While genetically encoded Ca2+ indicators are valuable for visualizing neural activity, their speed and sensitivity have had limited performance when compared to chemical dyes and electrophysiology, particularly at synaptic compartments. We addressed these limitations by engineering a suite of next-generation GCaMP8-based indicators, targeted to presynaptic boutons, active zones, and postsynaptic compartments at the Drosophila neuromuscular junction. We first validated these sensors to be superior to previous versions and synthetic dyes. Next, we developed a Python-based analysis program, CaFire, which enables the automated quantification of evoked and spontaneous Ca²+ signals. Using CaFire, we show a ratiometric presynaptic GCaMP8m sensor accurately captures physiologically relevant presynaptic Ca2+ changes with superior sensitivity and similar kinetics compared to chemical dyes. Moreover, we test the ability of an active zone-targeted, ratiometric GCaMP8m sensor to report differences in Ca²+ between release sites. Finally, a newly engineered postsynaptic GCaMP8m, positioned near glutamate receptors, detects quantal events with temporal and signal resolution comparable to electrophysiological recordings. These next-generation indicators and analytical methods demonstrate that GCaMP8 sensors, targeted to synaptic compartments, can now achieve the speed and sensitivity necessary to resolve Ca2+ dynamics at levels previously only attainable with chemical dyes or electrophysiology.

The iterative bleaching extends multiplexity (IBEX) Knowledge-Base is a central portal for researchers adopting IBEX and related 2D and 3D immunofluorescence imaging methods. The design of the Knowledge-Base is modeled after efforts in the open-source software community and includes three facets: a development platform (GitHub), static website, and service for data archiving. The Knowledge-Base facilitates the practice of open science throughout the research life cycle by providing validation data for recommended and non-recommended reagents, such as primary and secondary antibodies. In addition to reporting negative data, the Knowledge-Base empowers method adoption and evolution by providing a venue for sharing protocols, videos, datasets, software, and publications. A dedicated discussion forum fosters a sense of community among researchers while addressing questions not covered in published manuscripts. Together, scientists from around the world are advancing scientific discovery at a faster pace, reducing wasted time and effort, and instilling greater confidence in the resulting data.

 Prosapip1 is a brain-specific protein, encoded by Lzts3, localized to the postsynaptic density, where it promotes dendritic spine maturation in primary hippocampal neurons. However, nothing is known about the role of Prosapip1 in vivo. To examine this, we utilized the Cre-loxP system to develop a Prosapip1 neuronal knockout mouse. We found that Prosapip1 controls the synaptic localization of its binding partner SPAR, along with PSD-95 and the GluN2B subunit of the NMDA receptor (NMDAR) in the dorsal hippocampus (dHP). We next sought to identify the potential contribution of Prosapip1 to the activity and function of the NMDAR and found that Prosapip1 plays an important role in NMDAR-mediated transmission and long-term potentiation (LTP) in the CA1 region of the dHP. As LTP is the cellular hallmark of learning and memory, we examined the consequences of neuronal knockout of Prosapip1 on dHP-dependent memory. We found that global or dHP-specific neuronal knockout of Prosapip1 caused a deficit in learning and memor,y whereas developmental, locomotor, and anxiety phenotypes were normal. Taken together, Prosapip1 in the dHP promotes the proper localization of synaptic proteins which, in turn, facilitates LTP driving recognition, social, and spatial learning and memory.

The intrinsic ability of injured neurons to degenerate and regenerate their axons facilitates nervous system repair; however, this ability is not engaged in all neurons and injury locations. Here, we investigate the regulation of a conserved axonal injury response pathway with respect to the location of damage in branched motoneuron (MN) axons in Drosophila larvae. The dileucine zipper kinase (DLK; also known as MAP3K12 in mammals and Wallenda (Wnd) in Drosophila) is a key regulator of diverse responses to axonal injury. In three different populations of MNs, we observed the same striking result that Wnd/DLK signaling becomes activated only in response to injuries that remove all synaptic terminals. Injuries that spared even a small part of a synaptic terminal were insufficient to activate Wnd/DLK signaling, despite the presence of extensive axonal degeneration. The regulation of injury-induced Wnd/DLK signaling occurs independently of its previously known regulator, the Hiw/PHR ubiquitin ligase. We propose that Wnd/DLK signaling regulation is linked to the trafficking of a synapse-to-nucleus axonal cargo and that this mechanism enables neurons to respond to impairments in synaptic connectivity.

Signaling receptors often encounter multiple ligands and have been shown to respond selectively to generate appropriate, context-specific outcomes. At thermal equilibrium, ligand specificity is limited by the relative affinities of ligands for their receptors. Here, we present a non-equilibrium model in which receptors overcome thermodynamic constraints to preferentially signal from specific ligands while suppressing others. In our model, multi-site phosphorylation and active receptor degradation act in concert to regulate ligand specificity, with receptor degradation, a common motif in eukaryotes, providing a previously under-appreciated layer of control. Here, ligand-bound receptors undergo sequential phosphorylation, with progression restarted by ligand unbinding or receptor turnover. High-affinity complexes are kinetically sorted toward degradation-prone states, while low-affinity complexes are sorted toward inactivated states, both limiting signaling. As a result, network activity is maximized for ligands with intermediate affinities. This mechanism explains paradoxical experimental observations in receptor tyrosine kinase signaling, including non-monotonic dependence of signaling output on ligand affinity and kinase activity. Given the ubiquity of multi-site phosphorylation and ligand-induced degradation across signaling receptors, we propose that kinetic sorting may be a general non-equilibrium ligand-discrimination strategy used by multiple signaling receptors.

Therapeutic epigenetic modulation is currently being evaluated in the clinic to sensitize homologous recombination (HR)-proficient tumors to PARP inhibitors. To broaden its clinical applicability and identify more effective combination strategies, we conducted a drug screen combining PARP inhibitors with 74 well-characterized epigenetic modulators targeting five major classes of epigenetic enzymes. Notably, both type I PRMT inhibitors and PRMT5 inhibitors scored highly in combination efficacy and clinical prioritization. PRMT inhibition significantly enhanced PARP inhibitor-induced DNA damage in human HR-proficient ovarian and breast cancer cells. Mechanistically, PRMT suppression downregulates DNA damage repair genes and BRCAness-associated pathways, while also modulating intrinsic innate immune responses within cancer cells. Integrative analysis of large-scale genomic and functional datasets from TCGA and DepMap further supports PRMT1, PRMT4, and PRMT5 as promising therapeutic targets in oncology. Importantly, dual inhibition of PRMT1 and PRMT5 synergistically sensitizes tumors to PARP inhibitors. Collectively, our findings provide strong rationale for the clinical development of PRMT and PARP inhibitor combinations in HR-proficient ovarian and breast cancers.

ATAD2, a conserved protein which is predominantly expressed in embryonic stem (ES) cells and spermatogenic cells, emerges as a crucial regulator of chromatin plasticity. Our previous parallel studies conducted in both ES cells and S. pombe highlighted the fundamental role of ATAD2 in facilitating chromatin-bound histone chaperone turnover. Focusing on mouse spermatogenesis, we demonstrate here that ATAD2 regulates the HIRA-dependent localization of H3.3 on the genome and influences H3.3-mediated gene transcription. Moreover, by modulating histone eviction and the assembly of protamines, ATAD2 ensures proper chromatin condensation and genome packaging in mature sperm. Disruption of Atad2 function in mice leads to abnormal genome organization in mature spermatozoa. Together, these findings establish a previously overlooked level of chromatin dynamic regulation, governed by ATAD2-controlled histone chaperones binding to chromatin, which defines the balance between histone deposition and removal.

Microglia, the brain's resident macrophages, can be reconstituted by surrogate cells - a process termed 'microglia replacement'. To expand the microglia replacement toolkit, we here introduce estrogen-regulated (ER) homeobox B8 (Hoxb8) conditionally immortalized macrophages, a cell model for generation of immune cells from murine bone marrow, as a versatile model for microglia replacement. We find that ER-Hoxb8 macrophages are highly comparable to primary bone marrow-derived macrophages in vitro, and, when transplanted into a microglia-free brain, engraft the parenchyma and differentiate into microglia-like cells. Furthermore, ER-Hoxb8 progenitors are readily transducible by virus and easily stored as stable, genetically manipulated cell lines. As a demonstration of this system's power for studying the effects of disease mutations on microglia in vivo, we created stable, Adar1-mutated ER-Hoxb8 lines using CRISPR-Cas9 to study the intrinsic contribution of macrophages to Aicardi-Goutières syndrome (AGS), an inherited interferonopathy that primarily affects the brain and immune system. We find that Adar1 knockout elicited interferon secretion and impaired macrophage production in vitro, while preventing brain macrophage engraftment in vivo - phenotypes that can be rescued with concurrent mutation of Ifih1 (MDA5) in vitro, but not in vivo. Lastly, we extended these findings by generating ER-Hoxb8 progenitors from mice harboring a patient-specific Adar1 mutation (D1113H). We demonstrated the ability of microglia-specific D1113H mutation to drive interferon production in vivo, suggesting microglia drive AGS neuropathology. In sum, we introduce the ER-Hoxb8 approach to model microglia replacement and use it to clarify macrophage contributions to AGS.

Limbs execute diverse actions coordinated by the nervous system through multiple motor programs. The basic architecture of motor neurons that activate muscles which articulate joints for antagonistic flexion and extension movements is conserved from flies to vertebrates. While excitatory premotor circuits are expected to establish sets of leg motor neurons that work together, our study uncovered an instructive role for inhibitory circuits - including their ability to generate rhythmic leg movements. Using electron microscopy data in the Drosophila nerve cord, we categorized ~120 GABAergic inhibitory neurons from the 13 A and 13B hemilineages into classes based on similarities in morphology and connectivity. By mapping their connections, we uncovered pathways for inhibiting specific groups of motor neurons, disinhibiting antagonistic counterparts, and inducing alternation between flexion and extension. We tested the function of specific inhibitory neurons through optogenetic activation and silencing, using high-resolution quantitative analysis of leg movements during grooming. We combined findings from anatomical and behavioral analyses to construct a computational model that can reproduce major aspects of the observed behavior, demonstrating that these premotor inhibitory circuits can generate rhythmic leg movements.

Transition metals, such as iron and zinc, are indispensable trace elements for eukaryotic life, acting as co-factors in essential processes ranging from metabolism to DNA replication. These metals can be transported into cells by an evolutionary-conserved family of metal transporters; however, how the ubiquitous mammalian parasite Toxoplasma gondii acquires essential metals has been unknown. Here, we have identified and characterised the first iron and zinc importer in T. gondii. This transporter, named ZFT, localised to the parasite plasma membrane and is essential for the parasite's life cycle. We find ZFT is regulated by iron availability and overexpression sensitises cells to excess iron and zinc. Using a conditional knockdown system, we find that knockdown of ZFT leads to reduction in mitochondrial respiration and a switch to a more quiescent lifecycle stage. To confirm transport activity, we find that knockdown of ZFT leads to a reduction in parasite-associated zinc and iron, and ZFT expression complements loss of zinc transporter activity in a yeast model. Further, expression of ZFT in Xenopus oocytes demonstrates direct uptake of iron, which is outcompeted in the presence of zinc. Overall, we have identified the first metal uptake transporter in T. gondii and demonstrated the importance of iron and zinc to the parasite. This finding advances our understanding of how this obligate intracellular parasite acquires nutrients from its host.

Big-conductance, Ca²+-activated K+ (BK) channels consist of Ca²+- and voltage-sensing, pore-forming α (BKα) subunits and regulatory auxiliary β or γ subunits. Concatenated subunit constructs are powerful tools for elucidating subunit stoichiometry in ion channel gating and regulation, allowing control over subunit arrangement, stoichiometry, and mutation. However, the additional S0 transmembrane segment in BKα places its N- and C-termini on opposite sides of the membrane, preventing tandem BK channel subunit construction by conventional methods. To investigate the atypical 'all-or-none' modulatory function of γ subunits and the subunit stoichiometry of BK channel gating, we developed concatenated constructs containing 2 or 4 BKα subunits by splicing them into modular forms that can be co-expressed to form functional channels. These constructs retained voltage and Ca²+ gating properties similar to intact BK channels. By fusing the LRRC26 (γ1) subunit to the N-terminus of tandem BKα constructs, we found that a single γ1 subunit per α subunit tetramer is sufficient to fully modulate the channel. Furthermore, the L312A mutation in the deep pore region exhibited a stoichiometrically graded effect on voltage-gated BK channel activation. In contrast, a V288A mutation at the selectivity filter induced channel inactivation only when present in all four BKα subunits. Thus, by engineering concatenated BKα constructs, we identified three distinct stoichiometric modes of BK channel gating control by LRRC26, the pore, and the selectivity filter. This study offers new molecular tools and advances our understanding of subunit stoichiometry in BK channel gating and modulation.

Kinetoplastids, such as Trypanosoma brucei, are eukaryotes that likely separated from the main lineage at an exceptionally early point in evolution. Consequently, many aspects of kinetoplastid biology differ significantly from other eukaryotic model systems, including yeasts, plants, worms, flies, and mammals. As in many eukaryotes, the T. brucei genome contains repetitive elements at various chromosomal locations, including centromere- and telomere-associated repeats and interspersed retrotransposon elements. T. brucei also contains intermediate-sized and mini-chromosomes that harbour abundant 177 bp repeat arrays and 70 bp repeat elements implicated in Variable Surface Glycoprotein (VSG) gene switching. In many eukaryotes, repetitive elements are assembled in specialised chromatin such as heterochromatin; however, apart from centromere- and telomere-associated repeats, little is known about chromatin-associated proteins that decorate these and other repetitive elements in kinetoplastids. Here, we utilise affinity selection of synthetic TALE DNA binding proteins designed to target specific repeat elements to identify enriched proteins by proteomics. Validating the approach, a telomere repeat binding TelR-TALE identifies many proteins previously implicated in telomere function. Furthermore, the 70R-TALE designed to bind 70 bp repeats indicates that proteins involved in DNA repair are enriched on these elements that reside adjacent to VSG genes. Interestingly, the 177 bp repeat binding 177R-TALE enriches for many kinetochore proteins, suggesting that intermediate-sized and mini-chromosomes assemble kinetochores related in composition to those located on the main megabase chromosomes. This provides a first insight into the chromatin landscape of repetitive regions of the trypanosome genome with relevance for their mechanisms of chromosome integrity, immune evasion, and cell replication.

The macaque genus includes 25 species with diverse social systems, ranging from low to high social tolerance grades. Such interspecific behavioral variability provides a unique model to tackle the evolutionary foundation of primate social brain. Yet, the neuroanatomical correlates of these social tolerance grades remain unknown. To address this question, we expressed social tolerance grades within a novel cognitive framework and analyzed post-mortem structural scans from 12 macaque species. Our results show that amygdala volume is a subcortical predictor of macaques' social tolerance, with high tolerance species exhibiting larger amygdala than low tolerance ones. We further investigated the developmental trajectory of amygdala across social grades and found that intolerant species showed a gradual increase in relative amygdala volume across the lifespan. Unexpectedly, tolerant species exhibited a decrease in relative amygdala volume across the lifespan, contrasting with the age-related increase observed in intolerant species-a developmental pattern previously undescribed in primates. Taken together, these findings provide valuable insights into the cognitive, neuroanatomical, and evolutionary basis of primates' social behaviors.

DNA aptamers are short, single-stranded DNA molecules that bind specifically to a range of targets such as proteins, cells, and small molecules. Typically, they are utilized in the development of therapeutic agents, diagnostics, drug delivery systems, and biosensors. Although aptamers perform well in controlled extracellular environments, their intracellular use has been less explored due to challenges of expressing them in vivo. In this study, we employed the bacterial retron system Eco2 to express a DNA light-up aptamer in Escherichia coli. Our data confirms that structure-guided insertion of the aptamer domain into the non-coding region of the retron enables reverse transcription and biosynthesis of functional aptamer constructs in bacteria. The purified DNA aptamer synthesized under intracellular conditions shows comparable activity to a chemically synthesized control. Our findings demonstrate that retrons can be used to express short DNA aptamers within living cells, potentially broadening and optimizing their application in intracellular settings.

Distal renal tubular acidosis (dRTA) is a disorder characterized by the inability of the collecting duct system to secrete acids during metabolic acidosis. The pathophysiology of dominant or recessive SLC4A1 variant-related dRTA has been linked with the mis-trafficking defect of mutant kAE1 protein. However, in vivo studies in kAE1 R607H dRTA mice and humans have revealed a complex pathophysiology implicating a loss of kAE1-expressing intercalated cells and intracellular relocation of the H+-ATPase in the remaining type-A intercalated cells. These cells also displayed accumulation of ubiquitin and p62 autophagy markers. The highly active transport properties of collecting duct cells require the maintenance of cellular energy and homeostasis, a process dependent on intracellular pH. Therefore, we hypothesized that the expression of dRTA variants affects intracellular pH and autophagy pathways. In this study, we report the characterization of newly identified dRTA variants and provide evidence of abnormal autophagy and degradative pathways in mouse inner medullary collecting duct cells and kidneys from mice expressing kAE1 R607H dRTA mutant protein. We show that reduced transport activity of the kAE1 variants correlated with increased cytosolic pH, reduced ATP synthesis, attenuated downstream autophagic pathways pertaining to the fusion of autophagosomes and lysosomes and/or lysosomal degradative activity. Our study elucidated a close relationship between the expression of defective kAE1 proteins, reduced mitochondrial activity, and decreased autophagy and protein degradative flux.

Mycobacterium tuberculosis, the causative agent of tuberculosis, remains a major global health challenge. Nucleomodulins, bacterial effectors that target the host cell nuclei, are increasingly recognized as key virulence factors, but their roles in mycobacterial pathogenesis remain incompletely elucidated. Here, we characterize a hypothetical protein Rv2577 (designated MmpE) not only as a Fe³+/Zn²+-dependent metallophosphatase but also as a critical nucleomodulin involved in immune evasion and intracellular persistence. MmpE utilizes two nuclear localization signals, RRR20-22 and RRK460-462, to enter the host cell nucleus, where it binds to the promoter region of the vitamin D receptor (VDR) gene, thereby inhibiting host inflammatory gene expression. Additionally, MmpE regulates the PI3K-Akt-mTOR signaling pathway, thereby arresting lysosome maturation. These actions collectively facilitate immune suppression and promote mycobacterial survival in macrophages and in mice. Our findings identify MmpE as a conserved nucleomodulin in mycobacteria and reveal a novel mechanism of MmpE-mediated intracellular survival.

Human vision is characterized by frequent eye movements and constant shifts in visual input, yet our perception of the world remains remarkably stable. Here, we directly demonstrate image-specific foveal feedback to primary visual cortex in the context of saccadic eye movements. To this end, we used a gaze-contingent fMRI paradigm, in which peripheral saccade targets disappeared before they could be fixated. Despite no direct foveal stimulation, we were able to decode peripheral saccade targets from foveal retinotopic areas, demonstrating that image-specific feedback during saccade preparation may underlie this effect. Decoding was sensitive to shape but not semantic category of natural images, indicating feedback of only low-to-mid-level information. Cross-decoding to a control condition with foveal stimulus presentation indicates a shared representational format between foveal feedback and direct stimulation. Moreover, eccentricity-dependent analyses showed a U-shaped decoding curve, confirming that these results are not explained by spillover of peripheral activity or large receptive fields. Finally, fluctuations in foveal decodability covaried with activity in the intraparietal sulcus, thus providing a candidate region for driving foveal feedback. These findings suggest that foveal cortex predicts the features of incoming stimuli through feedback from higher cortical areas, which offers a candidate mechanism underlying stable perception.

Recent advances in electron microscopy (EM) and automated image segmentation have produced synaptic wiring diagrams of the Drosophila central nervous system. A limitation of existing fly connectome datasets is that most sensory neurons are excised during sample preparation, creating a gap between the central and peripheral nervous systems. Here, we bridge this gap by reconstructing wing sensory axons from the Female Adult Nerve Cord (FANC) EM dataset and mapping them to peripheral sensory structures using genetic tools and light microscopy. We confirm the location and identity of known wing mechanosensory neurons and identify previously uncharacterized axons, including a novel population of putative proprioceptors that make monosynaptic connections onto wing steering motor neurons. We also find that adjacent campaniform sensilla on the wing have distinct axon morphologies and postsynaptic partners, suggesting a high degree of specialization in axon pathfinding and synaptic partner matching. The peripheral location and central projections of wing sensory neurons are stereotyped across flies, allowing this wing proprioceptor atlas and genetic toolkit to guide analysis of other fly connectome datasets.

Adaptive goal-directed behavior requires dynamic coordination of movement, motivation, and environmental cues. Among these, cautious actions, where animals adjust their behavior in anticipation of predictable threats, are essential for survival. Yet, their underlying neural mechanisms remain less well understood than those of appetitive behaviors, where caution plays little role. Using calcium imaging in freely moving mice, we show that glutamatergic neurons in the subthalamic nucleus (STN) are robustly engaged by contraversive movement during cue-evoked avoidance and exploratory behavior. Model-based analyses controlling for movement and other covariates revealed that STN neurons additionally encode salient sensory cues, punished errors, and especially cautious responding, where their activity anticipates avoidance actions. Targeted lesions and optogenetic manipulations reveal that STN projections to the midbrain are necessary for executing cued avoidance. These findings identify a critical role for the STN in orchestrating adaptive goal-directed behavior by integrating sensory, motor, and punitive signals to guide timely, cautious actions via its midbrain projections.

Previously, we showed that adult human olfaction retains plasticity in the unilateral processing of molecular chirality (Feng and Zhou, 2019). Using a similar unilateral discrimination protocol across three experiments with human adults (n = 96; 1295 sessions), we now reveal distinct patterns of specificity, generalization, and persistence in olfactory learning, independent of adaptation or task difficulty. Training with binary odor mixtures at varying ratios consistently produced durable gains that transferred across nostrils and generalized to novel mixtures differing in both structure and perceptual quality. Conversely, training with odor enantiomers or concentration differences yielded neither transfer nor generalization, and concentration discrimination learning was short-lived. These results indicate that mixture configural quality is a distinct olfactory attribute from chirality or relative concentration, and that discrimination learning engages plasticity at different stages of olfactory processing depending on the task-relevant attribute. Moreover, they identify mixture discrimination training as a promising strategy for rehabilitating smell loss and cultivating olfactory expertise.