To select behaviors appropriate to their circumstances and needs, animals integrate information about the external environment with information about the state of their bodies, derived from sensing and processing internal signals (interoception). However, the brain-wide circuit activity underlying interoception and its integration with sensorimotor processing remains unclear, partly because of technical barriers to accessing whole-brain activity at the cellular level during physiological perturbations. We developed an all-optical system for whole-brain neuronal imaging in behaving larval zebrafish during optical uncaging of gut- or bloodstream-targeted nutrients and visuo-motor stimulation. Widespread neural activity throughout the brain encoded nutrient delivery, unfolding on multiple timescales across many peripheral and central regions. Evoked activity depended on delivery location and occurred in response to both amino acids and D-glucose, but not L-glucose. Many gut responsive neurons also responded to swimming and visual stimuli, with brainstem areas primarily integrating gut and motor signals and midbrain regions integrating jointly gut, visual, and motor signals. This platform links body-brain communication studies to brain-wide neural computation in awake, behaving vertebrates.

The lifetime of fluorescent molecules provides an orthogonal readout to fluorescence intensity, opening experimental possibilities of measuring changes in local molecular environments, mechanical tension, and metabolism, among other factors. These changes are best studied live and in vivo; however, limitations of slow imaging speeds, high phototoxicity, and increased data size and complexity have significantly impeded progress on this front. Here, we present a complete and transferable pipeline consisting of a light sheet FLIM microscope and an accompanying machine learning model for data processing that renders long-term and/or high-speed volumetric FLIM (vFLIM) tractable in living systems. We benchmark this pipeline across several biological use cases, model systems, lifetime ranges, and spatiotemporal scales, showcasing a suite of possibilities that our workflow enables. This comprehensive pipeline from imaging to analysis is a crucial step forward towards disseminating the power of live vFLIM to the broader bioimaging community.

The actin cytoskeleton is a fundamental and highly conserved structure that functions in diverse cellular processes, yet its direct contribution to organismal aging remains unclear. Here, we systematically interrogated how genetic and pharmacologic perturbations of actin structure and function influence lifespan and various hallmarks of aging in . Whole-animal and tissue-specific knockdown of actin and key actin-binding proteins (ABPs)- (Arp2/3), (cofilin), and (tropomyosin)-led to premature disruption of filament organization, reduced lifespan, and tissue-specific physiological defects. Actin dysfunction also displayed a more "aged" transcriptome using previously validated transcriptomics clocks, and broadly exacerbated many age-associated phenotypes, including mitochondrial dysfunction, lipid dysregulation, loss of proteostasis, impaired autophagy, and intestinal barrier failure. Pharmacological destabilization with Latrunculin A mirrored genetic knockdowns, while mild stabilization with Jasplakinolide modestly extended lifespan, emphasizing that optimal and finely tuned actin function is critical for healthy aging. Finally, analysis of human genome-wide association data revealed that common polymorphisms correlate with differences in age-related decline in gait speed, suggesting some links between aging and actin across organisms. Taken together, our results provide a comprehensive and publicly accessible resource that maps, for the first time, how changes in actin integrity correlate with diverse aging phenotypes across tissues. This descriptive framework is intended to enable future mechanistic discovery by offering a deep, unbiased dataset that can be integrated with emerging studies to define how actin dynamics can potentially influence aging.

To elicit voluntary movements and integrative reflexes underlying behavior, the brain sends command signals to the spinal cord via specialized long-range descending neurons, known as spinal-projecting neurons (SPNs). The vast and widespread distribution of SPNs, combined with their complex long-distance connectivity, poses a significant challenge for mapping their anatomical organization and associating specific populations with distinct functions. Here we took advantage of the transparency and genetic accessibility of larval zebrafish to uncover the fundamental principles of SPN anatomical organization in a Teleost. Using an optical backfilling method relying on photoactivable GFP, we generate a whole-brain map of all neurons sending axons towards the spinal cord. This approach reveals far more SPNs than previously described through conventional strategies, offering an unparalleled opportunity to revisit distinct spinal-projecting nuclei distributed across hindbrain, midbrain, and diencephalic structures. Combining information on cell location, morphology, and projection patterns, we propose tentative homological designations for zebrafish of SPN nuclei based on established descriptions in mammals and other vertebrates.

The activity-regulated cytoskeleton-associated protein (ARC/ARG3.1) is a key regulator of synaptic plasticity and has both synaptic and nuclear functions. ARC is known to undergo nuclear import and export, yet the dynamic transport behavior of individual ARC particles remains unknown. Using live-cell single-particle tracking, we directly visualize ARC nucleocytoplasmic transport and shuttling in primary hippocampal neurons. Synaptic activation by chemical long-term potentiation (cLTP) treatment increases shuttling behavior and reveals a previously underappreciated organization of ARC within the neuronal cell body cytoplasm, characterized by perinuclear ARC clusters. Disruption of the N-terminal ARC oligomerization motif markedly reduced both perinuclear cluster formation and nucleocytoplasmic shuttling. Together, these findings reveal an activity-dependent relationship between ARC self-assembly, perinuclear organization, and nucleocytoplasmic trafficking, providing a potential mechanism for coordinating the synaptic and nuclear functions of ARC during neuronal plasticity.

Animals actively sense their surroundings to acquire behaviorally relevant environmental cues and stimuli. This dynamic acquisition of sensory information is enabled by active positioning of sensors and helps guide behavioral responses in dynamic environments. Yet how these active movements are controlled during behavior and coordinated with ongoing sensory acquisition is not fully understood. In the fruit fly Drosophila melanogaster, the antennae are crucial sensors for extracting important information from the environment including mechanosensory, olfactory, and auditory signals. Just four distinct muscles command movement of the antennae, providing a tractable model system for understanding efferent control of sensation. This work characterizes motor neurons used by Drosophila to actively position the antennae. We first identify antennal motor neurons in the central brain, and map each one from a comprehensive connectomic dataset to its peripheral muscle target. Our analysis of presynaptic inputs to the entire antennal motor system reveals a diverse array of premotor neurons for antennal motor control. We then provide genetic access to each motor unit by building a library of genetic lines with expression in antennal motor neurons. Using this library of antennal motor neuron lines, we next characterize motor unit function with quantitative behavior and optogenetics, revealing that the antennal motor system produces two primary movements in the dorsal-ventral and medial-lateral axes. Together, this work provides a comprehensive framework for understanding the motor control of an active sensor.

Piconewton forces borne by individual proteins within complex assemblies underlie cell adhesion, migration, and tissue morphogenesis, yet remain difficult to image in living systems. Here, we introduce WHaloForce, a chemigenetic HaloTag-based tension sensor that converts force-dependent relief of tryptophan-mediated dye quenching into a fluorescence lifetime change. Optical tweezers revealed a switch-like unquenching transition near 5 pN, and the sensor responded reversibly to force changes in cells. WHaloForce enabled quantitative tension imaging of diverse force-bearing proteins (vinculin, E-cadherin, α-catenin, and laminin) in mammalian cells, mouse tissue, and C. elegans. Bright synthetic dyes made tension measurements possible at endogenous expression levels. In C. elegans, vinculin and laminin showed opposite tension patterns between tissues, revealing distinct force-transmission routes through adhesions and the extracellular matrix. During ovulation, laminin tension accumulated over repeated stretch-relaxation cycles, scaling with cumulative loading history. WHaloForce thus offers a modular platform for imaging spatiotemporal tension patterns in living systems.

Ribozymes capable of self-replication from nucleotides would have been central to the hypothesized RNA World. The leading laboratory models for such molecules were converted from a class I ligase by in vitro evolution but then developed without 3D structures. Here, scaffolded cryo-EM of the substrate-free tC19Z RNA polymerase ribozyme at 3.1 Å resolution shows how this conversion was achieved. An accessory domain evolved from random sequence grips the ancestral ligase through a loop-loop contact, a seam of magnesium ions, and a six-base stack, and rebuilds the ligase’s substrate binding site from different residues of its own. A previously unrecognized pairing, present before substrate binds, sequesters the 5' end that must otherwise pair with the template. Compensatory mutations to the ribozyme and template, designed to break this ectopic pairing, increase the extension rate. These results suggest that accelerating RNA structure determination may speed progress toward nucleotide-based self-replication.

Understanding molecular orientation and density distributions is essential for unveiling biological structure and function. Polarized fluorescence microscopy (PFM) offers valuable insights into the structural orientation information, yet existing methods typically retrieve only ensemble-averaged orientations within each voxel and struggle to resolve comprehensive three-dimensional (3D) orientation distributions, particularly in thick or densely labeled complex specimens. Here we introduce the efficient generalized Richardson-Lucy (eGRL) algorithm, a computational framework that reconstructs complete 3D position and 3D orientation (spatio-angular) distributions of fluorescent molecules from PFM measurements. eGRL statistically models the oriented-fluorophore imaging process, solves the inverse problem with an iterative maximum-likelihood solution, and integrates dimensionality reduction with angular-domain transformation to achieve accurate and efficient reconstruction on standard computational platforms. Validated on simulated and experimental data across diverse PFM implementations, eGRL resolves previously inaccessible spatio-angular structures and dynamics, including actin filament alignment, nanowire-guided cytoskeletal organization, rotational actin patterns, and membrane tension-induced anisotropy in live cells.

When sensory modalities yield conflicting information, animals must rapidly reassess stimuli to select their actions. We induced auditory–visual conflict in free-flying echolocating Egyptian fruit bats, by fitting animals with prisms that shifted the perceived visual location of a landing perch while echoes returned from its veridical location. Bats that course-corrected within a single goal-directed flight did so by decoupling sonar gaze from steering, to enable rapid reweighting of visual and auditory cues. We designed artificial agents that used Bayesian inference to construct estimates of goal locations in their environment. When competing estimates directed active-sensing behaviors distinctly from steering, agents course-corrected more rapidly. Consistent with this idea, when bats were fit with prisms and earplugs that attenuated auditory localization cues, they were unable to course-correct. Removing prisms produced no systematic after-effects. Our framework suggests that instead of correcting their behavior after failure, animals could efficiently employ active sensing to resolve sensory conflict before failure occurs.Cover figure. Bat wearing helmet with clear-glasses and tracking markers.