Optical recording of intricate molecular dynamics is becoming an indispensable technique for biological studies, accelerated by the development of new or improved biosensors and microscopy technology. This creates major computational challenges to extract and quantify biologically meaningful spatiotemporal patterns embedded within complex and rich data sources, many of which cannot be captured with existing methods. Here, we introduce activity quantification and analysis (AQuA2), a fast, accurate, and versatile data analysis platform built upon advanced machine-learning techniques. It decomposes complex live-imaging-based datasets into elementary signaling events, allowing accurate and unbiased quantification of molecular activities and identification of consensus functional units. We demonstrate applications across a wide range of biosensors, cell types, organs, animal models, microscopy techniques, and imaging approaches. As exemplar findings, we show how AQuA2 identified drug-dependent interactions between neurons and astroglia, as well as distinct sensorimotor signal propagation patterns in the mouse spinal cord.

To maintain tissue homeostasis, many cells reside in a quiescent state until prompted to divide. The reactivation of quiescent cells is perturbed with aging and may underlie declining tissue homeostasis and resiliency. The unfolded protein response regulators IRE-1 and XBP-1 are required for the reactivation of quiescent cells in developmentally L1-arrested C. elegans. Utilizing a forward genetic screen in C. elegans, we discovered that macroautophagy targets protein aggregates to lysosomes in quiescent cells, leading to lysosome damage. Genetic inhibition of macroautophagy and stimulation of lysosomes via the overexpression of HLH-30 (TFEB/TFE3) synergistically reduces lysosome damage. Damaged lysosomes require IRE-1/XBP-1 for their repair following prolonged L1 arrest. Protein aggregates are also targeted to lysosomes by macroautophagy in quiescent cultured mammalian cells and are associated with lysosome damage. Thus, lysosome damage is a hallmark of quiescent cells, and limiting lysosome damage by restraining macroautophagy can stimulate their reactivation.

The plasma proteome is maintained by the influx and efflux of proteins from surrounding organs and cells. To quantify the extent to which different organs and cells impact the plasma proteome in healthy and diseased conditions, we developed a mass-spectrometry-based proteomics strategy to infer the tissue origin of proteins detected in human plasma. We first constructed an extensive human proteome atlas from 18 vascularized organs and the 8 most abundant cell types in blood. The atlas was interfaced with previous RNA and protein atlases to objectively define proteome-wide protein-organ associations to infer the origin and enable the reproducible quantification of organ-specific proteins in plasma. We demonstrate that the resource can determine disease-specific quantitative changes of organ-enriched protein panels in six separate patient cohorts, including sepsis, pancreatitis, and myocardial injury. The strategy can be extended to other diseases to advance our understanding of the processes contributing to plasma proteome dynamics.

Neural reflexes to chemicals in the throat protect the airway from aspiration and infection. Mechanistic understanding of these reflexes remains premature, exemplified by chronic cough-a sensitized cough reflex-being a prevalent unmet clinical need. Here, in mice, a whole-body search for channel synapses-featuring CALHM1/3 channel-mediated neurotransmitter release-and single-cell transcriptomics uncovered subclasses of the Pou2f3+ chemosensory cell family in the throat communicating with vagal neurons via this synapse. They express G protein-coupled receptors (GPCRs) for noxious chemicals, T2Rs, which upon stimulation trigger swallow and cough-like expulsive reflexes in the hypopharynx and larynx, respectively. These reflexes were abolished by Calhm3 and Pou2f3 knockout and could be triggered by targeted optogenetic stimulation. Furthermore, aeroallergen exposure augmented CALHM3-dependent expulsive reflex. This study identifies Pou2f3+ epithelial cells with channel synapses as chemosensory end organs of airway protective reflexes and sites of their hyperresponsiveness, advancing mechanistic understanding of airway defense programs with distinct therapeutic potential.

Despite the fundamental importance of gut microbes, the genetic basis of their colonization remains largely unexplored. Here, by applying cross-species genotype-habitat association at the tree-of-life scale, we identify conserved microbial gene modules associated with gut colonization. Across thousands of species, we discovered 79 taxonomically diverse putative colonization factors organized into operonic and non-operonic modules. They include previously characterized colonization pathways such as autoinducer-2 biosynthesis and novel processes including tRNA modification and translation. In vivo functional validation revealed YigZ (IMPACT family) and tRNA hydroxylation protein-P (TrhP) are required for E. coli intestinal colonization. Overexpressing YigZ alone is sufficient to enhance colonization of the poorly colonizing MG1655 E. coli by >100-fold. Moreover, natural allelic variations in YigZ impact inter-strain colonization efficiency. Our findings highlight the power of large-scale comparative genomics in revealing the genetic basis of microbial adaptations. These broadly conserved colonization factors may prove critical for understanding gastrointestinal (GI) dysbiosis and developing therapeutics.

Western equine encephalitis virus (WEEV) is an arbovirus that historically caused large outbreaks of encephalitis throughout the Americas. WEEV binds protocadherin 10 (PCDH10) as a receptor, and highly virulent ancestral WEEV strains also bind low-density lipoprotein receptor (LDLR)-related proteins. As WEEV declined as a human pathogen in North America over the past century, isolates have lost the ability to bind mammalian receptors while still recognizing avian receptors. To explain shifts in receptor dependencies and assess the risk of WEEV re-emergence, we determined cryoelectron microscopy structures of WEEV bound to human PCDH10, avian PCDH10, and human very-low-density lipoprotein receptor (VLDLR). We show that one to three E2 glycoprotein substitutions are sufficient for a nonpathogenic strain to regain the ability to bind mammalian receptors. A soluble VLDLR fragment protects mice from lethal challenge by a virulent ancestral WEEV strain. Because WEEV recently re-emerged in South America after decades of inactivity, our findings have important implications for outbreak preparedness.

The structural basis for shifts in receptor usage remains poorly understood despite the implications for virus adaptation and emergence. Western equine encephalitis virus (WEEV) strains exhibit different patterns of engagement for two of their entry receptors: very-low-density lipoprotein receptor (VLDLR) and protocadherin 10 (PCDH10). Using structural and functional studies, we show that while all WEEV strains have a lipoprotein class A (LA) domain binding site near the E1 fusion loop, VLDLR engagement requires a second binding site in E2 that can vary with single nucleotide substitutions. We also resolve a structure of PCDH10 bound to WEEV, which reveals interactions near the E1 fusion loop with residues that also mediate LA domain binding. Evolutionary analysis enabled the generation of a PCDH10 decoy that protects in vivo against all WEEV strains tested. Our experiments demonstrate how viruses can engage multiple receptors using shared determinants, which likely impacts cellular tropism and virulence.

Claustrum orchestrates brain functions via its connections with numerous brain regions, but its molecular and cellular organization remains unresolved. Single-nucleus RNA sequencing of 227,750 macaque claustral cells identified 48 transcriptome-defined cell types, with most glutamatergic neurons similar to deep-layer insular neurons. Comparison of macaque, marmoset, and mouse transcriptomes revealed macaque-specific cell types. Retrograde tracer injections at 67 cortical and 7 subcortical regions defined four distinct distribution zones of retrogradely labeled claustral neurons. Joint analysis of whole-brain connectivity and single-cell spatial transcriptome showed that these four zones containing distinct compositions of glutamatergic (but not GABAergic) cell types preferentially connected to specific brain regions with a strong ipsilateral bias. Several macaque-specific glutamatergic cell types in ventral vs. dorsal claustral zones selectively co-projected to two functionally related areas-entorhinal cortex and hippocampus vs. motor cortex and putamen, respectively. These data provide the basis for elucidating the neuronal organization underlying diverse claustral functions.

The intestinal immune system maintains tolerance to harmless food proteins and gut microbiota through peripherally derived RORγt+ Tregs (pTregs), which prevent food intolerance and inflammatory bowel disease. Recent studies suggested that RORγt+ antigen-presenting cells (APCs), which encompass rare dendritic cell (DC) subsets and type 3 innate lymphoid cells (ILC3s), are key to pTreg induction. Here, we developed a mouse with reduced RORγt+ APCs by deleting a specific cis-regulatory element of Rorc encoding RORγt. Single-cell RNA sequencing and flow cytometry analyses confirmed the depletion of a RORγt+ DC subset and ILC3s. These mice showed a secondary reduction in pTregs, impaired tolerance to oral antigens, and an increase in T helper (Th)2 cells. Conversely, ILC3-deficient mice showed no pTregs or Th2 cell abnormalities. Lineage tracing revealed that RORγt+ DCs share a lymphoid origin with ILC3s, consistent with their similar phenotypic traits. These findings highlight the role of lymphoid RORγt+ DCs in maintaining intestinal immune balance and preventing conditions like food allergies.

Targeting ubiquitin E3 ligases is therapeutically attractive; however, the absence of an active-site pocket impedes computational approaches for identifying inhibitors. In a large, unbiased biochemical screen, we discover inhibitors that bind a cryptic cavity distant from the catalytic cysteine of the homologous to E6-associated protein C terminus domain (HECT) E3 ligase, SMAD ubiquitin regulatory factor 1 (SMURF1). Structural and biochemical analyses and engineered escape mutants revealed that these inhibitors restrict an essential catalytic motion by extending an α helix over a conserved glycine hinge. SMURF1 levels are increased in pulmonary arterial hypertension (PAH), a disease caused by mutation of bone morphogenetic protein receptor-2 (BMPR2). We demonstrated that SMURF1 inhibition prevented BMPR2 ubiquitylation, normalized bone morphogenetic protein (BMP) signaling, restored pulmonary vascular cell homeostasis, and reversed pathology in established experimental PAH. Leveraging this deep mechanistic understanding, we undertook an in silico machine-learning-based screen to identify inhibitors of the prototypic HECT E6AP and confirmed glycine-hinge-dependent allosteric activity in vitro. Inhibiting HECTs and other glycine-hinge proteins opens a new druggable space.

Amoebal predation exerts a strong evolutionary selection pressure on bacteria, thus driving the development of effective predator-defense strategies. However, little is known about the molecular interplay between bacteria and predators, particularly how bacteria can sense and kill their microbial predators. We show how the ubiquitous bacterium Pseudomonas syringae detects and kills the social amoeba Polysphondylium pallidum. Combining comparative genomics, molecular biology, and chemical analyses, we identified a chemical radar system. The system relies on P. syringae secreting the lipopeptide syringafactin, which is deacylated by the amoeba. The resulting peptides are sensed via the bacterial sensor protein chemical radar regulator (CraR) that activates genes for converting the predator-derived signal into the amoebicide pyrofactin. This system is widespread in P. syringae and enables bacteria to infect A. thaliana in the presence of amoebae. Our study advances the understanding of microbial sensing and opens new avenues for the discovery of natural products.

Single-cell proteomics (SCPs) has advanced significantly, yet it remains largely unidimensional, focusing primarily on protein abundances. In this study, we employed a pulsed stable isotope labeling by amino acids in cell culture (pSILAC) approach to simultaneously analyze protein abundance and turnover in single cells (SC-pSILAC). Using a state-of-the-art SCP workflow, we demonstrated that two SILAC labels are detectable from ∼4,000 proteins in single HeLa cells recapitulating known biology. We performed a large-scale time-series SC-pSILAC analysis of undirected differentiation of human induced pluripotent stem cells (iPSCs) encompassing 6 sampling times over 2 months and analyzed >1,000 cells. Protein turnover dynamics highlighted differentiation-specific co-regulation of protein complexes with core histone turnover, discriminating dividing and non-dividing cells. Lastly, correlating cell diameter with the abundance of individual proteins showed that histones and some cell-cycle proteins do not scale with cell size. The SC-pSILAC method provides a multidimensional view of protein dynamics in single-cell biology.

Ring-like structural maintenance of chromosome (SMC) complexes are crucial for genome organization and operate through mechanisms of DNA entrapment and loop extrusion. Here, we explore the DNA loading process of the bacterial SMC complex MukBEF. Using cryoelectron microscopy (cryo-EM), we demonstrate that ATP binding opens one of MukBEF's three potential DNA entry gates, exposing a DNA capture site that positions DNA at the open neck gate. We discover that the gp5.9 protein of bacteriophage T7 blocks this capture site by DNA mimicry, thereby preventing DNA loading and inactivating MukBEF. We propose a comprehensive and unidirectional loading mechanism in which DNA is first captured at the complex's periphery and then ingested through the DNA entry gate, powered by a single cycle of ATP hydrolysis. These findings illuminate a fundamental aspect of how ubiquitous DNA organizers are primed for genome maintenance and demonstrate how this process can be disrupted by viruses.

Anti-PD-(L)1 treatment is standard for non-small cell lung cancer (NSCLC), but patients show variable responses to the same regimen. The tumor immune microenvironment (TIME) is associated with immunotherapy response, yet the heterogeneous underlying therapeutic outcomes remain underexplored. We applied single-cell RNA and TCR sequencing (scRNA/TCR-seq) to analyze surgical tumor samples from 234 NSCLC patients post-neoadjuvant chemo-immunotherapy. Analyses revealed five distinct TIME subtypes with varying major pathological response (MPR) rates. MPR patients had elevated levels of FGFBP2+ NK/NK-like T cells, memory B cells, or effector T cells, while non-MPR patients showed higher CCR8+ Tregs. T cell clonal expansion analyses unveiled heterogeneity in non-MPR patients, marked by varying expansions of Tex-relevant cells and CCR8+ Tregs. Precursor exhausted T cells (Texp cells) correlated with recurrence-free survival, identifying a patient subgroup with reduced recurrence risk despite lack of MPR. Our study dissects TIME heterogeneity in response to chemoimmunotherapy, offering insights for NSCLC management.

Efforts to use genome-wide assays or brain scans to diagnose autism have seen diminishing returns. Yet the clinical intuition of healthcare professionals, based on longstanding first-hand experience, remains the gold standard for diagnosis of autism. We leveraged deep learning to deconstruct and interrogate the logic of expert clinician intuition from clinical reports to inform our understanding of autism. After pre-training on hundreds of millions of general sentences, we finessed large language models (LLMs) on >4,000 free-form health records from healthcare professionals to distinguish confirmed versus suspected autism cases. By introducing an explainability strategy, our extended language model architecture could pin down the most salient single sentences in what drives clinical thinking toward correct diagnoses. Our framework flagged the most autism-critical DSM-5 criteria to be stereotyped repetitive behaviors, special interests, and perception-based behaviors, which challenges today's focus on deficits in social interplay, suggesting necessary revision of long-trusted diagnostic criteria in gold-standard instruments.

Nipah virus recurrently spills over to humans, causing fatal infections. The viral receptor-binding protein (RBP or G) attaches to host receptors and is a major target of neutralizing antibodies. Here, we use deep mutational scanning to measure how all amino-acid mutations to the RBP affect cell entry, receptor binding, and escape from neutralizing antibodies. We identify functionally constrained regions of the RBP, including sites involved in oligomerization, along with mutations that differentially modulate RBP binding to its two ephrin receptors. We map escape mutations for six anti-RBP antibodies and find that few antigenic mutations are present in natural Nipah strains. Our findings offer insights into the potential for functional and antigenic evolution of the RBP that can inform the development of antibody therapies and vaccines.

How genomic DNA is folded during cell division to form the characteristic rod-shaped mitotic chromosomes essential for faithful genome inheritance is a long-standing open question in biology. Here, we use nanoscale DNA tracing in single dividing cells to directly visualize how the 3D fold of genomic DNA changes during mitosis at scales from single loops to entire chromosomes. Our structural analysis reveals a characteristic genome scaling minimum of 6-8 megabases in mitosis. Combined with data-driven modeling and molecular perturbations, we can show that very large and strongly overlapping loops formed by condensins are the fundamental structuring principle of mitotic chromosomes. These loops compact chromosomes locally and globally to the limit set by chromatin self-repulsion. The characteristic length, density, and increasingly overlapping structure of mitotic loops we observe in 3D fully explain how the rod-shaped mitotic chromosome structure emerges by self-organization during cell division.

Meningeal lymphatics serve as an outlet for cerebrospinal fluid, and their dysfunction is associated with various neurodegenerative conditions. Previous studies have demonstrated that dysfunctional meningeal lymphatics evoke behavioral changes, but the neural mechanisms underlying these changes have remained elusive. Here, we show that prolonged impairment of meningeal lymphatics alters the balance of cortical excitatory and inhibitory synaptic inputs, accompanied by deficits in memory tasks. These synaptic and behavioral alterations induced by lymphatic dysfunction are mediated by microglia, leading to increased expression of the interleukin 6 gene (Il6). IL-6 drives inhibitory synapse phenotypes via a combination of trans- and classical IL-6 signaling. Restoring meningeal lymphatic function in aged mice reverses age-associated synaptic and behavioral alterations. Our findings suggest that dysfunctional meningeal lymphatics adversely impact cortical circuitry through an IL-6-dependent mechanism and identify a potential target for treating aging-associated cognitive decline.

Microtubules are a hallmark of eukaryotes. Archaeal and bacterial homologs of tubulins typically form homopolymers and non-tubular superstructures. The origin of heterodimeric tubulins assembling into microtubules remains unclear. Here, we report the discovery of microtubule-forming tubulins in Asgard archaea, the closest known relatives of eukaryotes. These Asgard tubulins (AtubA/B) are closely related to eukaryotic α/β-tubulins and the enigmatic bacterial tubulins BtubA/B. Proteomics of Candidatus Lokiarchaeum ossiferum showed that AtubA/B were highly expressed. Cryoelectron microscopy structures demonstrate that AtubA/B form eukaryote-like heterodimers, which assembled into 5-protofilament bona fide microtubules in vitro. The additional paralog AtubB2 lacks a nucleotide-binding site and competitively displaced AtubB. These AtubA/B2 heterodimers polymerized into 7-protofilament non-canonical microtubules. In a sub-population of Ca. Lokiarchaeum ossiferum cells, cryo-tomography revealed tubular structures, while expansion microscopy identified AtubA/B cytoskeletal assemblies. Our findings suggest a pre-eukaryotic origin of microtubules and provide a framework for understanding the fundamental principles of microtubule assembly.

The organs of many female animals are remodeled by reproduction. Using the mouse intestine, a striking and tractable model of organ resizing, we find that reproductive remodeling is anticipatory and distinct from diet- or microbiota-induced resizing. Reproductive remodeling involves partially irreversible elongation of the small intestine and fully reversible growth of its epithelial villi, associated with an expansion of isthmus progenitors and accelerated enterocyte migration. We identify induction of the SGLT3a transporter in a subset of enterocytes as an early reproductive hallmark. Electrophysiological and genetic interrogations indicate that SGLT3a does not sustain digestive functions or enterocyte health; rather, it detects protons and sodium to extrinsically support the expansion of adjacent Fgfbp1-positive isthmus progenitors, promoting villus growth. Our findings reveal unanticipated specificity to physiological organ remodeling. We suggest that organ- and state-specific growth programs could be leveraged to improve pregnancy outcomes or prevent maladaptive consequences of such growth.