Microtubules have long been recognized as upstream mediators of intracellular signaling, but the mechanisms underlying this fundamental function remain elusive. Here, we identify the structural basis by which microtubules regulate the guanine nucleotide exchange factor H1 (GEFH1), a key activator of the Ras homolog family member A (RhoA) pathway. We show that specific features of the microtubule lattice bind the C1 domain of GEFH1, leading to the sequestration and inactivation of this signaling protein. Targeted mutations in C1 residues disrupt this interaction, triggering GEFH1 release and activation of RhoA-dependent immune responses. Building on this sequestration-and-release mechanism, we identify microtubule-binding C1 domains in additional signaling proteins, including other guanine nucleotide exchange factors (GEFs), kinases, a GTPase-activating protein (GAP), and a tumor suppressor, and show that microtubule-mediated regulation via C1 domains is conserved in the Ras association domain-containing protein 1A (RASSF1A). Our findings establish a structural framework for understanding how microtubules can function as spatiotemporal signal sensors, integrating and processing diverse signaling pathways to control important cellular processes.

Conventional hydrogel-based bioprinting methods often suffer from insufficient cell densities, which may limit crucial cell-cell interactions and impair overall tissue functions. Here, we present an approach that modifies cell membranes with acrylate bonds, allowing living cells at physiological densities (up to ∼109 cells mL-1) to serve directly as bioinks, demonstrating photoactivated bioprinting through digital light processing using purely cellular bioinks. Our cell-dense bioinks (CLINKs) rapidly produce tissue constructs that closely mimic native tissues, characterized by strong structural relevancy and robust functionality. The high cellularity and living nature of CLINKs enable the creation of advanced biological models such as connected neural circuits and rhythmically contracting mini-hearts derived entirely from stem cells, effectively capturing essential native-like behaviors. Implants created through this method showcase the capacity to integrate with the host, thereby promoting regeneration. Our CLINK technology holds substantial promise in tissue biofabrication, opening alternative avenues for biomedical applications.

Psilocybin holds promise as a treatment for mental illnesses. One dose of psilocybin induces structural remodeling of dendritic spines in the medial frontal cortex in mice. The dendritic spines would be innervated by presynaptic neurons, but the sources of these inputs have not been identified. Here, using monosynaptic rabies tracing, we map the brain-wide distribution of inputs to frontal cortical pyramidal neurons. We discover that psilocybin's effect on connectivity is network specific, strengthening the routing of inputs from perceptual and medial regions (homolog of the default mode network) to subcortical targets while weakening inputs that are part of cortico-cortical recurrent loops. The pattern of synaptic reorganization depends on the drug-evoked spiking activity because silencing a presynaptic region during psilocybin administration disrupts the rewiring. Collectively, the results reveal the impact of psilocybin on the connectivity of large-scale cortical networks and demonstrate neural activity modulation as an approach to sculpt the psychedelic-evoked neural plasticity.

Chronic kidney disease affects 1 in 10 people worldwide, with damage to specialized blood filter cells of the kidney, called podocytes, playing a critical role. In membranous nephropathy (MN), a major cause of nephrotic syndrome, circulating autoantibodies attack proteins on podocyte foot processes (FPs), damaging the kidney's filtration barrier. Our study shows that these autoantibodies trigger the formation of antigen-autoantibody aggregates on the podocyte FP plasma membrane. These aggregates bud off as stalked vesicles, termed autoimmunoglobulin-triggered extracellular vesicles (AIT-EVs), which are released into the urine. AIT-EVs carry disease-causing autoantibodies, their target antigens, essential FP proteins, and disease-associated stressors representing a mechanism for removing immune complexes (ICs) and waste. However, their excessive release leads to FP effacement and podocyte dysfunction. In MN patients, urinary AIT-EVs correspond to glomerular urinary-space aggregates. Enriching AIT-EVs enables detection and monitoring of pathogenic autoantibodies, suggesting a non-invasive approach for autoimmune kidney disease diagnosis and therapy.

Ferroptosis, driven by uncontrolled peroxidation of membrane phospholipids, is distinct from other cell death modalities because it lacks an initiating signal and is surveilled by endogenous antioxidant defenses. Glutathione peroxidase 4 (GPX4) is the guardian of ferroptosis, although its membrane-protective function remains poorly understood. Here, structural and functional analyses of a missense mutation in GPX4 (p.R152H), which causes early-onset neurodegeneration, revealed that this variant disrupts membrane anchoring without considerably impairing its catalytic activity. Spatiotemporal Gpx4 deletion or neuron-specific GPX4R152H expression in mice induced degeneration of cortical and cerebellar neurons, accompanied by progressive neuroinflammation. Patient induced pluripotent stem cell (iPSC)-derived cortical neurons and forebrain organoids displayed increased ferroptotic vulnerability, mirroring key pathological features, and were sensitive to ferroptosis inhibition. Neuroproteomics revealed Alzheimer's-like signatures in affected brains. These findings highlight the necessity of proper GPX4 membrane anchoring, establish ferroptosis as a key driver of neurodegeneration, and provide the rationale for targeting ferroptosis as a therapeutic strategy in neurodegenerative disease.

Renin synthesis and release is the rate-limiting step of the renin-angiotensin-aldosterone system (RAAS) that controls fluid homeostasis. A major activator of the RAAS is a decrease in perfusion pressure within the kidneys, suggesting a link between renal mechanotransduction and renin. However, the identity of the mechanosensor(s) in the kidneys and their physiological significance to the RAAS remain unclear. We find that loss of the force-gated nonselective cation channel PIEZO2 in cells of renin lineage dysregulates the RAAS by elevating renin. We observe that PIEZO2 is expressed in renin-producing juxtaglomerular granular cells and is required for their calcium dynamics in vivo. PIEZO2 deficiency in cells of renin lineage drives renin-dependent and MAS-receptor-dependent glomerular hyperfiltration and regulates the RAAS during acute and chronic blood volume challenges. Collectively, our study identifies PIEZO2 as an essential regulator of juxtaglomerular granular cell calcium activity and renin in vivo.

While non-mammalian embryos often rely on spatial pre-patterning, mammalian development has long been thought to begin with equivalent blastomeres. However, emerging evidence challenges this. Here, using multiplexed and label-free single-cell proteomics, we identify over 300 asymmetrically abundant proteins-many involved in protein degradation and transport-dividing mouse 2-cell-stage blastomeres into two distinct clusters, which we term alpha and beta. These proteomic asymmetries are detectable as early as the zygote stage, intensify by the 4-cell stage, and correlate with the sperm entry site, implicating fertilization as a symmetry-breaking event. Splitting 2-cell-stage embryos into halves reveals that beta blastomeres possess greater developmental potential than alpha blastomeres. Similar clustering and protein enrichment patterns found in human 2-cell embryos suggest this early asymmetry might be conserved. These findings uncover a previously unrecognized proteomic pre-patterning triggered by fertilization in mammalian embryos, with important implications for understanding totipotency and early lineage bias.

Bacteria exist in competitive and rapidly changing environments in which the nature of future threats cannot be easily predicted. Streptomyces coelicolor produces three antibacterial umbrella particles that harbor distinct polymorphic toxin domains and an overlapping set of six diversified lectins. Here, we show that the exquisite specificity of umbrella particles derives from lectin-mediated species-specific binding to previously undescribed hypervariable surface glycoconjugates. A cryo-electron microscopy (cryo-EM) structure of one such lectin in complex with its oligosaccharide substrate defines the molecular basis for targeting through the coordinated recognition of multiple glycan features. Biochemical and genetic studies of several target species, in conjunction with lectin-swapping experiments, support a model whereby S. coelicolor umbrella toxin diversification at the levels of lectin composition and toxin polymorphism represents a unique, two-tiered bet-hedging strategy. Bioinformatic analyses support this as a means by which the unusual architecture of umbrella toxins offers Streptomyces a generalizable strategy to antagonize an unpredictable array of competitors.

Mechanical forces influence cellular decisions to grow, die, or differentiate, through largely mysterious mechanisms. Separately, changes in resting membrane potential have been observed in development, differentiation, regeneration, and cancer. We demonstrate that membrane potential is an important mediator of cellular response to mechanical pressure. We show that mechanical forces acting on the cell change cellular biomass density, which, in turn, alters membrane potential. Membrane potential then regulates cell number density in epithelia by controlling cell growth, proliferation, and cell elimination. Mechanistically, we show that changes in membrane potential control signaling through the Hippo and mitogen-activated protein kinase (MAPK) pathways and potentially other signaling pathways that originate at the cell membrane. While many molecular interactions are known to affect Hippo signaling, the upstream signal that activates the canonical Hippo pathway at the membrane has previously been elusive. Our results establish membrane potential as an important regulator of growth and tissue homeostasis.

Using natural experiments, we have previously reported that live-attenuated herpes zoster (HZ) vaccination appears to have prevented or delayed dementia diagnoses in both Wales and Australia. Here, we find that HZ vaccination also reduces mild cognitive impairment diagnoses and, among patients living with dementia, deaths due to dementia. Exploratory analyses suggest that the effects are not driven by a specific dementia type. Our approach takes advantage of the fact that individuals who had their eightieth birthday just after the start date of the HZ vaccination program in Wales were eligible for the vaccine for 1 year, whereas those who had their eightieth birthday just before were ineligible and remained ineligible for life. The key strength of our natural experiments is that these comparison groups should be similar in all characteristics except for a minute difference in age. Our findings suggest that live-attenuated HZ vaccination prevents or delays mild cognitive impairment and dementia and slows the disease course among those already living with dementia.

Archaeal transcription is a hybrid of eukaryotic and prokaryotic features: an RNA polymerase II (RNAPII)-like polymerase transcribes genes organized in circular chromosomes within cells devoid of a nucleus. Consequently, archaeal genomes are depleted of transcriptional regulators found in other domains of life. Here, we outline the discovery of a cryptic, archaea-specific family of ligand-binding regulatory transcription factors (TFs), called AmzR (archaeal metabolite-sensing zipper-like regulators). We identify AmzR using an evolution-based genetic screen and show that it is a repressor of methanogenic growth on methylamines in the archaeon Methanosarcina acetivorans. AmzR binds its target promoters as an oligomer using paired basic α-helices akin to eukaryotic leucine zippers. AmzR also binds methylamines, which reduces its DNA-binding affinity and allows it to function as a one-component system commonly found in prokaryotes, while containing a eukaryotic-like DNA-binding motif. The AmzR family of TFs are widespread in archaea and broaden the scope of innovations at the prokaryote-eukaryote interface.

The combination of innate immune activation and metabolic disruption plays critical roles in many diseases, often leading to mitochondrial dysfunction and oxidative stress that drive pathogenesis. However, mechanistic regulation under these conditions remains poorly defined. Here, we report a distinct lytic cell death mechanism induced by innate immune signaling and metabolic disruption, independent of caspase activity and previously described pyroptosis, PANoptosis, necroptosis, ferroptosis, and oxeiptosis. Instead, mitochondria undergoing BAX/BAK1/BID-dependent oxidative stress maintained prolonged plasma membrane contact, leading to local oxidative damage, a process we termed mitoxyperiosis. This process then caused membrane lysis and cell death, termed mitoxyperilysis. mTORC2 regulated the cell death, and mTOR inhibition restored cytoskeletal activity for lamellipodia to retract and mobilize mitochondria away from the membrane, preserving integrity. Activating this pathway in vivo regressed tumors in an mTORC2-dependent manner. Overall, our results identify a lytic cell death modality in response to the synergism of innate immune signaling and metabolic disruption.

Stress has profound effects on health, yet how it damages tissues remains poorly understood. Here, we show that acute stress triggers rapid hair loss and initiates autoimmunity. Under stress, hyperactivated sympathetic nerves release excessive norepinephrine, causing necrosis in rapidly dividing hair follicle transit-amplifying cells (HF-TACs) while sparing most hair follicle stem cells (HFSCs). This differential sensitivity stems from differences in cell death pathways, metabolic strategies, and calcium homeostasis, which render HF-TACs more susceptible to norepinephrine-induced calcium surges. HF-TAC necrosis releases cellular debris that triggers macrophage-mediated clearance and dendritic cell activation, ultimately leading to the activation and amplification of autoreactive T cells that can attack the hair follicle under inflammatory insults. Our findings reveal mechanistically how stress causes immediate tissue damage in highly proliferative HF-TACs via sympathetic nerve-induced necrosis, which in turn fuels the activation of autoreactive T cells capable of mounting future attacks against the same tissue.

The molecular details governing transcription factor (TF) binding and the formation of accessible chromatin are not yet quantitatively understood-including how sequence context modulates affinity, how TFs search DNA, the kinetics of TF occupancy, and how motif grammars coordinate binding. To resolve these questions for a human TF, erythroid Krüppel-like factor (eKLF/KLF1), we quantitatively compare, in high throughput, in vitro TF binding rates and affinities with in vivo single-molecule TF and nucleosome occupancies and in vivo-derived deep learning models. We find that 40-fold flanking sequence effects on affinity are consistent with distal flanks tuning TF search parameters and captured by a linear energy model. Motif recognition probability, rather than time in the bound state, drives affinity changes, and in vitro and in nuclei measurements exhibit consistent, minutes-long TF residence times. Finally, in vitro biophysical parameters predict in vivo sequence preferences and single-molecule chromatin states for unseen motif grammars.

BAX is a pro-apoptotic BCL-2 protein that resides in the cytosol as a monomer until triggered by cellular stress to form an oligomer that permeabilizes mitochondria and induces apoptosis. The paradigm for apoptotic blockade involves heterodimeric interactions between pro- and anti-apoptotic monomers. Here, we find that full-length BCL-w forms a distinctive, symmetric dimer (BCL-wD) that dissociates oligomeric BAX (BAXO), inhibits mitochondrial translocation, promotes retrotranslocation, blocks membrane-porating activity, and influences apoptosis induction of cells. Structure-function analyses revealed discrete conformational changes upon BCL-w dimerization and reciprocal structural impacts upon BCL-wD and BAXO interaction. Small-angle X-ray scattering (SAXS) analysis demonstrated that BAXO disrupts membranes by inducing negative Gaussian curvature, which is reversed by positive Gaussian curvature exerted by BCL-wD. Systematic truncation and mutagenesis dissected the core features of BCL-wD activity-dimerization, BAXO engagement, and membrane interaction. Our studies reveal a downstream layer of apoptotic control mediated by protein and membrane interactions of higher-order BCL-2 family multimers.

Genome-wide assessment of genetic variation is becoming routine in genetics, yet functional interpretation of non-coding single nucleotide variants in both common and rare diseases remains a major challenge. Here, we used chromatin immunoprecipitation coupled to self-transcribing active regulatory region sequencing (ChIP-STARR-seq) to functionally annotate non-coding regulatory elements (NCREs) in cellular models of human brain development. This provides gene regulatory insights into neural stem cells and evidence of NCRE priming already in embryonic stem cells for later neural activity. Based on this functional genomics atlas, we developed BRAIN-MAGNET (brain-focused artificial intelligence method to analyze genomes for non-coding regulatory element mutation targets), a functionally validated convolutional neural network that predicts NCRE activity from DNA sequence composition and identifies nucleotides required for NCRE function. BRAIN-MAGNET allows fine-mapping of genome-wide association study (GWAS) loci for common neurological traits and prioritizing candidate disease-causing rare non-coding variants in genetically unexplained individuals with neurogenetic disorders. Together, this NCRE atlas and BRAIN-MAGNET represent a powerful resource for the interpretation of non-coding genetic variation, possibly aiding the identification of previously unrecognized enhanceropathies.

The ubiquitous metabolite heme has diverse enzymatic and signaling functions in most mammalian cells. Through integrated analyses of mouse models, human cell lines, and primary patient samples, we identify de novo heme biosynthesis as a selective dependency in acute myeloid leukemia (AML). The dependency is underpinned by a propensity of AML cells, and especially leukemic stem cells (LSCs), to downregulate heme biosynthesis enzymes (HBEs), which promotes their self-renewal. Inhibition of HBEs causes the collapse of mitochondrial Complex IV and dysregulates the copper-chaperone system, inducing cuproptosis, a form of programmed cell death brought about by the oligomerization of lipoylated proteins by copper. Moreover, we identify pathways that are synthetic lethal with heme biosynthesis, including glycolysis, which can be leveraged for combination strategies. Altogether, our work uncovers a heme rheostat that is connected to gene expression and drug sensitivity in AML and implicates HBE inhibition as a trigger of cuproptosis.

Human gut bacteria are routinely exposed to stresses, and community-level responses are difficult to predict. To interrogate these effects, we screened stool-derived in vitro communities with 707 clinically relevant drugs. Across ∼5,000 community-drug conditions, compositional and metabolomic responses were shaped by nutrient competition, with certain species expanding due to the suppression of competitors. Most compositional changes arose from strain extinction and were reversed by reintroducing extinct species, although certain drugs promoted alternative states long after treatment. Despite strong selection pressures, resistance emergence was infrequent. Responses to drugs were qualitatively conserved across communities, while nutrient competition quantitatively tuned species abundances, consistent with consumer-resource model predictions. Together, nutrient competition provides a predictive framework to anticipate and potentially mitigate drug side effects on the gut microbiota.