and various functional assays. The commonly reported metabolic modes include glycolysis or both glycolysis and oxidative phosphorylation (i.e., metabolic bivalency) in pluripotent stem cells. However, besides the presence of metabolic bivalency, RSeT hESCs exhibited a unique metabolome with additional fatty acid oxidation and imbalanced nucleotide metabolism. This metabolic quadrivalency is linked to hESC growth independent of oxygen tension and restricted capacity for naive reprogramming in these cells. Thus, this study provides new insights into pluripotent state transitions and metabolic stress-associated hPSC growth in vitro.

biochemistry↗

bioRxiv2023

Nuclear Localization of Argonaute is affected by Cell Density and May Relieve Repression by microRNAs

Argonaute protein is associated with post-transcriptional control of cytoplasmic gene expression through miRNA-induced silencing complexes (miRISC). Specific cellular and environmental conditions can trigger AGO protein to accumulate in the nucleus. Localization of AGO is central to understanding miRNA action, yet the consequences of AGO being in the nucleus are undefined. We show nuclear enrichment of AGO2 in HCT116 cells grown in two-dimensional culture to high density, HCT116 cells grown in three-dimensional tumor spheroid culture, and human colon tumors. The shift in localization of AGO2 from cytoplasm to nucleus de-represses cytoplasmic AGO2-eCLIP targets that were candidates for canonical regulation by miRISC. Constitutive nuclear localization of AGO2 using an engineered nuclear localization signal increases cell migration. Critical RNAi factors also affect the localization of AGO2. Knocking out an enzyme essential for miRNA biogenesis, DROSHA, depletes mature miRNAs and restricts AGO2 localization to the cytoplasm, while knocking out the miRISC scaffolding protein, TNRC6, results in nuclear localization of AGO2. These data suggest that AGO2 localization and miRNA activity can be regulated depending on environmental conditions, expression of mature miRNAs, and expression of miRISC cofactors. Localization and expression of core miRISC protein machinery should be considered when investigating the roles of miRNAs.

biochemistry↗

bioRxiv2023

Multi-modal Proteomic Characterization of Lysosomal Function and Proteostasis in Progranulin-Deficient Neurons

Progranulin (PGRN) is a lysosomal protein implicated in various neurodegenerative diseases. Over 70 mutations discovered in the GRN gene all result in reduced expression of PGRN protein. However, the detailed molecular function of PGRN within lysosomes and the impact of PGRN deficiency on lysosomal biology remain unclear. Here we leveraged multifaceted proteomic techniques to comprehensively characterize how PGRN deficiency changes the molecular and functional landscape of neuronal lysosomes. Using lysosome proximity labeling and immuno-purification of intact lysosomes, we characterized lysosome compositions and interactomes in both human induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (i3Neurons) and mouse brains. Using dynamic stable isotope labeling by amino acids in cell culture (dSILAC) proteomics, we measured global protein half-lives in i3Neurons for the first time and characterized the impact of progranulin deficiency on neuronal proteostasis. Together, this study indicated that PGRN loss impairs the lysosomes degradative capacity with increased levels of v-ATPase subunits on the lysosome membrane, increased catabolic enzymes within the lysosome, elevated lysosomal pH, and pronounced alterations in neuron protein turnover. Collectively, these results suggested PGRN as a critical regulator of lysosomal pH and degradative capacity, which in turn influences global proteostasis in neurons. The multi-modal techniques developed here also provided useful data resources and tools to study the highly dynamic lysosome biology in neurons.

neuroscience↗

bioRxiv2022

Psychosis-associated Valine to Leucine mutation in Neuregulin1 disrupts expression of a schizophrenia susceptibility gene network within the hippocampus and alters adult hippocampal neurogenesis.

Neuregulin1 (Nrg1) signaling is critical for aspects of neuronal development and function from fate specification to synaptic plasticity. Type III Nrg1 is a synaptic protein which engages in bi-directional signaling with its receptor ErbB4. Forward signaling engages ErbB4 phosphorylation, whereas back signaling engages two known mechanisms: 1. local axonal PI3K-AKT signaling, and 2. cleavage by gamma secretase resulting in cytosolic release of the intracellular domain (ICD), which can traffic to the nucleus (Bao, Wolpowitz et al. 2003, Hancock, Canetta et al. 2008). To dissect the contribution of these alternate signaling strategies to neuronal development we generated a transgenic mouse with a missense mutation (V321L) in the Nrg1 transmembrane domain that disrupts nuclear back signaling with minimal effects on forward signaling or local back-signaling and was previously found to be associated with psychosis (Walss-Bass, Liu et al. 2006). We combined RNA sequencing, retroviral fate mapping of neural stem cells, behavioral analyses, and various network analyses of transcriptomic data to investigate the effect of disrupting Nrg1 nuclear back-signaling in the dentate gyrus (DG) of male and female mice. The V321L mutation impairs nuclear translocation of the Nrg1 ICD and alters gene expression in the DG. V321L mice show reduced stem cell proliferation, altered cell cycle dynamics, fate specification defects, and dendritic dysmorphogenesis. Orthologs of known schizophrenia (SCZ)-susceptibility genes were dysregulated in the V321L DG. These genes coordinated a larger network with other dysregulated genes. WGCNA and protein-interaction network analyses revealed striking similarity between DG transcriptomes of V321L mouse and humans with schizophrenia. SIGNIFICANCE STATEMENTSynaptic contact is predicted to be a regulator of the generation of nuclear signaling by Nrg1. Here we show that a schizophrenia-associated mutation in Nrg1 disrupts its ability to communicate extracellular signals to the neuronal genome which results in altered expression of a gene network enriched for orthologs of schizophrenia-susceptibility genes. The striking overlap in functional and molecular alterations between a single rare homozygous missense mutation (V321L) and schizophrenia patient data (complex polygenic and environmental burden) underscores potential convergence of rare and common variants on the same cellular and molecular phenotypes. Furthermore, our data indicate that the evolutionarily conserved gene networks that form the basis for this risk are necessary for coordinating neurodevelopmental events in the DG.

neuroscience↗

bioRxiv2021

KSR1-and ERK-dependent Translational Regulation of the Epithelial-to-Mesenchymal Transition

The epithelial-to-mesenchymal transition (EMT) is considered a transcriptional process that induces a switch in cells from a polarized state to a migratory phenotype. Here we show that KSR1 and ERK promote EMT through the preferential translation of Epithelial-Stromal Interaction 1 (EPSTI1), which is required to induce the switch from E-to N-cadherin and coordinate migratory and invasive behavior. EPSTI1 is overexpressed in human colorectal cancer (CRC) cells. Disruption of KSR1 or EPSTI1 significantly impairs cell migration and invasion in vitro, and reverses EMT, in part, by decreasing the expression of N-cadherin and the transcriptional repressors of E-cadherin expression, ZEB1 and Slug. In CRC cells lacking KSR1, ectopic EPSTI1 expression restored the E-to N-cadherin switch, migration, invasion, and anchorage-independent growth. KSR1-dependent induction of EMT via selective translation of mRNAs reveals its underappreciated role in remodeling the translational landscape of CRC cells to promote their migratory and invasive behavior.

cancer biology↗

bioRxiv2020

Multivariate Meta-Analysis of Differential Principal Components underlying Human Primed and Naive-like Pluripotent States

The ground or naive pluripotent state of human pluripotent stem cells (hPSCs), which was initially established in mouse embryonic stem cells (mESCs), is an emerging and tentative concept. To verify this important concept in hPSCs, we performed a multivariate meta-analysis of major hPSC datasets via the combined analytic powers of percentile normalization, principal component analysis (PCA), t-distributed stochastic neighbor embedding (t-SNE), and SC3 consensus clustering. This vigorous bioinformatics approach has significantly improved the predictive values of the current meta-analysis. Accordingly, we were able to reveal various similarities between some naive-like hPSCs (NLPs) and their human and mouse in vitro counterparts. Moreover, we also showed numerous fundamental inconsistencies between diverse naive-like states, which are likely attributed to interlaboratory protocol differences. Collectively, our meta-analysis failed to provide global transcriptomic markers that support a bona fide human naive pluripotent state, rather suggesting the existence of altered pluripotent states under current naive-like growth protocols.

bioinformatics↗

bioRxiv2020

Image-based pooled whole genome CRISPR screening for Parkin and TFEB subcellular localization

Genome-wide CRISPR screens have transformed our ability to systematically interrogate human gene function, but are currently limited to a subset of cellular phenotypes. We report a novel pooled screening approach for a wider range of cellular and subtle subcellular phenotypes. Machine learning and convolutional neural network models are trained on the subcellular phenotype to be queried. Genome-wide screening then utilizes cells stably expressing dCas9 (CRISPRi), photoactivatable fluorescent protein (PA-mCherry), and a lentiviral guide RNA (gRNA) pool. Cells are screened by microscopy and classified by artificial intelligence (AI) algorithms, which precisely identify the genetically altered phenotype. Cells with the phenotype of interest are photoactivated, isolated via flow cytometry, and the gRNAs are identified by sequencing. A proof-of-concept screen accurately identified PINK1 as essential for Parkin recruitment to mitochondria. A genome-wide screen identified factors mediating TFEB relocation from the nucleus to the cytosol upon prolonged starvation. Twenty of the sixty-four hits called by the neural network model were independently validated, revealing new effectors of TFEB subcellular localization. This approach, AI-Photoswitchable Screening (AI-PS) offers a novel screening platform capable of classifying a broad range of mammalian subcellular morphologies, an approach largely unattainable with current methodologies at genome-wide scale.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2019

Interlaboratory Data Variability Contributes to the Differential Principal Components of Human Primed and Naïve-like Pluripotent States in Multivariate Meta-Analysis

Currently, genome-wide data analyses have revealed significant differences between various human naive-like pluripotent states derived from different laboratory protocols, confounding the establishment of defining criteria of human naive pluripotency. Thus, it is imperative to understand the concept concerning the ground or naive pluripotent state of pluripotent stem cells, which was initially established in mouse embryonic stem cells (mESCs). Putative human pluripotency has been proposed, largely based on comparing genome-wide transcriptomic signatures of human pluripotent stem cells (hPSCs) with human pre-implantation embryos and mESCs by several research groups. Current bioinformatics approaches, however, have inevitable conceptual biases and technological limitations, including the choices of datasets, analytic methods, and interlaboratory data variability. In this report, we performed a multivariate meta-analysis of major hPSC datasets via the combined analytic powers of percentile normalization, principal component analysis (PCA), t-distributed stochastic neighbor embedding (t-SNE), and SC3 consensus clustering. This vigorous bioinformatics approach has significantly improved the predictive values of the current meta-analysis. Accordingly, we were able to reveal various fundamental inconsistencies between naive-like hPSCs and their human and mouse in vitro counterparts, which are likely attributed to interlaboratory protocol differences. Moreover, our meta-analysis failed to provide global transcriptomic markers that support the putative in vitro human naive pluripotent state, rather suggesting the existence of altered pluripotent states under current naive-like hPSC growth protocols.

bioinformatics↗

bioRxiv2019

Constructing a Timescale of Biotic Recovery across the Cretaceous Paleogene Boundary, Corral Bluffs, Denver Basin, Colorado

ABSTRACTThe Cretaceous–Paleogene (K–Pg) boundary interval represents one of the most significant mass extinctions and ensuing biotic recoveries in Earth history. Earliest Paleocene fossil mammal faunas corresponding to the Puercan North American Land Mammal Age (NALMA) are thought to be highly endemic and potentially diachronous, necessitating precise chronostratigraphic controls at key fossil localities to constrain recovery dynamics in continental biotas following the K–Pg mass extinction. The Laramide synorgenic sedimentary deposits within the Denver Basin preserve one of the most continuous and fossiliferous records of the K–Pg boundary interval in North America. However, poor exposure in much of the Denver Basin makes it difficult to correlate between outcrops. In order to constrain fossil localities across the basin, previous studies have relied upon chronostratigraphic methods such as magnetostratigraphy. Here we present a new high-resolution magnetostratigraphy of 10 lithostratigraphic sections spanning the K–Pg boundary interval at Corral Bluffs located east of Colorado Springs in the southern part of the Denver Basin. Fossil localities from Corral Bluffs have yielded limited dinosaur remains, mammal fossils assigned to the Puercan NALMA, and numerous fossil leaf localities. Palynological analysis identifying the K–Pg boundary in three sections and two independent but nearly identical 206Pb/238U age estimates for the same volcanic ash, provide key temporal calibration points. Our paleomagnetic analysis has identified clear polarity reversal boundaries from Chron C30n to Chron C28r across the sections. It is now possible to place the fossil localities at Corral Bluffs within the broader basin-wide chronostratigraphic framework and evaluate them in the context of K–Pg boundary extinction and recovery.View Full Text

paleontology↗

bioRxiv2017

ELMOD1 stimulates ARF6-GTP hydrolysis to stabilize apical structures in developing vestibular hair cells

Sensory hair cells require control of physical properties of their apical plasma membranes for normal development and function. Members of the ARF small GTPase family regulate membrane trafficking and cytoskeletal assembly in many cells. We identified ELMOD1, a guanine nucleoside triphosphatase activating protein (GAP) for ARF6, as the most highly enriched ARF regulator in hair cells. To characterize ELMOD1 control of trafficking, we used a mouse strain lacking functional ELMOD1 (roundabout; rda). In rda/rda mice, cuticular plates of utricle hair cells initially formed normally, then degenerated after postnatal day 5 (P5); large numbers of vesicles invaded the compromised cuticular plate. Hair bundles initially developed normally, but the cells apical membrane lifted away from the cuticular plate, and stereocilia elongated and fused. Membrane trafficking in type I hair cells, measured by FM1-43 dye labeling, was altered in rda/rda mice. Consistent with the proposed GAP role for ELMOD1, the ARF6 GTP/GDP ratio was significantly elevated in rda/rda utricles as compared to controls, and the level of ARF6-GTP was correlated with the severity of the rda/rda phenotype. These results suggest that conversion of ARF6 to its GDP-bound form is necessary for final stabilization of the hair bundle.\n\nSignificance StatementAssembly of the mechanically sensitive hair bundle of sensory hair cells requires growth and reorganization of apical actin and membrane structures. Hair bundles and apical membranes in mice with mutations in the Elmod1 gene degenerate after formation, suggesting that the ELMOD1 protein stabilizes these structures. We show that ELMOD1 is a GTPase-activating protein in hair cells for the small GTP-binding protein ARF6, known to participate in actin assembly and membrane trafficking. We propose that conversion of ARF6 into the GDP-bound form in the apical domain of hair cells is essential for stabilizing apical actin structures like the hair bundle and ensuring that the apical membrane forms appropriately around the stereocilia.

neuroscience↗