bioRxiv @biorxivpreprint@qoto.org

The question of how interactions between the gut microbiome and vertebrate hosts contribute to host adaptation and speciation is one of the major problems in current evolutionary research. Using bacteriome and mycobiome metabarcoding, we examined how these two components of the gut microbiota vary with the degree of host admixture in secondary contact between two house mouse subspecies (Mus musculus musculus and M. m. domesticus). We used a large dataset collected at two replicates of the hybrid zone and model-based statistical analyses to ensure the robustness of our results. Assuming that the microbiota of wild hosts suffers from spatial autocorrelation, we directly compared the results of statistical models that were spatially naive with those that accounted for spatial autocorrelation. We showed that neglecting spatial autocorrelation can drastically affect the results and lead to misleading conclusions. The spatial analyses showed little difference between subspecies, both in microbiome composition and in individual bacterial lineages. Similarly, the degree of admixture had minimal effects on the gut bacteriome and mycobiome and was caused by changes in a few microbial lineages that correspond to the common symbionts of free-living house mice. In contrast to previous studies, these data do not support the hypothesis that the microbiota plays an important role in host reproductive isolation in this particular model system. ### Competing Interest Statement The authors have declared no competing interest.

Sarcomas are a family of rare malignancies composed of over 100 distinct histological subtypes. The rarity of sarcoma poses significant challenges in conducting clinical trials to identify effective therapies, to the point that many rarer subtypes of sarcoma do not have standard-of-care treatment. Even for established regimens, there can be substantial heterogeneity in responses. Overall, novel, personalized approaches for identifying effective treatments are needed to improve patient outcomes. Patient-derived tumor organoids (PDTOs) are clinically relevant models representative of the physiological behavior of tumors across an array of malignancies. Here, we use PDTOs as a tool to better understand the biology of individual tumors and characterize the landscape of drug resistance and sensitivity in sarcoma. We collected n=194 specimens from n=126 sarcoma patients, spanning 24 distinct subtypes. We characterized PDTOs established from over 120 biopsy, resection, and metastasectomy samples. We leveraged our organoid high-throughput drug screening pipeline to test the efficacy of chemotherapeutics, targeted agents, and combination therapies, with results available within a week from surgery. Sarcoma PDTOs showed patient-specific growth characteristics and subtype-specific histopathology. Organoid sensitivity correlated with diagnostic subtype, patient age at diagnosis, lesion type, prior treatment history, and disease trajectory for a subset of the compounds screened. We found 90 biological pathways that were implicated in response to treatment of bone and soft tissue sarcoma organoids. By comparing functional responses of organoids and genetic features of the tumors, we show how screenings can provide an orthogonal set of information to facilitate optimal drug selection, avoid ineffective therapies, and mirror patient outcomes in sarcoma. In aggregate, we could identify at least one effective FDA-approved or NCCN-recommended regimen for 59% of the specimens tested, providing an estimate of the proportion of immediately actionable information identified through our pipeline. ### Competing Interest Statement The authors have declared no competing interest.

Summary Glioma is the most common malignancy in the skull, accounting for approximately 2% of adult systemic tumors. It is characterized by a high recurrence rate, high disability rate, and poor sensitivity to chemotherapy. Different subtypes of glioma exhibit varying prognosis and chemotherapy sensitivities. Currently, researchers extensively study the molecular classification of glioma based on transcriptomic features, facilitating the evaluation of prognosis. However, identification of tumor subtypes based on the single-layer-omics data have several limitations. In this regard, the present study aimed to identify new subtypes of glioma using three omics datasets for prognosis prediction. As a result, cluster A subtype was identified to be associated with lower activation of cell proliferation and better prognosis, while cluster B subtype exhibited higher infiltration of M2-type macrophages and higher activation of the epithelial-mesenchymal transition (EMT) pathway, potentially leading to a poorer prognosis. Furthermore, we employed the Weighted Gene Co-expression Network Analysis (WGCNA) algorithm and limma R package to identify driver genes for clusters A and B. Consequently, nine genes were identified as gene signatures for cluster A. Based on this finding, we quantified the two subtypes using the single-sample gene set enrichment analysis (ssGSEA) algorithm, where a higher score were linked to elevated tumor mutation burden (TMD) and signaling pathways related to cell proliferation. Low scores indicated enrichment of tumorigenesis-related pathways and poor prognosis. In silico drug screening suggested that AGI-6780 could be an effective compound for high cluster subtypes, based on brain tumor cell lines. Consequently, our study determined that the score can serve as an effective index to predict the prognosis of glioma. ### Competing Interest Statement The authors have declared no competing interest.

Aberrant alternative splicing can generate neoantigens, which can themselves stimulate immune responses and surveillance. Previous methods for quantifying splicing-derived neoantigens are limited by independent references and potential batch effects. Here, we introduce SpliceMutr, a bioinformatics approach and pipeline for identifying splicing derived neoantigens from paired tumor normal data. SpliceMutr facilitates the identification of tumor-specific antigenic splice variants, predicts MHC-binding affinity, and estimates splicing antigenicity scores per gene. By applying this tool to genomic data from The Cancer Genome Atlas (TCGA), we generate splicing-derived neoantigens and neoantigenicity scores per sample and across all cancer types and find numerous correlations between splicing antigenicity and well-established biomarkers of anti-tumor immunity. Notably, carriers of mutations within splicing machinery genes have higher splicing antigenicity, which provides support for our approach. Further analysis of splicing antigenicity in cohorts of melanoma patients treated with mono- or combined immune checkpoint inhibition suggest that the abundance of splicing antigens is reduced post-treatment from baseline in patients who progress, likely because of an immunoediting process. We also observe increased splicing antigenicity in responders to immunotherapy, which may relate to an increased capacity to mount an immune response to splicing-derived antigens. This new computational tool provides novel analytical capabilities for splicing antigenicity and is openly available for further immuno-oncologic analysis. ### Competing Interest Statement EJF is on the Scientific Advisory Board for Resistance Bio/Viosera Therapeutics and a paid consultant for Mestag Therapeutics and Merck. NZ receives research support from BMS. NZ has served on advisory board for Genentech. NZ is consultant for and receives other support from Adventris Pharmaceuticals. NZ is co-inventor on filed patents related to KRAS peptide vaccines. EMJ reports other support from Abmeta, other support from Adventris, personal fees from Achilles, personal fees from DragonFly, personal fees from Parker Institute, personal fees from Surge, grants from Lustgarten, grants from Genentech, personal fees from Mestag, personal fees from Medical Home Group, grants from BMS, and grants from Break Through Cancer outside the submitted work. MY received grant/research support (to Johns Hopkins) from Bristol-Myers Squibb, Incyte, Genentech. Honoraria from Genentech, Exelixis, Eisai, AstraZeneca, Replimune, Hepion. Equity interest in Adventris Pharmaceuticals NA is a paid consultant for Tempus, Mirati and QED. NA receives institutional funding from Agios, Inc., Array, Atlas, Bayer HealthCare, BMS, Celgene, Debio, Eli Lilly and Company, EMD Serono, Incyte Corporation, Intensity, Merck & Co., Inc. and Taiho Pharmaceuticals Co., Ltd. NA participates on advisory boards for Incyte, QED, and Glaxo Smith Kline.

The NRAS-mutant subset of melanoma represents the most aggressive and the deadliest types associated with poorer overall survival. Unfortunately, for more than 40 years, no therapeutic agent directly targeting NRAS mutations have been clinically approved yet. Herein, based on microsecond scale molecular dynamics simulations, the concept of NRAS-Q61 mutation classification was firstly proposed. NRAS Q61 positively charged mutations (Q61R and Q61K) was classified together, with a specific targetable pocket, while NRAS-Q61L classified into another category. Moreover, the isomer-sourced structure iteration (ISSI) method was developed for the in silico design of potential inhibitors (HM-516) targeting NRAS mutations. Overall, through this article, we hope to provide the academic and clinical community with new perspective and understanding of NRAS oncoproteins, and propose possible solution to this challenge. ### Competing Interest Statement The authors have declared no competing interest.

Cytoplasmic dynein is the primary motor that drives the motility and force generation functions towards the microtubule minus end. The activation of dynein motility requires its assembly with dynactin and a cargo adaptor. This process is facilitated by two dynein-associated factors, Lis1 and Nde1/Ndel1. Recent studies proposed that Lis1 rescues dynein from its autoinhibited conformation, but the physiological function of Nde1/Ndel1 remains elusive. Here, we investigated how human Nde1 and Lis1 regulate the assembly and subsequent motility of the mammalian dynein/dynactin complex using in vitro reconstitution and single molecule imaging. We found that Nde1 promotes the assembly of active dynein complexes by competing with the inhibitor of Lis1, PAFAH-α2, and recruiting Lis1 to dynein. However, excess Nde1 inhibits dynein, presumably by competing against dynactin to bind the dynein intermediate chain. The association of dynactin with dynein triggers Nde1 dissociation before the initiation of dynein motility. Our results provide a mechanistic explanation for how Nde1 and Lis1 synergistically activate the dynein transport machinery. ### Competing Interest Statement The authors have declared no competing interest.

Super-resolution structured illumination microscopy (SR-SIM) is a method in optical fluorescence microscopy which is suitable for imaging a wide variety of cells and tissues in biological and biomedical research. Typically, SIM methods use high spatial frequency illumination patterns generated by laser interference. This approach provides high resolution but is limited to thin samples such as cultured cells. Using a different strategy for processing the raw data and coarser illumination patterns, we imaged through a 150 micrometer thick coronal section of a mouse brain expressing GFP in a subset of neurons. The resolution reached 144 nm, an improvement of 1.7-fold beyond conventional widefield imaging. ### Competing Interest Statement The authors have declared no competing interest.

Motor function is a critical aspect of communication in a wide range of taxa. The transcription factor FoxP2 plays an important role in coordinating the development of motor areas related to vocal communication in humans, mice, and songbirds. However, the role of FoxP2 in regulating motor coordination of non-vocal communication behaviors in other vertebrate taxa is unclear. Here, we test the hypothesis that FoxP2 is associated with begging behavior in tadpoles of the Mimetic poison frog (Ranitomeya imitator). In this species, mothers provide unfertilized egg meals to tadpoles that perform a begging display to communicate hunger by vigorously dancing back and forth. We mapped the neural distribution of FoxP2-positive neurons in the tadpole brain, where its wide distribution paralleled that of mammals, birds, and fishes. We next evaluated the activity of FoxP2-positive neurons during tadpole begging and found that FoxP2-positive neurons showed increased activation in the striatum, preoptic area and cerebellum. Overall, this work suggests a generalizable function of FoxP2 in social communication across terrestrial vertebrates. ### Competing Interest Statement The authors have declared no competing interest.

During C. elegans oocyte meiosis I, cortical actomyosin is locally remodeled to assemble a contractile ring near the spindle. In contrast to mitosis, when most cortical actomyosin converges into a contractile ring, the small oocyte ring forms within and remains part of a much larger and actively contractile cortical actomyosin network. This network both mediates contractile ring dynamics and generates shallow ingressions throughout the oocyte cortex during polar body extrusion. Based on our analysis of requirements for CLS-2, a member of the CLASP family of proteins that stabilize microtubules, we recently proposed that a balance of actomyosin-mediated tension and microtubule-mediated stiffness are required for contractile ring assembly within the oocyte cortical actomyosin network. Here, using live cell imaging and fluorescent protein fusions, we show that CLS-2 is part of a complex of kinetochore proteins, including the scaffold KNL-1 and the kinase BUB-1, that also co-localize to patches distributed throughout the oocyte cortex during meiosis I. By reducing their function, we further show that KNL-1 and BUB-1, like CLS-2, are required for cortical microtubule stability, to limit membrane ingression throughout the oocyte, and for meiotic contractile ring assembly and polar body extrusion. Moreover, nocodazole or taxol treatment to destabilize or stabilize oocyte microtubules, respectively, leads to excess or decreased membrane ingression throughout the oocyte and defective polar body extrusion. Finally, genetic backgrounds that elevate cortical microtubule levels suppress the excess membrane ingression in cls-2 mutant oocytes. These results support our hypothesis that CLS-2, as part of a sub-complex of kinetochore proteins that also co-localize to patches throughout the oocyte cortex, stabilizes microtubules to stiffen the oocyte cortex and limit membrane ingression throughout the oocyte, thereby facilitating contractile ring dynamics and the successful completion of polar body extrusion during meiosis I. ### Competing Interest Statement The authors have declared no competing interest.

Endothelial cells (ECs) in the descending aorta are exposed to high laminar shear stress, which supports an anti-inflammatory phenotype that protects them from atherosclerosis. High laminar shear stress also supports flow-aligned cell elongation and front-rear polarity, but whether this is required for athero-protective signaling is unclear. Here, we show that Caveolin-1-rich microdomains become polarized at the downstream end of ECs exposed to continuous high laminar flow. These microdomains are characterized by higher membrane rigidity, filamentous actin (F-actin) and lipid accumulation. Transient receptor potential vanilloid-type 4 (Trpv4) ion channels, while ubiquitously expressed, mediate localized Ca2+ entry at these microdomains where they physically interact with clustered Caveolin-1. The resultant focal bursts in Ca2+ activate the anti-inflammatory factor endothelial nitric oxide synthase (eNOS) within the confines of these domains. Importantly, we find that signaling at these domains requires both cell body elongation and sustained flow. Finally, Trpv4 signaling at these domains is necessary and sufficient to suppress inflammatory gene expression. Our work reveals a novel polarized mechanosensitive signaling hub that induces an anti-inflammatory response in arterial ECs exposed to high laminar shear stress. ### Competing Interest Statement The authors have declared no competing interest.

Exosomes are small extracellular vesicles important in health and disease. Syntenin is thought to drive the endosomal biogenesis of CD63 exosomes by recruiting Alix and the ESCRT machinery to endosome-localized CD63. We find instead that syntenin blocks CD63 endocytosis, allowing CD63 to accumulate at the plasma membrane, thereby increasing its direct budding from the plasma membrane. Consistent with this model, we show that specific and general inhibitors of endocytosis induce the exosomal secretion of CD63 and that endocytosis signals inhibit the vesicular secretion of exosome cargo proteins. Furthermore, we show that CD63 is itself a competitive inhibitor of AP-2-mediated endocytosis, driving the plasma membrane accumulation and exosomal secretion of itself and other lysosome membrane proteins. Our results support the hypothesis that highly-enriched exosome cargo proteins bud primarily from the plasma membrane, that endocytosis inhibits their loading into exosomes, and that syntenin and CD63 regulate the loading of lysosomal proteins into exosomes by an Alix-independent modulation of endocytosis. ### Competing Interest Statement The authors have declared no competing interest.

Decline in protein homeostasis (proteostasis) is a hallmark of cellular aging and aging-related diseases. Maintaining a balanced proteostasis requires a complex network of molecular machineries that govern protein synthesis, folding, localization, and degradation. Under proteotoxic stress, misfolded proteins that accumulate in cytosol can be imported into mitochondria for degradation via 'mitochondrial as guardian in cytosol' (MAGIC) pathway. Here we report an unexpected role of yeast Gas1, a cell wall-bound glycosylphosphatidylinositol (GPI)-anchored β-1,3-glucanosyltransferase, in differentially regulating MAGIC and ubiquitin-proteasome system (UPS). Deletion of Gas1 inhibits MAGIC but elevates polyubiquitination and UPS-mediated protein degradation. Interestingly, we found that Gas1 exhibits mitochondrial localization attributed to its C-terminal GPI anchor signal. But this mitochondria-associated GPI anchor signal is not required for mitochondrial import and degradation of misfolded proteins via MAGIC. By contrast, catalytic inactivation of Gas1 via the gas1E161Q mutation inhibits MAGIC but not its mitochondrial localization. These data suggest that the glucanosyltransferase activity of Gas1 is important for regulating cytosolic proteostasis. ### Competing Interest Statement The authors have declared no competing interest.

Jedi1, a chemical activator of the mechanosensitive cation channel Piezo1, shares structural similarities with the uremic solute 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF). We explored the hypothesis that CMPF at a concentration seen in uremia activates Piezo1 located on red blood cells (RBC). We incubated RBC from five healthy individuals with either Jedi1 or CMPF (both 87 μM), with or without the Piezo1 inhibitor GsMTx-4 (2 μM), and quantified the cells osmotic fragility. Our results indicate that compared to controls (i.e., RBC incubated with buffer), both Jedi1 and CMPF increase the osmotic fragility of RBC (i.e., reduce their resistance to osmotic stress). Effects of Jedi1 and CMPF were reversed to the control level by GsMTx-4. These results indicate a role of Piezo1 in augmenting RBC osmotic fragility and modulation of this effect by Jedi1 and CMPF. Our findings open the possibility that CMPF may act as an endogenous chemical activator of Piezo1. ### Competing Interest Statement The authors have declared no competing interest.

The cholinergic innervation of the cortex originates almost entirely from populations of neurons in the basal forebrain. Structurally, the ascending basal forebrain cholinergic projections are highly branched, with individual cells targeting multiple different cortical regions. However, it is not known whether the structural organization of basal forebrain projections reflects their functional integration with the cortex. We therefore used high resolution 7T diffusion and resting state functional MRI in humans to examine multimodal gradients of forebrain cholinergic connectivity with the neocortex. Moving from anteromedial to posterolateral BF, structural and functional gradients became progressively detethered, with the most pronounced dissimilarity localized in the nucleus basalis of Meynert (NbM). Structure-function tethering was shaped in part by the distance of cortical parcels from the BF and their myelin content. Functional but not structural connectivity with the BF grew stronger at shorter geodesic distances, with weakly myelinated transmodal cortical areas most strongly expressing this divergence. We then used an in vivo cell type-specific marker of the presynaptic cholinergic nerve terminals, [18F] FEOBV PET, to demonstrate that the transmodal cortical areas exhibiting highest structure-function detethering with BF gradients are also among the most densely innervated by its cholinergic projections. Altogether, multimodal gradients of basal forebrain connectivity reveal inhomogeneity in structure-function tethering which becomes most pronounced in the transition from anteromedial to posterolateral BF. Cortical cholinergic projections emanating from the NbM in particular may exhibit a broad repertoire of connections with key transmodal cortical areas associated with the ventral attention network. ### Competing Interest Statement The authors have declared no competing interest.

A tool to map changes in synaptic strength during a defined time window could provide powerful insights into the mechanisms governing learning and memory. We developed a technique, Extracellular Protein Surface Labeling in Neurons (EPSILON), to map alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) insertion in vivo by pulse-chase labeling of surface AMPARs with membrane-impermeable dyes. This approach allows for single-synapse resolution maps of plasticity in genetically targeted neurons during memory formation. We investigated the relationship between synapse-level and cell-level memory encodings by mapping synaptic plasticity and cFos expression in hippocampal CA1 pyramidal cells upon contextual fear conditioning (CFC). We observed a strong correlation between synaptic plasticity and cFos expression, suggesting a synaptic mechanism for the association of cFos expression with memory engrams. The EPSILON technique is a useful tool for mapping synaptic plasticity and may be extended to investigate trafficking of other transmembrane proteins. ### Competing Interest Statement The authors have declared no competing interest.

The fluid movement of an arm is controlled by multiple parameters that can be set independently. Recent studies argue that arm movements are generated by the collective dynamics of neurons in motor cortex. But how these collective dynamics simultaneously encode and control multiple parameters of movement is an open question. Using a task where monkeys made sequential, varied arm movements, we show that the direction and urgency of arm movements are simultaneously encoded in the low dimensional trajectories of population activity: each movement's direction by a fixed, looped neural trajectory and its urgency by how quickly that trajectory was traversed. Network models showed this latent coding is potentially advantageous as it allows the direction and urgency of arm movement to be independently controlled. Our results suggest how low-dimensional neural dynamics can define multiple parameters of goal-directed movement simultaneously. ### Competing Interest Statement The authors have declared no competing interest.

Thalamocortical (TC) neurons within the ventrolateral thalamus (VL) receive projections from the cerebellum and the basal ganglia (BG) to facilitate motor and non-motor functions. Tonic and rebound firing patterns in response to excitatory cerebellar and inhibitory BG inputs, respectively, are a canonical feature of TC neurons and plays a key role in signal processing. The intrinsic excitability of TC neurons has a strong influence on how they respond to synaptic inputs, however, it is unknown whether their afferents influence their firing properties. Understanding the input-specific firing patterns could shed light into movement disorders with cerebellar or BG involvement. Here, we used whole-cell electrophysiology in brain slices from C57BL/6 mice to investigate the firing of TC neurons with optogenetic confirmation of cerebellar or BG afferents. TC neurons with cerebellar afferents exhibited higher tonic and rebound firing rates than those with BG afferents. This increased firing was associated with faster action potential depolarization kinetics and a smaller afterhyperpolarization potential. We also found differences in the passive membrane properties and sag currents during hyperpolarization. Despite higher rebound firing in TC neurons with cerebellar afferents, there were no differences in T-type calcium channel function compared to those with BG inputs. These data suggest input-specific differences in sodium and SK, but not T-type calcium channels, impact firing properties in TC populations. Altogether, we showed that the pronounced divergence observed in TC neuron firing properties correlate with its heterogeneous anatomical connectivity, which could signify a distinct signal integration and processing by these neurons. ### Competing Interest Statement The authors have declared no competing interest.

Applications of mammalian synthetic biology increasingly require the ability to express multiple proteins at user-determined stoichiometries from single, compactly encoded transcripts. Here we present an approach for expressing multiple open reading frames (ORFs) from a single transcript, taking advantage of the leaky scanning model of translation initiation. In this method, adjacent ORFs are translated from a single messenger RNA at tunable ratios determined by their order in the sequence and the strength of their translation initiation sites. We call this approach Stoichiometric Expression of Messenger Polycistrons by Eukaryotic Ribosomes (SEMPER). We demonstrate the principles of this approach by expressing up to three fluorescent proteins from one plasmid in two different cell lines. We then use it to encode a stoichiometrically tuned polycistronic construct encoding gas vesicle acoustic reporter genes, showing that enforcing the optimal ratio in every cell enables efficient formation of the multi-protein complex while minimizing cellular toxicity. Finally, we demonstrate the polycistronic expression of two fluorescent proteins from single mRNAs made through in vitro transcription and delivered to cells. SEMPER will enable a broad range of applications requiring tunable expression from compact eukaryotic constructs. ### Competing Interest Statement The authors have declared no competing interest.

In tandem with the expanding obesity pandemic, the prevalence of nonalcoholic steatohepatitis (NASH)-associated hepatocellular carcinoma (HCC) is predicted to rise globally, creating a significant need for therapeutic agents to treat the disease. In this study, we aim to examine the hepatoprotective effect of curcumin and elucidate the mechanism by which curcumin ameliorates NASH and prevents tumorigenesis. A preclinical murine model that recapitulates human NASH-HCC in which mice were exposed to a very low dose of carbon tetrachloride (CCl4- 0.2 ul/g) once per week with administration of chow diet and normal water (CDNW) or high-fat western diet and ad lib sugar water (WDSW) was used. Compared to mice on WDSW/CCl4 and vehicle controls, mice receiving WDSW/CCl4 and curcumin showed significant reductions in liver enzymes and dyslipidemia and ameliorated steatosis, lobular inflammation, and hepatocellular ballooning in WDSW/CCl4 mice. In addition, curcumin treatment markedly reduced the hepatic expression of inflammatory markers, fibrogenic genes, and oncogenic markers. Interestingly, curcumin inhibited apoptosis antagonizing transcription factor (AATF), an oncogene overexpressed in NASH-HCC. Furthermore, the anti-tumor effects of curcumin via AATF were validated using human HCC cells. Kruppel-like factor 4 (KLF4) was significantly upregulated by curcumin in NASH liver and HCC cells. Curcumin treatment effectively inhibited the progression of NASH to HCC by downregulating the expression of AATF via the KLF4-Sp1 (specificity protein-1) signaling pathway. Herein, we report the novel molecular link between curcumin and AATF in reducing hepatocarcinogenesis, and these preclinical data provide a strong rationale for developing curcumin for the treatment of NASH-HCC in humans. ### Competing Interest Statement The authors have declared no competing interest.

The endoplasmic reticulum acts as a protein quality control center where a range of chaperones and foldases facilitates protein folding. IRE1 is a sensory trans- membrane protein that transduces signals of proteotoxic stress by forming clusters and activating a cellular program called the unfolded protein response (UPR). Recently, membrane thickness variation due to membrane compositional changes have been shown to drive IRE1 cluster formation, activating the UPR even in the absence of proteotoxic stress. Here, we demonstrate a direct relationship between bilayer tension and UPR activation based on IRE1 dimer stability. The stability of the IRE1 dimer in a (50%DOPC-50%POPC) membrane at different applied bi- layer tensions was analyzed via molecular dynamics simulations. The potential of mean force for IRE1 dimerization predicts a higher concentration of IRE1 dimers for both tensed and compressed ER membranes. This study shows that IRE1 may be a mechanosensitive membrane protein and establishes a direct biophysical relationship between bilayer tension and UPR activation. ### Competing Interest Statement The authors have declared no competing interest.