Protein assemblies, including aggregates and condensates, are closely linked to health and diseases. We demonstrate boxcar-enhanced Fluorescence-detected mid-Infrared photothermaL Microscopy (FILM), using two model species, Caenorhabditis elegans and Saccharomyces cerevisiae, to quantitatively resolve these protein states in vivo by imaging β-sheet and α-helix secondary structures and analyzing their ratios. This method directly distinguishes polyglutamine (PolyQ) protein aggregates, α-synuclein protein condensates, and P-granule condensates implicated in neurodegenerative diseases and embryonic development in live organisms. It further enables the unraveling of protein assembly dynamics and their physio-pathological roles, such as age-related progression of PolyQ from condensates to aggregates.

Sleep is regulated by a homeostatic process and associated with an increased arousal threshold, but the genetic and neuronal mechanisms that implement these essential features of sleep remain poorly understood.To address these fundamental questions, we performed a zebrafish genetic screen informed by human genome-wide association studies.We found that mutation of serine/threonine kinase 32a (stk32a) results in increased sleep and impaired sleep homeostasis in both zebrafish and mice, and that stk32a acts downstream of neurotensin signaling and the serotonergic raphe in zebrafish. stk32a mutation reduces phosphorylation of neurofilament proteins, which are co-expressed with stk32a in neurons that regulate motor activity and in lateral line hair cells that detect environmental stimuli, and ablating these cells phenocopies stk32a mutation. Neurotensin signaling inhibits specific sensory and motor populations, and blocks stimulus-evoked responses of neurons that relay sensory information from hair cells to the brain.Our work thus shows that stk32a is an evolutionarily conserved sleep regulator that links neuropeptidergic and neuromodulatory systems to homeostatic sleep drive and changes in arousal threshold, which are implemented through suppression of specific sensory and motor systems.

In natural environments, animals must allocate choices across multiple concurrently available resources when foraging, a complex decision-making process not fully captured by existing models. To understand how rodents learn to navigate this challenge, we developed a novel paradigm in which naive, water-restricted mice freely sampled six options of varying quality arranged around a large (∼2 m) arena. Mice exhibited rapid learning, matching their choices to integrated reward probabilities across six options within tens of minutes. A reinforcement learning model with distinct states for staying vs. leaving an option, as well as a dynamic global learning rate, accurately reproduced behavior. Fiber photometry recordings revealed that dopamine in the nucleus accumbens core (NAcC), but not the dorsomedial striatum (DMS), reflected this learning rate. Moreover, optogenetic manipulation of NAcC dopamine bidirectionally altered learning in quantitative agreement with model predictions. Together, we identified a neural substrate of a learning algorithm enabling efficient multi-option foraging in large spatial environments.

From work emerging through the middle of the 20th century, the essence of meaning has become widely accepted as being described by the three orthogonal dimensions of valence, arousal, and dominance. These essential dimensions have become the cornerstone of sentiment analysis across many fields. By reexamining first types and then tokens for the English language, and through the use of automatically annotated histograms-"ousiograms"-we find here that the essence of meaning conveyed by words is instead best described by a goodness-power-aggression-danger-structure (GPADS) circumplex framework; that large-scale English language corpora reveal a systematic bias toward safe, low-danger words; and that the power-danger-structure framework is the minimal framework that represents essential meaning. We find remarkable congruences between the GPADS framework and other spaces including mental states and fictional archetypes, and we construct and demonstrate a prototype ousiometer.

Fluorescence microscopy is constrained by optical limits, fluorophore chemistry and finite photon budgets, imposing trade-offs between imaging speed, resolution and phototoxicity. Here we introduce MicroSplit, a deep learning-based computational multiplexing method that enables multiple cellular structures to be imaged simultaneously in a single fluorescent channel and then computationally unmixed. We show that MicroSplit separates up to four superimposed noisy structures into distinct, denoised image channels, enabling faster and more photon-efficient imaging. Built on Variational Splitting Encoder-Decoder networks,  MicroSplit models a posterior distribution over solutions, allowing uncertainty-aware predictions and the estimation of spatially resolved prediction errors from posterior variability. We demonstrate robust performance across diverse datasets, noise levels and imaging conditions, and show that  MicroSplit improves downstream analysis while reducing photon exposure. All methods, data and trained models are released as open resources, enabling immediate adoption of computational multiplexing in biological imaging.

Understanding how nervous systems generate coordinated movement requires precise measurement of body kinematics during natural behavior. The fruit fly, Drosophila, is a model organism with sophisticated behavior and well-studied neural circuits, but tracking fly movements in 3D remains challenging because of their teeny bodies, rapid movements, and frequent self-occlusions. Here we present a pipeline for markerless, full-body 3D pose estimation of fly terrestrial behavior, combining seven synchronized high-speed cameras to capture whole-body kinematics at 800 frames per second. We trained a hybrid 2D/3D deep learning model to track 50 keypoints, then refined them to produce anatomically feasible kinematic trajectories through a retargeting process that solved an inverse kinematics problem constrained by a biomechanical body model. Analysis of 3D kinematics revealed that flies perform grounded running across their full speed range, without transitioning between discrete gaits. Using multi-animal tracking, we found that courting males coordinate both wings during song and modulate body pitch to track the female’s vertical position. Our open-source pipeline and 3D kinematic dataset of fly behavior provide a foundation for neuromechanical modeling and mechanistic studies of motor control in a genetically tractable model organism.

Cells depend on the spatial organization of proteins, RNA, and DNA into discrete subcellular compartments. Previous methods have largely centered on measuring spatial organization based on only one of these biomolecular classes at a time. Here, we demonstrate that POCA photocatalytic proximity labeling can serve as a unified photosensitizer-based platform for profiling the proximal proteomes of protein, RNA, and DNA targets within a single experimental framework. We show that POCA can harness standard immunofluorescence or in situ hybridization workflows to specifically target organic fluorophore photosensitizers to intracellular targets for proximity labeling in fixed cells. POCA-targeted proximity labeling requires minimal cellular input and does not require genetic engineering. Additionally, POCA photosensitizers are selected to also be fluorescent, enabling direct confirmation of on-target localization by imaging prior to proteomic analysis. To demonstrate broad utility, we apply POCA across multiple molecular targets spanning protein, RNA, and genomic DNA, including components of the nuclear pore complex, nucleolus, nuclear speckles, telomeres, and pericentromeric heterochromatin. By anchoring proximity labeling to both a protein and an RNA within the same nuclear compartment, we resolve shared and distinct proximal proteomes from orthogonal molecular perspectives.Competing Interest StatementD.K.S. is a collaborator with Thermo Fisher Scientific, Genentech, Calico Labs, Matchpoint Therapeutics, and AI Proteins. K.M.B is a collaborator with Thermo Fisher Scientific and on the advisory board for Matchpoint Therapeutics. B.J.B. has filed a patent application covering aspects of this work (US Patent App. 18/728,937). B.J.B. is listed as an inventor on patent applications related to the SABER technology related to this work (US Patent 11,492,661; US Patent App. 18/607,269). E.L.H. also collaborates with Thermo Fisher Scientific, Genentech, and Xaira Therapeutics and consults for Calico Labs, Matchpoint Therapeutics, and Flagship Pioneering. Patents and patent applications covering azetidine-containing rhodamine dyes (with inventors J.B.G. and L.D.L.) are assigned to HHMI. L.D.L. is a scientific cofounder, consultant, and shareholder of Eikon Therapeutics. The other authors declare no conflicts.National Institutes of Health, R35GM137916, R35GM150919, DP2GM146246, P30 CA015704, U24HG006673, T32HL007093W. M. Keck Foundation, https://ror.org/000dswa46Pew Charitable Trusts, https://ror.org/02xhk2825Andy Hill CARE FoundationDavid and Lucile Packard Foundation, https://ror.org/032atxq54Damon Runyon Cancer Research Foundation, https://ror.org/01gd7b947

Fructose-1,6-bisphosphate (FBP) is the product of the first committed step of glycolysis, and its concentration is tightly correlated with glycolytic flux. Glycolytic activity varies across tissues and cell types: some tissues, such as the brain, dynamically regulate glycolysis in response to demand, while others, such as the liver have characterized spatial heterogeneity. Here, we report HYlight2, an improved sensor for FBP developed through random whole-gene mutagenesis in E. coli lysate. After four rounds of screening, we isolated HYlight2, which retains its binding affinity while displaying a ΔR/R \~9 in vitro, a three-fold improvement in mammalian cells, and a two-fold improvement in detecting glycolytic responses during stimulated neuronal activity. We further demonstrate its use in vivo to detect altered glycolytic activity in C. elegans neurons, zebrafish pancreatic islets, and mouse liver.

Phagocytosis requires coordinated remodeling of the actin cytoskeleton to generate protrusive and contractile forces that drive target engulfment. Class I myosins Myo1e and Myo1f (Myo1e/f) have been implicated in linking the plasma membrane to the actin network, but their specific roles during Fc-receptor-mediated phagocytosis remain unclear. Using CRISPR-edited RAW 264.7 macrophages lacking Myo1e and Myo1f, we show that double knockout (dKO) cells exhibit markedly reduced uptake of IgG-coated beads, a phenotype that is partially rescued by re-expression of either myosin. Lattice-light-sheet and confocal imaging revealed distinct F-actin architectures corresponding to the various stages of cup progression, including basal podosome-like adhesions, individual phagocytic podosomes (actin teeth) along the rim of the cup, and a contractile phagocytic ring formed by the reorganization of podosomes into a higher-order network. In Myo1e/f- deficient cells, podosome formation was diminished, actin teeth were largely absent, and the phagocytic ring formed prematurely, which was often accompanied by stalled cup progression and repeated engulfment attempts. Myo1e/f localized both to podosomes and to the inner surface of the phagocytic ring, non-muscle myosin II (NM2) localized to the outer surface, and the absence of Myo1e/f correlated with the diffuse distribution of NM2. In addition, Myo1e/f-deficient macrophages exhibited increased trogocytosis of antibody-opsonized HL-60 cells, indicating a shift from whole-target engulfment toward partial target ingestion. These results suggest that Myo1e/f coordinate spatial and temporal transitions between protrusive and contractile actin networks, thereby ensuring efficient phagocytic cup progression. Our findings highlight a dual role for Myo1e/f in adhesion regulation and force balance during macrophage phagocytosis.

In Candida albicans, potassium (K) channels fine-tune ionic balance under stress, contributing to host colonization. Fungal two-pore domain, outwardly rectifying potassium (TOK) channels remain insufficiently characterized despite evidence implicating them in growth and viability. Here, we describe the atomic-resolution structure of a fungal potassium channel, TOK1 from C. albicans (CaTOK), revealing an architecture defined by eight transmembrane helices and a membrane topology distinct from previously characterized K⁺ channel classes. The first four helices form a tetraspanin-like bundle resembling auxiliary subunits of human neuronal ion channels. The pore features an inner helical gating movement analogous to mammalian dimeric K channels, while the K selectivity filter exhibits atypical ion coordination. A cytosolic C-terminal bundle forms an intramolecular network that likely stabilizes CaTOK and may mediate gating. These findings provide a framework for understanding TOK channel function and facilitate future studies of fungal ion homeostasis, pathogenicity, and therapeutic development.