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The blood-brain barrier (BBB) protects the brain from circulating metabolites and plays central roles in neurological diseases. Endothelial cells (ECs) of the BBB are enwrapped by mural cells including pericytes and vascular smooth muscle cells (vSMCs) that regulate angiogenesis, vessel stability and barrier function. To explore mural cell control of the BBB, we investigated neurovascular phenotypes in zebrafish pdgfrb mutants that lack brain pericytes and vSMCs. As expected, mutants showed an altered cerebrovascular network with mispatterned capillaries. Unexpectedly, mutants displayed no BBB leakage at larval stages of development. This suggests that pericytes and vSMCs are not essential for normal BBB function in developing zebrafish. Instead, we observed juvenile and adult BBB disruption occurring at 'hotspot' focal hemorrhages at large vessel aneurysms. ECs at leakage hotspots showed induction of caveolae on abluminal surfaces and structural defects including basement membrane thickening and disruption. Our work suggests that capillary pericytes primarily regulate cerebrovascular patterning in development and vSMCs of major arteries protect from hemorrhage and BBB breakdown in older zebrafish. The fact that young zebrafish have a functional BBB in the absence of mural cells calls for renewed interrogation of mural cell control of the BBB throughout vertebrate evolution.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection poses a major threat to public health, and understanding the mechanism of viral replication and virion release would help identify therapeutic targets and effective drugs for combating the virus. Herein, we identified E3 ubiquitin protein ligase Itchy homolog (ITCH) as a central regulator of SARS-CoV-2 at multiple steps and processes. ITCH enhances the ubiquitination of viral envelope and membrane proteins and mutual interactions of structural proteins, thereby aiding in virion assembly. ITCH-mediated ubiquitination also enhances the interaction of viral proteins to the autophagosome receptor p62, promoting their autophagosome-dependent secretion. Additionally, ITCH disrupts the trafficking of the protease furin and the maturation of cathepsin L, thereby suppressing their activities in cleaving and destabilizing the viral spike protein. Furthermore, ITCH exhibits robust activation during the SARS-CoV-2 replication stage, and SARS-CoV-2 replication is significantly decreased by genetic or pharmacological inhibition of ITCH. These findings provide new insights into the mechanisms of the SARS-CoV-2 life cycle and identify a potential target for developing treatments for the virus-related diseases.

Collagen is the most abundant protein in the human body and has an important role in healthy tissue as well as in a range of prevalent diseases. Medical research and diagnostics, hence, call for means of mapping collagen in vivo. Magnetic resonance imaging (MRI) is a natural candidate for this task, offering full 3D capability and versatile contrast non-invasively. However, collagen has so far been invisible to MRI due to extremely short lifetime of its resonances. Here, we report the direct imaging of collagen in vivo by magnetic resonance on the microsecond scale. The dynamics of resonance signals from collagen were first assessed in samples of bovine tendon and cortical bone. On this basis, imaging was performed at echo times down to 10 microseconds, yielding collagen-specific depiction by echo subtraction. The same approach was then extended for use in vivo, enabling direct collagen imaging of a human forearm. This capability suggests significant promise for biomedical science and clinical use. Magnetic resonance imaging (MRI) is a well-established clinical technique for examining the body non-invasively. It uses magnetic fields and radio waves to disturb hydrogen atoms in the body, which then emit signals as they return to a normal state. A computer analyses these signals to create detailed images of different tissues inside the body. Because these signals have a limited lifetime, the duration of the imaging process is critical in determining which tissues can be detected. Conventional MRI primarily measures signals from bulk water in soft tissues, which decay over tens to hundreds of milliseconds. However, a substantial fraction of signals in the body decays on the microsecond timescale, making them inaccessible to standard MRI methods. One important source of such rapidly decaying signals is collagen, the most abundant protein in the human body and a key structural component of tissues including skin, cartilage, tendons and bone. Because of its extremely short signal lifetime, collagen has traditionally been assessed only indirectly through MRI of the surrounding water. Direct collagen MRI could offer greater specificity than indirect approaches and support both research and clinical applications. For example, it could improve understanding of tissue changes associated with disease and injury or enable bone density measurements without exposure to ionising radiation. To find out if MRI could image collagen directly, van Schoor et al. used a combination of recently developed custom hardware and specialised imaging methodology designed to detect and spatially encode the extremely short-lived collagen signal. The approach was first validated in bovine tendon and bone samples and subsequently extended to imaging of the human forearm. The images obtained not only visualised collagen directly but also captured the rapid decay of its signal over time. This demonstrates that direct MRI of collagen is indeed feasible. Direct MRI of collagen could have important applications in fields such as musculoskeletal medicine, tissue engineering, and fibrosis research, where collagen content and organisation are central to tissue function and pathology. Although the current method still relies on custom-built hardware, these findings provide a foundation for developing clinical MRI systems capable of imaging rapidly decaying signals in a wider range of tissues, diseases and patient populations. With further refinement, this approach could complement existing imaging techniques, provide new non-invasive insights into tissue structure, and potentially enable direct MRI of other macromolecules.

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Tyrosine kinase 2 (TYK2) is a genetically defined target for autoimmune disease, with first-generation inhibitors showing clinical success in some but not all associated indications. A deeper understanding of TYK2 structure-function relationships, protein-ligand interactions, and the impact of human variants could inform next-generation therapeutics. Here, we applied deep mutational scanning (DMS) to assess >23,000 amino acid substitutions across two TYK2 functions: interferon alpha (IFN-α) signaling and protein abundance. This enabled high-resolution structure-function mapping and the identification of novel allosteric sites. By coupling DMS with inhibitor treatment, we uncovered variants that modulate compound potency. We also show that human variants - both common and rare - that are protective against autoimmune phenotypes reduce TYK2 protein abundance. Together, these findings demonstrate that DMS can prospectively reveal novel druggable sites, clarify structure-activity relationships (SAR), and highlight TYK2 degradation as a potential therapeutic strategy in autoimmunity.

Organisms have evolved protective strategies that are geared toward limiting cellular damage and enhancing organismal survival in the face of environmental stresses, but how these protective mechanisms are coordinated remains unclear. Here, we define a requirement for neural activity in mobilizing the antioxidant defenses of the nematode Caenorhabditis elegans both during chronic oxidative stress and prior to its onset. We show that acetylcholine-deficient mutants are particularly vulnerable to chronic oxidative stress. We find that extended oxidative stress mobilizes a broad transcriptional response which is strongly dependent on both cholinergic signaling and activation of the muscarinic G-protein acetylcholine-coupled receptor (mAChR) GAR-3. Gene enrichment analysis revealed a lack of upregulation of proteasomal proteolysis machinery in both cholinergic-deficient and gar-3 mAChR mutants, suggesting that muscarinic activation is critical for stress-responsive upregulation of protein degradation pathways. Further, we find that GAR-3 overexpression in cholinergic motor neurons prolongs survival during chronic oxidative stress. Our studies demonstrate neuronal modulation of antioxidant defenses through cholinergic activation of G protein-coupled receptor signaling pathways, defining new potential links between cholinergic signaling, oxidative damage, and neurodegenerative disease.

Functional brain network organisation, measured by functional connectivity (FC), reflects key neurodevelopmental processes for healthy development. Early exposure to adversity, for example undernutrition, affects neurodevelopment, observable via disrupted FC, and leads to poorer outcomes from preschool age onwards. We assessed longitudinally the impact of early growth trajectories on developmental FC in a rural Gambian population from age 5-24 months. To investigate how these early trajectories relate to later childhood outcomes, we assessed cognitive flexibility at 3-5 years. We observed that early physical growth before the fifth month of life drove optimal developmental trajectories of FC that in turn predicted cognitive flexibility at pre-school age. In contrast to previously studied developmental populations, this Gambian sample exhibited long-range interhemispheric FC that decreased with age. Our results highlight the measurable effects that poor growth in early infancy has on brain development and the possible subsequent impact on pre-school age cognitive development, underscoring the need for early life interventions throughout global settings of adversity.

Calcium-sensitive fluorescent indicators enable monitoring of spiking activity in large neuronal populations in animal models. Despite the plethora of algorithms developed over the past decades, accurate spike-time inference methods for spike rates exceeding 20 Hz are lacking. More importantly, little attention has been devoted to the quantification of statistical uncertainties in spike time estimation, which is essential for assigning confidence levels to inferred spike patterns. To address these challenges, we introduce (1) a statistical model that accounts for bursting neuronal activity and baseline fluorescence modulation and (2) apply a Monte Carlo strategy (particle Gibbs with ancestor sampling) to estimate the joint posterior distribution of spike times and model parameters. Our method is competitive with state-of-the-art supervised and unsupervised algorithms, as evaluated on the CASCADE benchmark datasets. Analysis of fluorescence transients recorded with the ultrafast genetically encoded calcium indicator GCaMP8f demonstrates that our method can resolve interspike intervals as short as 5 ms. Overall, our study describes a Bayesian inference method for detecting neuronal spiking patterns and quantifying their uncertainty. The use of particle Gibbs samplers enables unbiased estimates of spike times and all model parameters, providing a flexible statistical framework for testing more specific models of calcium indicators.

RAB5-GTP activation of the multiprotein VPS34 complex II (VPS34-CII) is critical for endosomal sorting and maturation, phagocytosis, and receptor downregulation. RAB5-GTP activates VPS34-CII by binding to a helical insertion in the C2 domain of VPS34 on the BECLIN1/UVRAG-containing adaptor arm of the complex. The autophagy complex, VPS34 complex I (VPS34-CI), features a unique ATG14L subunit in place of the VPS34-CII UVRAG subunit, and we found that this distorts the adaptor arm to alter the VPS34 RAB-GTPase binding pocket so that it preferentially binds RAB1-GTP. Surprisingly, our higher-resolution single-particle cryo-EM structure of VPS34-CII showed a second RAB5-GTP binding site on the VPS15 solenoid region. This site (VPS15-RAB5-site) appears to be the primordial RAB5-binding region. A mutant in the helical insertion of the C2 domain of human VPS34 that mimics the Saccharomyces cerevisiae sequence abolishes RAB5 binding to VPS34. Mutation of the VPS15-RAB5-site ortholog in S. cerevisiae VPS15 resulted in defective CPY sorting, loss of colocalisation with the RAB5 ortholog Vps21, and loss of binding to Vps21 in vitro. Evolutionary expansion from one to two RAB5-orthologue binding sites may have increased membrane binding and VPS34-CII activity to adapt to more complex endocytic systems.

Perception relies on the neural representation of sensory stimuli. Primary sensory cortical representations have been extensively studied, but how sensory information propagates to memory-related multisensory areas has not been well described. We studied this question in the olfactory cortico-hippocampal pathway in mice. We recorded single units in the anterior olfactory nucleus (AON), the anterior piriform cortex (aPCx), the lateral entorhinal cortex (LEC), the hippocampal CA1 subfield, and the subiculum (SUB) while animals performed a non-associative learning paradigm involving novel and familiar stimuli. In the AON, neurons were broadly tuned to different chemicals, and their responses were strongly modulated by experience. From the AON to hippocampal structures, the selectivity of neurons for specific odorants increased, concurrent with the development of population-level odor representations, which became independent of novelty and familiarity. While both stimulus identity and experience were thus reflected in all regions, their neural representations progressively separated. Our findings provide a potential mechanism for how sensory representations are transformed to support stimulus identification and implicit memories.

The glutamine-binding protein GlnBP is part of an ATP-binding cassette transporter system in Escherichia coli and uses two well-characterized conformational states, an open ligand-free and a closed-liganded state, to facilitate active amino-acid uptake. Existing literature on its ligand-binding mechanism lacked sufficient evidence to univocally assign the kinetic type of binding mechanism for GlnBP: ligand binding prior to conformational change, that is an induced fit, or the conformational selection, in which the ligand binds the matching conformation from a pre-existing ensemble. Since such mechanistic questions are relevant for our fundamental understanding of how this and other biomacromolecules regulate cellular processes, we here revisit the question for GlnBP. We present a biochemical and biophysical analysis using a combination of calorimetry, single-molecule and surface-plasmon resonance spectroscopy, and molecular dynamics simulations. We found that both apo- and holo-GlnBP show no detectable exchange between open and (semi-)closed conformations on timescales between 100 ns and 10 ms and that ligand binding and conformational changes in GlnBP are correlated. A global analysis of our experimental results suggests that the conformational selection model is only compatible with GlnBP for the extreme scenario of very fast conformational exchange between the open and closed states on timescales <100 ns. In contrast, all data remains compatible with an induced-fit mechanism, where the ligand binds GlnBP prior to conformational rearrangements. Importantly, our work demonstrates that it is an intricate task to identify the type of kinetic binding mechanism and that this requires not only a sufficient set of data, but also an integrative experimental and theoretical framework to address the question. Based on this concept, we propose that various protein systems, for which so far only insufficient kinetic data are available, should be revisited.

Visual motion information is essential to guiding the movements of many animals. The establishment of direction-selective signals, a hallmark of motion detection, is considered a core neural computation and has been characterized extensively in primates, mice, and fruit flies. In flies, the circuits that produce direction-selective signals rely on feedforward visual pathways that connect peripheral visual inputs to the dendrites of the ON and OFF-direction-selective cells. Here, we describe a novel role for feedback inhibition in motion computation. Two GABAergic neurons, C2 and C3, connect to neurons upstream of the direction-selective T4 and T5 cells, and blocking C2 and C3 affects direction selectivity in T4/T5. In the ON pathway, this is likely achieved by C2-mediated suppression of responses in the major T4 input neuron Mi1. Together, C2 and C3 suppress responses to non-preferred stimuli in both T4 and T5. At the behavioral level, feedback inhibition temporally sharpens responses to ON-moving stimuli, enhancing the fly's ability to discriminate visual stimuli that occur in quick succession. GABAergic inhibitory feedback neurons thus constitute an essential component within the circuitry that computes visual motion.

Bacteria precisely regulate the place and timing of their cell division. One of the best-understood systems for division site selection is the Min system in Escherichia coli. In E. coli, the Min system displays remarkable pole-to-pole oscillation, creating a time-averaged minimum at the cell's geometric center, which marks the future division site. Interestingly, the Gram-positive model species Bacillus subtilis also encodes homologous proteins: the cell division inhibitor MinC and the Walker-ATPase MinD. However, B. subtilis lacks the activating protein MinE, which is essential for Min dynamics in E. coli. We have shown before that the B. subtilis Min system is highly dynamic and quickly relocalizes to active sites of division. This raised questions about how Min protein dynamics are regulated on a molecular level in B. subtilis. Here, we show with a combination of in vitro experiments and in vivo single-molecule imaging that the ATPase activity of B. subtilis MinD is activated by membrane binding. Additionally, both monomeric and dimeric MinD bind to the membrane, and binding of ATP to MinD is a prerequisite for fast membrane detachment. Single-molecule localization microscopy data confirm membrane binding of monomeric MinD variants. However, only wild-type MinD enriches at cell poles and sites of ongoing division, likely due to interaction with MinJ. Monomeric MinD variants and locked dimers remain distributed along the membrane and lack the characteristic pattern formation. Single-molecule tracking data further support that MinD has a freely diffusive population, which is increased in the monomeric variants and a membrane-binding defective mutant. Thus, MinD dynamics in B. subtilis under the tested conditions do not require any unknown protein component and can be fully explained by MinD's binding and unbinding kinetics with the membrane. The spatial organization of MinD relies on the short-lived temporal residence of MinD dimers at the membrane.

The claustrum, with its extensive reciprocal connections to nearly all cortical regions, has long been hypothesized as a key hub for integrating diverse cognitive, sensory and motor information. However, despite its anatomical connectivity, whether and how it functionally integrates different inputs to generate coherent representations has remained unclear. Here, we developed a recurrent neural network (RNN) trained via supervised learning on behavioral metrics of delayed escape-a behavioral paradigm that requires integration of temporally separated task-relevant signals. A subset of RNN neurons exhibited dynamics similar to those of anterior claustral neurons during this behavior. These neurons formed a recurrent cluster, a structure supported by in vitro stimulation experiments in claustral brain slices. We analyzed the computational properties of this claustrum-like cluster via dimensionality reduction of population activity. The network showed nonlinear integration of temporally distributed inputs and increased synergistic information. Rather than settling into attractors, integrated information was dynamically encoded along continuously evolving neural trajectories. Notably, similar trajectory patterns associated with dynamic integration were observed in claustral recordings, suggesting the model's biological plausibility. We propose that the anterior claustrum dynamically integrates task-relevant input signals over time and broadcasts the evolving representation to downstream brain regions capable of reading and interpreting it in a context-dependent manner.

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Value-based decision making is regulated by a delicate interplay of instrumental and Pavlovian controllers. Here, we assessed the role of catecholamines in this interplay. We investigated the effects of the catecholamine reuptake inhibitor methylphenidate (MPH) in 100 healthy subjects using a combined appetitive and aversive Pavlovian-to-instrumental transfer (PIT) paradigm, including approach and withdrawal actions. By administering the drug after learning, our design allowed us to establish that MPH can also bias action outside a learning context by directly modulating the interaction of Pavlovian cues with instrumental action. Previously we showed that the effect of MPH on bias varied across these individuals as a function of their working memory (WM) span capacity (Swart et al., 2017). Here, we show by assessing both approach and withdrawal actions that MPH enhanced not only the invigorating effect of appetitive cues on active approach but also the inhibitory effect of appetitive Pavlovian cues on active withdrawal and the invigorating effect of aversive cues on active withdrawal. Thus, in participants with high WM capacity, MPH boosted both approach and withdrawal PIT. Taken together, this pattern of effects is most consistent with the hypothesis that MPH modulates the cognitive control of Pavlovian biasing in a baseline-state-dependent manner, in line with the well-established inverted U-shaped relationship between catecholamine receptor stimulation in prefrontal cortex and cognitive control.

Chromosomes must efficiently and properly interact with the mitotic spindle during prometaphase for correct segregation in anaphase. Chromosomes at the nuclear periphery or behind the spindle poles interact less efficiently with the mitotic spindle, increasing the risk of missegregation. The mechanisms that mitigate such risks in unperturbed cells are unknown. An actomyosin network (PANEM) forms around the nucleus during prophase. While the myosin-II-dependent PANEM contraction immediately after nuclear envelope breakdown (NEBD) facilitates chromosome interaction with the mitotic spindle, the mechanism by which it does so remains unclear. Here, using human cell lines, we show that immediately after NEBD, PANEM contraction directly pushes chromosomes at the nuclear periphery or behind spindle poles toward the center of cells. Detailed tracking of kinetochore movements following light-induced activation of a myosin II inhibitor reveals that this inward movement of chromosomes facilitates kinetochores' initial interaction with spindle microtubules. It also promotes the onset of kinetochores' congression toward the spindle mid-plane, but not congression itself once it starts. Thus, PANEM contraction ensures high-fidelity chromosome segregation by relocating chromosomes from unfavorable locations. Since some chromosomally unstable cancer cells fail to establish PANEM during early mitosis, the absence of PANEM may contribute to numerical chromosomal instability in these cells.

Multiplexed assays of variant effects (MAVEs) make it possible to measure the functional impact of all possible single amino acid residue substitutions in a protein in a single experiment. Combination of variant effect data from several such experiments provides the opportunity to conduct large-scale analyses of variant effect scores measured across proteins, but can be complicated by variations in the phenotypes that are probed across experiments. Thus, using variant effect datasets obtained with similar MAVE techniques can help reveal general rules governing the effects of amino acid variation for a single molecular phenotype. In this work, we accordingly combined data from six individual variant abundance by massively parallel sequencing (VAMP-seq) experiments and analysed a total of 31,614 variant effect scores reporting solely on the impact of single amino acid residue substitutions on the cellular abundance of proteins. Using our combined variant effect dataset, we derived and analysed a collection of amino acid substitution matrices describing the average impact on cellular abundance of all residue substitution types in different structural environments. We found that the substitution matrices predict the cellular abundance of protein variants with surprisingly high accuracy when given structural information only in the form of whether a residue is buried or exposed. We thus propose our substitution matrix-based predictions as strong baselines for future abundance model development.

Insulin degrading enzyme (IDE) is a dimeric M16A zinc metalloprotease that degrades amyloidogenic peptides diverse in shape and sequence, including insulin and amyloid-&#x3b2;, to prevent toxic amyloid fibril formation. IDE has a hollow catalytic chamber formed by two ~55 kDa N- and C- domains (IDE-N and IDE-C, respectively), in which peptides bind, unfold, and are repositioned for proteolysis. IDE is known to transition between a closed state, poised for catalysis, and an open state, able to release cleavage products and bind a new substrate. Here, we present six cryo-EM structures of the IDE dimer at 3.0-5.1 &#xc5; resolution, obtained in the presence of a sub-saturating concentration of insulin. Combining cryo-EM heterogeneity analysis with all-atom molecular dynamics (MD) simulations, we identified the structural basis and key residues for IDE conformational dynamics that were not previously revealed by IDE static structures. Notably, R668 serves as a molecular latch mediating the open-close transition and facilitates key protein motions through charge-swapping interactions at the IDE-N/C interface. Our small-angle X-ray scattering analysis and enzymatic assays of an R668A mutant indicate a profound alteration of conformational dynamics and catalytic activity. By integrating coarse-grained MD simulations, our analysis reveals that IDE unfolds its substrates through the coordinated motion between IDE-N and IDE-C, as well as &#x3b2;-sheet formation between IDE and insulin. Additionally, our time-resolved cryo-EM analysis uncovers IDE allostery within the IDE dimer. Collectively, our findings demonstrate the strength of combining experimental and computational approaches to probe protein dynamics and pave the way for developing substrate-specific modulators of IDE activity.