Primary cilia are micron-scale cellular compartments with key roles in cell signaling and disease. Cilium assembly has been delineated as a multi-step process that is disrupted in ciliopathies, yet many features of cilia disassembly remain incompletely understood. Here, we review recent insights into cilia disassembly. In addition to discussing the longstanding link between cilia disassembly and the cell cycle, we consider new findings that broaden our understanding of the molecular mechanisms and physiological contexts that govern cilia disassembly. We also synthesize recent evidence that mutual antagonism between assembly and disassembly pathways governs ciliation state. Together, these studies reveal that both excessive and impaired disassembly can perturb ciliary signaling and function, with consequences for development, tissue homeostasis and tumorigenesis. Motivated by these advances, we propose a new ciliopathy subclassification - disassembly-related ciliopathies - to encompass diseases in which aberrant cilia disassembly contributes to pathogenesis. Continued exploration of the mechanisms controlling cilia disassembly will provide both fundamental biological insights and open new avenues for targeted treatment of diseases caused by ciliary dysfunction.

Synaptic plasticity is often guided by instructive inputs to neural circuits, but learning only succeeds when these instructions reach neurons that mediate relevant outputs. This creates the credit assignment problem: how does a neuron receive instructions suited to its own behavioral function? Here we report a developmental mechanism that enables credit assignment by using instructive inputs to organize the downstream architecture through which learning is expressed. In the olivocerebellar learning system, the inferior olive provides instructive inputs that guide plasticity within the cerebellum. During circuit assembly in zebrafish, we find these same inputs regulate long-range cerebellar projections during a highly plastic, two-day critical period. Developmental experience caused specific maturation of projections to targets that were coactivated with olivary inputs. Mathematical theory and computational modeling show how the resulting architecture constrains later learning, such that a single input can sculpt downstream connectivity and then leverage that circuit to assign credit. Simulated learning became paradoxically more robust if we reduced plasticity after a developmental critical period, protecting key architecture from corruption during learning. Thus, instructive inputs can first build the circuits they later teach, coordinating development and learning to enable effective credit assignment.

Bone marrow endothelial cells (ECs) form specialized vascular niches that support hematopoietic stem and progenitor cells (HSPC), yet the molecular regulators of this function remain incompletely defined. Here, we identify the Rho GTPase-activating protein Dlc1 as an essential regulator of developmental vasculogenesis, and adult bone marrow vascular niche integrity. Endothelial-specific deletion of Dlc1 caused embryonic lethality with severe vascular defects. In adult mice, inducible EC-specific Dlc1 deletion disrupted the bone marrow vascular architecture, associated with a significant reduction of multipotent hematopoietic progenitors and myeloid-lineage cells. Single-cell RNA sequencing revealed transcriptional reprogramming of Dlc1-deficient ECs, with the emergence of cell subsets displaying altered transcriptional profiles and disrupted expression of key niche signals, including Kitl, Cxcl12, and Pdgfb. Ligand-receptor interactome analysis demonstrated impaired EC-to-HSPC communication, and the hematopoietic cells exhibited transcriptional features of metabolic stress and reduced biosynthetic activity. These findings position Dlc1 as a central regulator of developmental vasculogenesis, adult vascular architecture in the bone marrow, and EC-derived hematopoietic cell support. This work uncovers a previously unrecognized role for Dlc1 in coupling vessel function to hematopoietic cell output, with implications for understanding bone marrow failure syndromes and targeting endothelial dysfunction in hematologic diseases.

Animals efficiently learn to navigate their environment. In the laboratory, naive mice can localize new spatial targets within a handful of trials. It is unclear how such efficiency is possible, as existing models require far more experience to achieve comparable performance. Taking inspiration from behavior as mice learned to intercept hidden spatial targets, we designed agents that generate structured behavioral trajectories by controlling their speed and angular velocity between anchor points. To rapidly learn good anchors, agents use Bayesian inference on past trajectories to infer the probability that an anchor will be successful and active sampling to refine hypothesized anchors. Agents learn within tens of trials to intercept a hidden target, capturing the evolution of behavioral structure and the upper limits of learning efficiency observed in mice. This algorithm further explains how mice avoid obstacles and rapidly adapt to target switches, and it naturally encompasses both egocentric and allocentric strategies for navigation.

How mature cells actively defend their identity against alternative fates is poorly understood. Here we show that two types of fully differentiated Drosophila somatosensory neurons, derived from distinct lineages, are continuously prevented from transdifferentiating into one another by a post-mitotic bistable switch. Two homeodomain transcription factors, Hmx and Cut, are co-expressed in both neuronal types but adopt opposite stoichiometric dominances through mutual repression, each defining one identity. Disrupting this balance in mature neurons triggers deterministic, lineage-crossing reprogramming of transcriptome, dendritic and axonal architecture, and sensory behavior without reversion to a progenitor state. This regulatory logic is evolutionarily conserved: Drosophila sensory identities share transcriptomic signatures with distinct human Aδ mechanoreceptors, and human CUX and HMX proteins functionally replace their fly counterparts. Mature neuronal identity is thus not a fixed endpoint but a poised equilibrium actively maintained by ongoing competition between opposing selectors.TEASER Mature somatosensory identity is a poised equilibrium actively maintained by continuous competition between opposing terminal selectors.

Genetically encoded calcium indicators (GECIs) are vital tools for fluorescence-based visualization of neuronal activity with high spatial and temporal resolution. However, current highest-performance GECIs are predominantly green or red fluorescent, limiting multiplexing options and efficient excitation with fixed-wavelength femtosecond lasers operating at 1030 nm. In an effort to overcome these limitations, we developed OCaMP, an orange fluorescent GECI engineered from O-GECO1 through targeted substitutions to improve calcium affinity while retaining the favorable photophysical properties of mOrange2. OCaMP exhibits improved two-photon cross-section, responsiveness, photostability, and calcium affinity relative to O-GECO1. In cultured neurons, zebrafish, and mouse cortex, OCaMP outperforms the red GECIs jRCaMP1a and jRGECO1a in sensitivity, photostability, and signal-to-noise ratio. Here we show that OCaMP, an orange fluorescent GECI, is a robust tool for high-fidelity neural imaging optimized for wavelengths above 1000 nm and a practical option within the spectral gap between existing green and red GECIs.

The cellular circuits through which the internal state of an organism is sensed and relayed to the brain, and through which they bring about appropriate physiological and behavioral responses, remain largely unelucidated. Using whole-animal electron microscopy reconstruction of a Drosophila larva, we previously identified an Aorta sensory organ whose somata lie on the periphery of the brain, with dendritic arborizations in the wall of the anterior aorta, and whose axons project to the protocerebrum and synapse onto the insulin-producing cells (IPCs). Here we characterize the full anatomy and connectivity of this sensory organ. We show that the aorta sensory neurons make their strongest direct synaptic contacts onto IPCs and the neurosecretory cells producing Dromyosuppressin (DMS), with only weaker direct contacts to diuretic hormone 44 (DH44). They also make direct contacts to the enteric serotonergic neurons modulating swallowing (Se0ens), and to two single mushroom body neurons, DAN-j1 and MBON-d3, of the learning and memory center of Drosophila. Calcium imaging shows that the neurons are activated by fructose, and optogenetic manipulation reveals that their activity influences peptide content in IPCs, DH44 and DMS cells. Polysynaptic paths to the brain run through two morphologically distinct interneuron families. One, formed by the Hugin protocerebral neurons, targets the neuroendocrine system and is associated with bitter taste and pathogen induced feeding suppression. The other, formed by the BAmas projection neurons, targets dopaminergic input neurons and output neurons of the mushroom body. The Aorta sensory pathway therefore couples internal hemolymph state to two parallel central systems — endocrine state control and mushroom body-associated value updating — and provides a candidate substrate for a metabolic memory through which post-ingestive state is fed back into future feeding choice.Competing Interest StatementThe authors have declared no competing interest.

Terminally misfolded or damaged proteins in the endoplasmic reticulum (ER) are degraded by the proteasome in a process termed endoplasmic reticulum-associated degradation (ERAD). To reach the proteasome in the cytosol, cargo proteins must be dislocated across the ER lipid bilayer. Retrograde transport is performed by a set of membrane-spanning multi-protein complexes that are notoriously difficult to study. Despite decades of genetic and biochemical analysis, there is little consensus regarding its molecular mechanism. Here, we characterize the dynamic assembly of the mammalian Hrd1 complex, one of the most thoroughly studied mammalian dislocons, using in situ multicolor single-particle tracking. Surprisingly, quantitative dual-color tracking reveals that the majority of Hrd1 is assembled into stable homo-tetramers in situ. Using a herein developed single-molecule assay based on binding competition, we show that this tetramerization is driven by a short stretch (Hrd1479-530, HAF-H) within the cytosolic domain of Hrd1. Combining classical purification assays, quantitative imaging, and structural predictions, we demonstrate that Hrd1479-530 forms a highly stable tetrameric helix bundle via a conserved hydrophobic coiled-coil motif. While higher order assemblies of Hrd1 have been previously implicated, this work offers direct evidence of their role in dislocation, demonstrating that quantitative single-molecule imaging can yield information on species previously hidden from classical biochemistry.

The lung, one the largest branching organs in our body and vital for survival at first breath, is commonly known as the gas-exchange organ. Recent novel findings on lung sensing cells, molecules, and lung-brain crosstalk in the emerging discipline of interoception have highlighted the consideration of the lung as a sensory organ. Acknowledging the central importance of lung sensing to human health and disease, NHLBI convened a workshop to synthesize the past, current, and future of lung sensing research, with in-person presentations by ∼20 interdisciplinary experts and a large virtual audience. Here, we highlight the key topics discussed and summarize a blueprint for the future in this important field.

Opsins are known as proteins that bind retinal to form light-sensing rhodopsins, which mediate diverse forms of photoreception and are widely used for optogenetic control of cellular activity. Yet many opsins lack canonical retinal-binding features or, in their native biological contexts, are expressed where chromophore availability is limited, raising the question whether retinal-free opsins are silent. Here we show that retinal-free ion-transporting microbial opsins are not inert but adopt intrinsic, light-independent functional states alongside their canonical light-driven activity. Using developmental phenotyping in transgenic Drosophila melanogaster, electrophysiology in Xenopus oocytes and mammalian cells, together with structural and computational analyses, we demonstrate robust light-independent ion conductance in multiple channel-type opsins and an ion-pumping opsin. Structural modeling and molecular dynamics simulations reveal conformational rearrangements in transmembrane helices and internal water networks that are consistent with stabilization of apo-conductive states. Together, these findings establish apo-opsin activity as a defined functional state of microbial opsins, expand the current framework of rhodopsin biology, and highlight the need to consider chromophore-independent activity in both basic research and optogenetic applications.