Science Journals

Synthetic nanobodies—also called sybodies—have proven valuable for stabilizing conformations of purified proteins, advancing structural and functional studies for example of transmembrane proteins. However, their utility in modulating protein function in living cells has remained less well explored. Structural Maintenance of Chromosomes (SMC) complexes facilitate chromosome organization, a fundamental process in all domains of life. In this study, we target the bacterial SMC complex, Smc-ScpAB, in <i>Bacillus subtilis</i> with synthetic nanobodies, aiming to identify key functional regions of the protein complex in a largely unbiased manner. We first isolate sybodies that specifically bind purified Smc-ScpAB and then express them in <i>B. subtilis</i> to select binders capable of disrupting Smc-ScpAB function, leading to chromosome segregation defects and cell death. Mapping and biochemical characterization show that the 14 disruptive sybodies belong to one of three library designs, target the Smc subunit near the same coiled coil arm interface and modulate its ATPase activity in two principal ways, highlighting the mid-region of the Smc coiled coil as critical feature of the SMC-DNA folding process. These findings underscore the potential of sybodies—and, by extension, designed binders—as versatile tools for probing dynamic protein function in living cells.

<i>Trypanosoma brucei,</i> the causal agent of Human and Animal African trypanosomiasis proliferates in the extracellular milieu of mammals. It acquires host macromolecular nutrients by receptor-mediated endocytosis. The best characterised cell surface receptor is for transferrin (TfR), and it has been reported to be preferentially localised in the flagellar pocket domain of the plasma membrane, the sole site of endocytosis. In this location, the TfR may be inaccessible to adaptive immune system effectors. The <i>T. brucei</i> genome encodes ~15 TfR variants, and here we compared two, the first attached to the plasma membrane by a single glycosylphosphatidylinositol (GPI)-anchor and the other by two. Transferrin uptake kinetics were similar and rapid for both. Unexpectedly, initial binding of transferrin occurred over the whole cell surface suggesting the TfR was not localised solely in the flagellar pocket. This localisation was confirmed by immunofluorescence assays and was independent of the number of GPI-anchors. Two other GPI-anchored receptors were investigated to determine whether localisation to the whole cell surface was a general property of GPI-anchored receptors. Haptoglobin-haemoglobin uptake assays and immunofluorescence localisation of complement factor H receptor showed both were also whole cell surface localised. The mechanisms by which trypanosome receptors are protected from antibody-mediated attack are more complex than hiding in a pocket.

Animals routinely need to make decisions about what to eat and when. These decisions are influenced not only by the availability and quality of food but also by the internal state of the animal, which needs to compute and give weights to these different variables before making a choice. Feeding preferences of female mosquitoes exemplify this behavioural plasticity. Both male and female mosquitoes usually feed on carbohydrate-rich sources of nectar or sap but the female also feeds on blood, which is essential for egg development. This blood-appetite is modulated across the female’s reproductive cycle, yet little is known about the factors that bring it about. We show that mated, but not virgin <i>Anopheles stephensi</i> females, a major vector of urban malaria in the Indian subcontinent and West Africa, suppress blood feeding between a blood meal and oviposition. We identify several candidate genes through transcriptomics of blood-deprived and -sated <i>An. stephensi</i> central brains that could modulate this behaviour. We show that <i>short neuropeptide F (sNPF</i>) and <i>RYamide (RYa</i>) act together to promote blood feeding and identify a cluster of cells in the subesophageal zone that expresses <i>sNPF</i> transcripts only in the blood-hungry state. Such females also have more <i>sNPF</i> transcripts in their midguts. Based on these data, we propose a model where increased <i>sNPF</i> levels in the brain and gut promote a state of blood-hunger, which drives feeding behaviour either by <i>sNPF’s</i> action in the two tissues independently or via a communication between them. This occurs in the context of the action of <i>RYa</i> in the brain.

Isogenic cells can break symmetry and adopt different fates, even when exposed to a seemingly identical environment. This deeply conserved phenomenon allows unicellular organisms to pre-empt dynamically changing environments and is central to the evolution of multicellularity. It is thought that cells are primed towards different lineages by cell-cell variation, although the underlying mechanisms are poorly understood. To address this, we exploit the tractability of the social amoeba <i>Dictyostelium discoideum</i>, where cell fate choice also does not depend on spatial cues. We develop and test a model to explain quantitative experimental single-cell observations of probabilistic differentiation. The model suggests that cell cycle position affects lineage choice, as previously shown but that stochastic cell-cell variation also plays a key role. Single cell sequencing reveals genes that exhibit cell type-specific expression or genes that affect fate choice exhibit extensive stochastic cell-cell expression variation. Like lineage priming genes in ESCs, they are associated with H3K4 methylation, which when perturbed affects their expression and disrupt fate choice. We suggest the integration of stochastic and deterministic inputs represents an adaptive mechanism to increase developmental robustness against perturbations that affect deterministic signals.

Constructing combinatorially complete species assemblages is often necessary to dissect the complexity of microbial interactions and to find optimal microbial consortia. At the moment, this is accomplished through either painstaking, labor-intensive liquid handling procedures, or through the use of state-of-the-art microfluidic devices. Here, we present a simple, rapid, low-cost, and highly accessible liquid handling methodology for assembling all possible combinations of a library of microbial strains, which can be implemented with basic laboratory equipment. To demonstrate the usefulness of this methodology, we construct a combinatorially complete set of consortia from a library of eight <i>Pseudomonas aeruginosa</i> strains, and empirically measure the community-function landscape of biomass productivity, identify the highest-yield community, and dissect the interactions that lead to its optimal function. This easy-to-implement, inexpensive methodology will make the assembly of combinatorially complete microbial consortia easily accessible for all laboratories.

An endogenous circadian clock controls many of the behavioral traits of <i>Drosophila melanogaster</i>. This ‘clock’ relies on the activity of interconnected clusters of neurons that harbor the clock machinery. The hierarchy among clusters involved in the control of rest-activity cycles has been extensively studied. Sexually dimorphic behaviors, on the other hand, have received less attention. Even though egg-laying, a female characteristic behavior, has been shown to be rhythmic, it remains largely unexplored possibly due to methodological constraints. The current study provides the first steps towards determining the neural substrates underlying the circadian control of egg-laying. We show that, whereas the lateral ventral neurons (LNvs) and the dorsal neurons (DNs) are dispensable, the lateral dorsal neurons (LNds) are necessary for rhythmic egg-laying. Systematically probing the <i>Drosophila</i> connectome for contacts between circadian clusters and oviposition-related neurons, we found no evidence of direct connections between LNvs or DNs and neurons recruited during oviposition. Conversely, we did find bidirectional connections between two Cryptochrome (Cry) expressing LNd (Cry + LNds) and oviposition-related neurons. Taken together, these results reveal that Cry + LNd neurons have a leading role in the control of the egg-laying rhythm in <i>Drosophila</i> females.

The ability to record the real-time activity of specialized neurons in the brains of female mice is providing new insights into the hormonal control of ovulation.

A large fraction of newly transcribed RNA is degraded in the nucleus, but nuclear mRNA degradation pathways remain largely understudied. The yeast nuclear endoribonuclease Rnt1 has a well-characterized role in the maturation of many ncRNA precursors. However, the scope and consequence of its function in mRNA degradation pathways are much less defined. Here, we take a whole-transcriptome approach to identify Rnt1 cleavage sites throughout the yeast transcriptome in vivo, at single-nucleotide resolution. We discover previously unknown Rnt1 cleavage sites in many protein-coding regions and find that the sequences and structures necessary for cleavage mirror those required for the cleavage of known targets. We show that the nuclear localization of Rnt1 functions as an additional layer of target selection control, and that cleaved mRNAs are likely exported to the cytoplasm to be degraded by Xrn1. Further, we find that several cleavage products are much more abundant in our degradome sequencing libraries than decapping products, and strikingly, mutations in one Rnt1 target, <i>YDR514C</i>, suppress the growth defect of an <i>RNT1</i> deletion. Overexpression of <i>YDR514C</i> results in slow growth, further suggesting that Rnt1 may limit the expression of <i>YDR514C</i> to maintain proper cell growth. This study uncovers a broader target range and function for the well-known RNase III enzyme.

The greatest mass extinction at the end of the Permian, ca. 252 million years ago, led to a tropical dead zone on land and sea. The speed of recovery of life has been debated, whether fast or slow, and terrestrial ecosystems are much less understood than marine. Here, we show fast reestablishment of riparian ecosystems in low-latitude North China as little as ~2 million years after the end-Permian mass extinction. The initial ichnoassemblages in shallow lacustrine and fluvial facies of late Smithian age are monospecific, devoid of infaunalization, with apparent size reduction. In the following Spathian, relatively complex, multi-level, structured riverain ecosystems had been rebuilt including medium-sized carnivores, plant stems, root traces, increased ichnological complexity, and significantly increased infaunalization. Specifically, burrowing behavior had re-emerged as a key life strategy not only to minimize stressful climatic conditions, but possibly to escape predation.

The ion channel (IC) genes encoded in the human genome play fundamental roles in cellular functions and disease, and are one of the largest classes of druggable proteins. However, limited knowledge of the diverse molecular and cellular functions carried out by ICs presents a major bottleneck in developing selective chemical probes for modulating their functions in disease states. The wealth of sequence data available on ICs from diverse organisms provides a valuable source of untapped information for illuminating the unique modes of channel regulation and functional specialization. However, the extensive diversification of IC sequences and the lack of a unified resource present a challenge in effectively using existing data for IC research. Here, we perform integrative mining of available sequence, structure, and functional data on 419 human ICs across disparate sources, including extensive literature mining by leveraging advances in LLMs to annotate and curate the full complement of the ‘channelome’. We employ a well-established orthology inference approach to identify and extend the IC orthologs across diverse organisms to above 48,000. We show that the depth of conservation and taxonomic representation of IC sequences can further be translated to functional similarities by clustering them into functionally relevant groups, which can be used for downstream functional prediction on understudied members. We demonstrate this by delineating co-conserved patterns characteristic of the understudied family of the calcium homeostasis modulator (CALHM) family of ICs. Through mutational analysis of co-conserved residues altered in human diseases and electrophysiological studies, we show that these evolutionarily constrained residues play an important role in channel gating functions. Thus, by providing new tools and resources for performing large comparative analyses on ICs, this study addresses the unique needs of the IC community and provides the groundwork for acceler…

The rapid evolution of SARS-CoV-2 and other respiratory RNA viruses limits the success of current vaccines and antibody-based therapies. Engineered decoy receptors based on soluble angiotensin-converting enzyme 2 (sACE2) offer promising alternatives but show limited clinical success. This study conducted functional and mechanistic analyses using an optimized sACE2 mutant fused to human IgG1 Fc (B5-D3) as a representative, revealing redirection of virus–decoy complexes from epithelial infection to lysosomal degradation in phagocytes beyond viral neutralization. Intranasal prophylactic delivery of B5-D3 confers complete protection in SARS-CoV-2-infected K18-hACE2 mice, regardless of age. Abrogation of Fc effector functions compromises antiviral protection, indicating that Fc-mediated uptake of virus–decoy complexes is critical. Transcriptomic analysis suggests that B5-D3 induces early immune activation in the lungs of infected mice. Bio-distribution and flow cytometry reveal selective targeting of airway phagocytes. In vitro assays confirm lysosomal degradation of virus–decoy complexes by macrophages without productive infection. These findings reveal a distinct antiviral mechanism via phagocytic clearance, supporting refined regimens for decoy treatments against SARS-CoV-2 and potentially other respiratory viruses.

Neutrophils are short-lived cells of the innate immune system that play numerous roles in defense against infection, regulation of immune responses, tissue damage and repair, autoimmunity, and other non-communicable diseases. Understanding neutrophil function at a mechanistic level has been hampered by the difficulty of working with primary neutrophils, which die rapidly upon isolation, and the relative paucity of neutrophil cell lines. Here, we report the creation of a Cas9+ER-Hoxb8 neutrophil progenitor cell line that enables both forward and reverse genetic analysis of neutrophils. By editing progenitors via transduction with sgRNAs and then withdrawing estrogen, Cas9-edited neutrophils are produced with high efficiency. Importantly, neutrophil differentiation of edited progenitors occurs both <i>in vitro</i> in cell culture and when transferred into murine recipients. To demonstrate the utility of Cas9+ER-Hoxb8 progenitors for forward genetics, we performed a pooled CRISPR screen to identify factors required for survival during neutrophil differentiation. This screen identified hundreds of genes, including <i>Cebpe</i>, a transcription factor known to be required for neutrophil differentiation from pre-neutrophils to immature neutrophils. Using this progenitor cell line, we confirmed that <i>Cebpe</i> is required for neutrophil differentiation <i>in vivo</i>, validating the utility of this line for studying <i>in vivo</i> phenotypes. The screen also identified all components of the WASH complex as being required for neutrophil differentiation, extending its known role in hematopoietic stem cell differentiation to later stages of neutrophil development. Taken together, this resource enables the analysis of the role of neutrophils in numerous disease states using genetics for the first time.

Extremely preterm birth (at &lt;28 postconceptional weeks) leads to brain injury and represents the leading cause of childhood-onset neuropsychiatric diseases. No effective therapeutics exist to reduce the incidence and severity of brain injury of prematurity. Hypoxic events are the most important environmental factor, along with inflammation. Among other developmental processes, the second half of in utero fetal development coincides with the migration of cortical interneurons from the ganglionic eminences into the cortex; this process is thus prone to disruptions following extremely preterm birth. To date, no studies have directly investigated the migration of human cortical inhibitory neurons under hypoxic conditions. Using multi-day confocal live imaging in human forebrain assembloids (hFA) derived from human-induced pluripotent stem cells (hiPSCs) and ex vivo developing human brain tissue, we found a substantial reduction in the migration of hypoxic interneurons. Using transcriptomics, we identified adrenomedullin (<i>ADM</i>) as the gene with the highest fold change increase in expression. Based on previous literature about the protective role of supplemental ADM for other injuries, here, we demonstrated that addition of exogenous ADM to the hypoxic media restores the migration defects of interneurons. Lastly, we showed that one of the mechanisms of protection by ADM is through the activation of the cAMP/PKA pathway and subsequent pCREB-dependent rescued expression of a subset of GABA receptors, which are known to promote migration. Overall, in this manuscript, we provide the first direct evidence for hypoxia-induced deficits in the migration of human cortical interneurons and identify ADM as a possible target for therapeutic development.

Although it is well established that stem cells maintain tissue homeostasis while tumors disrupt it, the mechanisms by which tumors influence the development of nearby stem cells remain poorly understood. Using <i>Drosophila</i> ovaries as a model system, here we discovered that <i>bam</i> or <i>bgcn</i> mutant germline tumors inhibit the differentiation of neighboring wild-type germline stem cells (GSCs). Mechanistically, these tumor cells mimic the stem cell niche by secreting the bone morphogenetic protein (BMP) ligands Dpp and Gbb, but at reduced levels, resulting in moderate BMP signaling activation in adjacent GSCs. Such BMP signaling activation is sufficient to repress <i>bam</i> transcription, thereby blocking GSC differentiation. To our knowledge, this is the first example that tumors can functionally mimic a stem cell niche to inhibit the differentiation of neighboring wild-type stem cells. Similar regulatory paradigms may operate in mammalian tissues, including humans, during tumorigenesis.

Addictive drugs cause long-lasting changes in connectivity from inputs onto ventral tegmental area dopamine cells (VTA^DA) that contribute to drug-induced behavioral adaptations. However, it is not known which inputs are altered. Here, we used a rabies virus (RABV)-based mapping strategy to quantify RABV-labeled inputs to VTA cells after a single exposure to one of a variety of misused drugs – cocaine, amphetamine, methamphetamine, morphine, and nicotine – and compared the relative global input labeling across conditions. We observed that all tested addictive drugs elicited similar input changes onto VTA^DA cells, in particular onto DA cells projecting to the lateral shell of the nucleus accumbens and amygdala. In addition, repeated administration of ketamine/xylazine to induce anesthesia induces a change in inputs to VTA^DA cells that is similar to but different from those elicited by a single exposure to addictive drugs, suggesting that caution should be taken when using ketamine/xylazine-based anesthesia in rodents when assessing motivated behaviors. Furthermore, comparison of viral tracing data to an atlas of gene expression in the adult mouse brain showed that the basal expression patterns of several gene classes, especially calcium channels, were highly correlated with the extent of both addictive drug- or ketamine/xylazine-induced changes in RABV-labeled inputs to VTA^DA cells. Reducing expression levels of the voltage-gated calcium channel <i>Cacna1e</i> in cells in the nucleus accumbens lateral shell reduced RABV-mediated input labeling of these cells into VTA^DA cells. These results directly link genes controlling cellular excitability and the extent of input labeling by RABV.

Here, we identify the subunit e of F₁F₀-ATP synthase (ATP5I) as a target of metformin, a first-in-class antidiabetic biguanide. ATP5I maintains the stability of F₁F₀-ATP synthase dimers, which is crucial for shaping cristae morphology. We demonstrate that ATP5I interacts with a biguanide analogue in vitro, and disabling its expression by CRISPR–Cas9 in pancreatic cancer cells leads to the same phenotype as biguanide-treated cells, including mitochondrial morphology alterations, reduction of the NAD⁺/NADH ratio, inhibition of oxidative phosphorylation (OXPHOS), rescue of respiration by uncouplers, and a compensatory increase in glycolysis. Notably, metformin disrupts F₁F₀-ATP synthase oligomerization, leading to the accumulation of vestigial assembly intermediates in pancreatic and osteosarcoma cancer cells, a phenotype also observed upon ATP5I inactivation in pancreatic cancer cells. Moreover, ATP5I knockout (KO) cells exhibit resistance to the antiproliferative effects of biguanides, but reintroduction of ATP5I rescues the metabolic and antiproliferative effects of metformin and phenformin. Finally, a genome-wide CRISPR screening in NALM-6 lymphoma cells revealed that metformin-treated cells exhibit genetic interaction profiles similar to those observed with the F₁F₀-ATP synthase inhibitor oligomycin, but not with the complex I inhibitor rotenone. This provides unbiased support for the relevance of the newly proposed target.

Odorants stimulate olfactory sensory neurons (OSNs) to create a bilateral sensory map defined by a set of glomeruli present in the left and right olfactory bulbs. Using <i>Xenopus tropicalis</i> tadpoles, we challenged the notion that glomerular activation is exclusively determined ipsilaterally. Glomerular responses evoked by unilateral stimulation were potentiated following transection of the contralateral olfactory nerve. The gain of function was observed as early as 2 hr after injury and faded away with a time constant of 4 days. Potentiation was mediated by the presence of larger and faster calcium transients driving glutamate release from OSN axon terminals. The cause was the reduction of the tonic presynaptic inhibition exerted by dopamine D₂ receptors. Inflammatory mediators generated by injury were not involved. These findings reveal the presence of a bilateral modulation of glomerular output driven by dopamine that compensates for imbalances in the number of operative OSNs present in the two olfactory epithelia. Considering that the constant turnover of OSNs is an evolutionarily conserved feature of the olfactory system and determines the innervation of glomeruli, the compensatory mechanism described here may represent a general property of the vertebrate olfactory system to establish an odor map.

The universal features that define genomic regions acting as replication origins remain unclear. In this study, we mapped a set of origins in <i>Trypanosoma brucei</i> using stranded short nascent strand sequencing methods. Our results showed that DNA replication predominantly initiates in intergenic regions between poly(dA)- and poly(dT)-enriched sequences. G4 structures were detected in the vicinity of some origins and were embedded in poly(dA)-enriched sequences in a strand-specific manner: G4s on the plus strand were located upstream while those on the minus strand were located downstream of the centre. The origins' centres were found to be areas of low nucleosome occupancy, surrounded by regions of high nucleosome occupancy. Furthermore, our results demonstrate that 90% of replication origins overlap with a minor proportion of the previously reported RNA: DNA hybrids. These findings shed new light on the sequence and structural features that define the topology of replication origins in <i>T. brucei</i>. To further characterise replication dynamics at the single-molecule level, we employed DNA combing analysis.

Engaging in prosocial behavior requires effort, yet people are often averse to exerting effort for others’ benefit. However, it remains unclear how effort exertion affects subsequent reward evaluation during prosocial acts. Here, we combined high-temporal-resolution electroencephalography with a paradigm that independently manipulated physical effort and monetary reward for self and others to elucidate the neural mechanisms underlying the reward after-effect of prosocial effort expenditure. We found dissociable reward after-effects for self-benefiting and other-benefiting effort. For self-benefiting rewards, the reward positivity (RewP) increased with effort demand, suggesting an effort-enhancement effect. In contrast, for other-benefiting rewards, the RewP decreased as effort increased, demonstrating an effort-discounting effect. Critically, this dissociation was contingent upon high reward magnitude and modulated by individual differences in effort discounting, yet remained distinct from performance evaluation. Our findings reveal distinct neural computations for self- and other-benefiting efforts, offering new insights into how prior effort expenditure shapes reward evaluation during prosocial behavior.

Short-chain fatty acylations establish connections between cell metabolism and regulatory pathways. Lysine acetoacetylation (Kacac) was recently identified as a new histone mark. However, regulatory elements, substrate proteins, and epigenetic functions of Kacac are not yet fully understood, hindering further in-depth understanding of acetoacetate-modulated (patho)physiological processes. Here, we created a chemo-immunological approach for reliable detection of Kacac, and demonstrated that acetoacetate serves as the primary precursor for histone Kacac. We report the enzymatic addition of the Kacac mark by the acyltransferases GCN5, p300, and PCAF, and its removal by the deacetylase HDAC3. Furthermore, we establish acetoacetyl-CoA synthetase as a key regulator of cellular Kacac levels. A comprehensive proteomic analysis has identified 139 Kacac sites on 85 human proteins. Bioinformatics analysis of Kacac substrates and RNA sequencing data reveal the broad impacts of Kacac on multifaceted cellular processes. These findings unveil pivotal regulatory mechanisms for the acetoacetate-mediated Kacac pathway, opening a new avenue for further investigation into ketone body functions in various pathophysiological states.

Neural activity in the dentate gyrus (DG) supports the detection and discrimination of novelty, context, and patterns. Granule cell activation differs between the supra- and infrapyramidal blades across hippocampal-dependent tasks, yet how excitatory dynamics shape this blade-specific bias under varying cognitive demands remains unclear. Here, we combined an automated touchscreen pattern separation task in mice with temporally controlled tagging of active neurons to determine how increasing cognitive demand influences spatial activity patterns in the DG. As task difficulty increased, activation became progressively biased toward the suprapyramidal blade and was accompanied by structured distributions of active mature granule cells (mGCs) along both the radial and transverse axes. Selective inhibition of mGCs did not alter these spatial patterns, but profoundly impaired performance, as mice were no longer able to discriminate between closely spaced locations. In contrast, chemogenetic inhibition of adult-born dentate granule cells (abDGCs) beyond a critical maturation window impaired performance under high-demand conditions, increased overall mGC activity, and disrupted blade-specific organization even in animals that successfully completed the task. These findings demonstrate that high cognitive demand recruits spatially organized mGC activity and support a modulatory role for abDGCs in shaping dentate circuit dynamics.

Episodic memory is critically dependent on the hippocampal network and is frequently impaired in many clinical disorders. Recent findings highlight Hippocampal Indirectly Targeted Stimulation (HITS) as a promising, network-guided non-invasive transcranial magnetic stimulation (TMS) procedure to enhance episodic memory performance. Here, we report the first comprehensive meta-analysis of HITS effects on episodic memory, encompassing both healthy individuals and clinical populations. HITS using parieto-occipital network targets robustly improved episodic memory, with effects selective for episodic memory versus other non-memory cognitive domains. Efficacy was significantly greater when memory performance was assessed using memory tasks sensitive to recollection, which is strongly linked to hippocampal network function, compared to recognition or other types of episodic memory tasks. Efficacy was also significantly greater when HITS was delivered before the memory tasks were administered versus in the period between study and test phases of tasks. No serious adverse events were reported. These findings establish HITS as a robust approach for episodic memory enhancement, suggesting potential for clinical translation in memory disorders. Selectivity of effects for episodic memory generally and for recollection-format tests in particular indicates cognitive and mechanistic specificity, supporting the potential for targeted and selective neuromodulation of hippocampal networks and their associated functions.

The ryanodine receptor (RYR) genes encode evolutionarily conserved calcium release channels involved in a wide range of calcium-dependent biological processes. Here, we show that the sole <i>Drosophila</i> RYR gene (<i>dRyR</i>) functions in differentiated somatic and cardiac muscle as well as in developing embryonic myotubes. In the larval body wall muscles, dRyR protein localizes at the SR membranes, and <i>dRyR</i> knockdown adversely affects muscle contractility, suggesting its conserved role in calcium-triggered E-C coupling. After <i>dRyR</i> attenuation, sarcomere, and mitochondrial patterns are severely impaired, showing <i>dRyR</i> involvement in structural muscle properties. However, <i>dRyR</i> is also prominently expressed and functionally required in growing embryonic muscles. <i>dRyR</i> loss of function leads to myotube growth defects and thin myofiber phenotypes, while its overexpression induces myofiber splitting. Given the structural and functional conservation of <i>dRyR</i>, we used <i>Drosophila</i> to test the impact of one human <i>RYR1</i> variant of unknown significance (VUS). Larvae carrying <i>p.Met4881Ile RYR1</i> VUS showed impaired mobility and altered structural muscle properties reminiscent of those seen in <i>dRyR</i> knockdown, thus indicating it is likely pathogenic. Overall, we show that <i>Drosophila dRyR</i> plays a conserved role in setting muscle contractility and structural muscle features. Our findings underline the still under-investigated role of <i>dRyR</i> as a promyogenic factor and provide a first example of the impact assessment of a human <i>RYR1</i> VUS in <i>Drosophila</i>.

Behavioral flexibility, the ability to adjust behavioral strategies in response to changing environmental contingencies and internal demands, is fundamental to cognitive functions. Despite a large body of pharmacology and lesion studies, the precise neurophysiological mechanisms that underlie behavioral flexibility are still under active investigations. This work is aimed to determine the role of a brainstem-to-prefrontal cortex circuit in flexible rule switching. We trained mice to perform a set-shifting task in which they learned to switch attention to distinguish complex sensory cues. Using chemogenetic inhibition, we selectively targeted genetically defined locus coeruleus (LC) neurons or their input to the medial prefrontal cortex (mPFC). We revealed that suppressing either the LC or its mPFC projections severely impaired switching behavior, establishing the critical role of the LC-mPFC circuit in supporting attentional switching. To uncover the neurophysiological substrates of the behavioral deficits, we paired endoscopic calcium imaging of the mPFC with chemogenetic inhibition of the LC in task-performing mice. We found that mPFC prominently responded to attentional switching and that LC inhibition not only enhanced the engagement of mPFC neurons but also broadened single-neuron tuning in the task. At the population level, LC inhibition disrupted mPFC dynamic changes and impaired the encoding capacity for switching. Our results highlight the profound impact of the ascending LC input on modulating prefrontal dynamics and provide new insights into the cellular and circuit-level mechanisms that support behavioral flexibility.

The meninges, which envelop and protect the brain, host a dense network of resident macrophages with diverse roles in regulating homeostasis and neuroinflammation. Despite their importance, we have a limited understanding of their behavior in vivo. Many dynamic cellular functions of macrophages involve intracellular Ca²⁺ signaling. However, virtually nothing is known about the spatiotemporal Ca²⁺ dynamics of meningeal macrophages in vivo. We developed a chronic intravital two-photon imaging approach and related computational analysis tools to interrogate meningeal macrophage Ca²⁺ dynamics, at subcellular resolution, in a novel Pf4-Cre:Ai162 conditional GCaMP6s reporter mouse model. Using imaging in awake mice, we characterized Ca²⁺ activity in meningeal macrophages at steady state and in response to cortical spreading depolarization (CSD), an aberrant pro-inflammatory brain hyperexcitability event implicated in migraine, traumatic brain injury, and stroke. In homeostatic meninges, macrophages in the dural perivascular niche exhibited several Ca²⁺ dynamic features, including event duration and signal frequency spectrum, distinct from those localized to the interstitial, non-perivascular niche. Simultaneous tracking of macrophage Ca²⁺ dynamics and local vasomotion revealed a subset of dural perivascular macrophages whose activity was coupled to locomotion-driven diameter fluctuations of their associated vessels. Most perivascular and non-perivascular meningeal macrophages displayed propagating intracellular Ca²⁺ activity and synchronized intercellular Ca²⁺ elevations, potentially driven by extrinsic factors. In response to CSD, the majority of perivascular and non-perivascular meningeal macrophages showed a persistent decrease in Ca²⁺ activity, while a smaller subset displayed Ca²⁺ elevations. Mechanistically, calcitonin gene-related peptide receptor signal…