The efficient knock-in of large DNA fragments to label endogenous proteins remains especially challenging in non-dividing cells such as neurons. We developed Targeted Knock-In with Two (TKIT) guides as a novel CRISPR/Cas9 based approach for efficient, and precise, genomic knock-in. Through targeting non-coding regions TKIT is resistant to INDEL mutations. We demonstrate TKIT labeling of endogenous synaptic proteins with various tags, with efficiencies up to 42% in mouse primary cultured neurons. Utilizing in utero electroporation or viral injections in mice TKIT can label AMPAR subunits with Super Ecliptic pHluorin, enabling visualization of endogenous AMPARs in vivo using two-photon microscopy. We further use TKIT to assess the mobility of endogenous AMPARs using fluorescence recovery after photobleaching. Finally, we show that TKIT can be used to tag AMPARs in rat neurons, demonstrating precise genome editing in another model organism and highlighting the broad potential of TKIT as a method to visualize endogenous proteins.

Since the Black Lives Matter movement rose to mainstream prominence, the academic enterprise has started recognizing the systematic racism present in science. However, there have been relatively few efforts to make sure that the language used to communicate science is inclusive. Here, I quantify the number of research articles published between 2000 and 2020 that contained non-inclusive terms with racial connotations, such as "blacklist" and "whitelist", or "master" and "slave". This reveals that non-inclusive language is being increasingly used in the life sciences literature, and I urge the global academic community to expunge these archaic terms to make science inclusive for everyone.

Mammalian target of rapamycin complex 1 (TORC1) is controlled by the GATOR complex composed of the GATOR1 subcomplex and its inhibitor, the GATOR2 subcomplex, sensitive to amino acid starvation. Previously, we identified fission yeast GATOR1 that prevents deregulated activation of TORC1 (Chia et al., 2017). Here, we report identification and characterization of GATOR2 in fission yeast. Unexpectedly, the GATOR2 subunit Sea3, an ortholog of mammalian WDR59, is physically and functionally proximal to GATOR1, rather than GATOR2, attenuating TORC1 activity. The fission yeast GATOR complex is dispensable for TORC1 regulation in response to amino acid starvation, which instead activates the Gcn2 pathway to inhibit TORC1 and induce autophagy. On the other hand, nitrogen starvation suppresses TORC1 through the combined actions of the GATOR1-Sea3 complex, the Gcn2 pathway, and the TSC complex, another conserved TORC1 inhibitor. Thus, multiple, parallel signaling pathways implement negative regulation of TORC1 to ensure proper cellular starvation responses.

The enhancement of associative synaptic plasticity often results in impaired rather than enhanced learning. Previously, we proposed that such learning impairments can result from saturation of the plasticity mechanism (Nguyen-Vu et al., 2017), or, more generally, from a history-dependent change in the threshold for plasticity. This hypothesis was based on experimental results from mice lacking two class I major histocompatibility molecules, MHCI H2-Kb and H2-Db (MHCI KbDb-/-), which have enhanced associative long-term depression at the parallel fiber-Purkinje cell synapses in the cerebellum (PF-Purkinje cell LTD). Here, we extend this work by testing predictions of the threshold metaplasticity hypothesis in a second mouse line with enhanced PF-Purkinje cell LTD, the Fmr1 knockout mouse model of Fragile X syndrome (FXS). Mice lacking Fmr1 gene expression in cerebellar Purkinje cells (L7-Fmr1 KO) were selectively impaired on two oculomotor learning tasks in which PF-Purkinje cell LTD has been implicated, with no impairment on LTD-independent oculomotor learning tasks. Consistent with the threshold metaplasticity hypothesis, behavioral pre-training designed to reverse LTD at the PF-Purkinje cell synapses eliminated the oculomotor learning deficit in the L7-Fmr1 KO mice, as previously reported in MHCI KbDb-/-mice. In addition, diazepam treatment to suppress neural activity and thereby limit the induction of associative LTD during the pre-training period also eliminated the learning deficits in L7-Fmr1 KO mice. These results support the hypothesis that cerebellar LTD-dependent learning is governed by an experience-dependent sliding threshold for plasticity. An increased threshold for LTD in response to elevated neural activity would tend to oppose firing rate stability, but could serve to stabilize synaptic weights and recently acquired memories. The metaplasticity perspective could inform the development of new clinical approaches for addressing learning impairments in autism and other disorders of the nervous system.

Perceiving biological motion (BM) is crucial for human survival and social interaction. Many studies have reported impaired BM perception in autism spectrum disorder, which is characterised by deficits in social interaction. Children with attention deficit hyperactivity disorder (ADHD) often exhibit similar difficulties in social interaction. However, few studies have investigated BM perception in children with ADHD. Here, we compared differences in the ability to process local kinematic and global configurational cues, two fundamental abilities of BM perception, between typically developing and ADHD children. We further investigated the relationship between BM perception and social interaction skills measured using the Social Responsiveness Scale and examined the contributions of latent factors (e.g. sex, age, attention, and intelligence) to BM perception. The results revealed that children with ADHD exhibited atypical BM perception. Local and global BM processing showed distinct features. Local BM processing ability was related to social interaction skills, whereas global BM processing ability significantly improved with age. Critically, general BM perception (i.e. both local and global BM processing) may be affected by sustained attentional ability in children with ADHD. This relationship was primarily mediated by reasoning intelligence. These findings elucidate atypical BM perception in ADHD and the latent factors related to BM perception. Moreover, this study provides new evidence that BM perception is a hallmark of social cognition and advances our understanding of the potential roles of local and global processing in BM perception and social cognitive disorders.

Asymmetric cell divisions (ACDs) generate two daughter cells with identical genetic information but distinct cell fates through epigenetic mechanisms. However, the process of partitioning different epigenetic information into daughter cells remains unclear. Here, we demonstrate that the nucleosome remodeling and deacetylase (NuRD) complex is asymmetrically segregated into the surviving daughter cell rather than the apoptotic one during ACDs in Caenorhabditis elegans. The absence of NuRD triggers apoptosis via the EGL-1-CED-9-CED-4-CED-3 pathway, while an ectopic gain of NuRD enables apoptotic daughter cells to survive. We identify the vacuolar H+-adenosine triphosphatase (V-ATPase) complex as a crucial regulator of NuRD's asymmetric segregation. V-ATPase interacts with NuRD and is asymmetrically segregated into the surviving daughter cell. Inhibition of V-ATPase disrupts cytosolic pH asymmetry and NuRD asymmetry. We suggest that asymmetric segregation of V-ATPase may cause distinct acidification levels in the two daughter cells, enabling asymmetric epigenetic inheritance that specifies their respective life-versus-death fates.

The lateral parafacial area (pFL) is a crucial region involved in respiratory control, particularly in generating active expiration through an expiratory oscillatory network. Active expiration involves rhythmic abdominal (ABD) muscle contractions during late-expiration, increasing ventilation during elevated respiratory demands. The precise anatomical location of the expiratory oscillator within the ventral medulla's rostro-caudal axis is debated. While some studies point to the caudal tip of the facial nucleus (VIIc) as the oscillator's core, others suggest more rostral areas. Our study employed bicuculline (a γ-aminobutyric acid type A [GABA-A] receptor antagonist) injections at various pFL sites (-0.2 mm to +0.8 mm from VIIc) to investigate the impact of GABAergic disinhibition on respiration. These injections consistently elicited ABD recruitment, but the response strength varied along the rostro-caudal zone. Remarkably, the most robust and enduring changes in tidal volume, minute ventilation, and combined respiratory responses occurred at more rostral pFL locations (+0.6/+0.8 mm from VIIc). Multivariate analysis of the respiratory cycle further differentiated between locations, revealing the core site for active expiration generation with this experimental approach. Our study advances our understanding of neural mechanisms governing active expiration and emphasizes the significance of investigating the rostral pFL region.

Multicellular organisms are composed of specialized cell types with distinct proteomes. While recent advances in single-cell transcriptome analyses have revealed differential expression of mRNAs, cellular diversity in translational profiles remains underinvestigated. By performing RNA-seq and Ribo-seq in genetically defined cells in the Drosophila brain, we here revealed substantial post-transcriptional regulations that augment the cell-type distinctions at the level of protein expression. Specifically, we found that translational efficiency of proteins fundamental to neuronal functions, such as ion channels and neurotransmitter receptors, was maintained low in glia, leading to their preferential translation in neurons. Notably, distribution of ribosome footprints on these mRNAs exhibited a remarkable bias toward the 5' leaders in glia. Using transgenic reporter strains, we provide evidence that the small upstream open-reading frames in the 5' leader confer selective translational suppression in glia. Overall, these findings underscore the profound impact of translational regulation in shaping the proteomics for cell-type distinction and provide new insights into the molecular mechanisms driving cell-type diversity.

Immune checkpoint inhibitors have produced encouraging results in cancer patients. However, the majority of ß-catenin-mutated tumors have been described as lacking immune infiltrates and resistant to immunotherapy. The mechanisms by which oncogenic ß-catenin affects immune surveillance remain unclear. Herein, we highlighted the involvement of ß-catenin in the regulation of the exosomal pathway and, by extension, in immune/cancer cell communication in hepatocellular carcinoma (HCC). We showed that mutated ß-catenin represses expression of SDC4 and RAB27A, two main actors in exosome biogenesis, in both liver cancer cell lines and HCC patient samples. Using nanoparticle tracking analysis and live-cell imaging, we further demonstrated that activated ß-catenin represses exosome release. Then, we demonstrated in 3D spheroid models that activation of β-catenin promotes a decrease in immune cell infiltration through a defect in exosome secretion. Taken together, our results provide the first evidence that oncogenic ß-catenin plays a key role in exosome biogenesis. Our study gives new insight into the impact of ß-catenin mutations on tumor microenvironment remodeling, which could lead to the development of new strategies to enhance immunotherapeutic response.

Rapid eye movement sleep (REMs) is characterized by activated electroencephalogram (EEG) and muscle atonia, accompanied by vivid dreams. REMs is homeostatically regulated, ensuring that any loss of REMs is compensated by a subsequent increase in its amount. However, the neural mechanisms underlying the homeostatic control of REMs are largely unknown. Here, we show that GABAergic neurons in the preoptic area of the hypothalamus projecting to the tuberomammillary nucleus (POAGAD2→TMN neurons) are crucial for the homeostatic regulation of REMs in mice. POAGAD2→TMN neurons are most active during REMs, and inhibiting them specifically decreases REMs. REMs restriction leads to an increased number and amplitude of calcium transients in POAGAD2→TMN neurons, reflecting the accumulation of REMs pressure. Inhibiting POAGAD2→TMN neurons during REMs restriction blocked the subsequent rebound of REMs. Our findings reveal a hypothalamic circuit whose activity mirrors the buildup of homeostatic REMs pressure during restriction and that is required for the ensuing rebound in REMs.

Imidacloprid is a global health threat that severely poisons the economically and ecologically important honeybee pollinator, Apis mellifera. However, its effects on developing bee larvae remain largely unexplored. Our pilot study showed that imidacloprid causes developmental delay in bee larvae, but the underlying toxicological mechanisms remain incompletely understood. In this study, we exposed bee larvae to imidacloprid at environmentally relevant concentrations of 0.7, 1.2, 3.1, and 377 ppb. There was a marked dose-dependent delay in larval development, characterized by reductions in body mass, width, and growth index. However, imidacloprid did not affect on larval survival and food consumption. The primary toxicological effects induced by elevated concentrations of imidacloprid (377 ppb) included inhibition of neural transmission gene expression, induction of oxidative stress, gut structural damage, and apoptosis, inhibition of developmental regulatory hormones and genes, suppression of gene expression levels involved in proteolysis, amino acid transport, protein synthesis, carbohydrate catabolism, oxidative phosphorylation, and glycolysis energy production. In addition, we found that the larvae may use antioxidant defenses and P450 detoxification mechanisms to mitigate the effects of imidacloprid. Ultimately, this study provides the first evidence that environmentally exposed imidacloprid can affect the growth and development of bee larvae by disrupting molting regulation and limiting the metabolism and utilization of dietary nutrients and energy. These findings have broader implications for studies assessing pesticide hazards in other juvenile animals.

The complex of methyltransferase-like proteins 3 and 14 (METTL3-14) is the major enzyme that deposits N6-methyladenosine (m6A) modifications on messenger RNA (mRNA) in humans. METTL3-14 plays key roles in various biological processes through its methyltransferase (MTase) activity. However, little is known about its substrate recognition and methyl transfer mechanism from its cofactor and methyl donor S-adenosylmethionine (SAM). Here, we study the MTase mechanism of METTL3-14 by a combined experimental and multiscale simulation approach using bisubstrate analogues (BAs), conjugates of a SAM-like moiety connected to the N6-atom of adenosine. Molecular dynamics simulations based on crystal structures of METTL3-14 with BAs suggest that the Y406 side chain of METTL3 is involved in the recruitment of adenosine and release of m6A. A crystal structure with a BA representing the transition state of methyl transfer shows a direct involvement of the METTL3 side chains E481 and K513 in adenosine binding which is supported by mutational analysis. Quantum mechanics/molecular mechanics (QM/MM) free energy calculations indicate that methyl transfer occurs without prior deprotonation of adenosine-N6. Furthermore, the QM/MM calculations provide further support for the role of electrostatic contributions of E481 and K513 to catalysis. The multidisciplinary approach used here sheds light on the (co)substrate binding mechanism, catalytic step, and (co)product release, and suggests that the latter step is rate-limiting for METTL3. The atomistic information on the substrate binding and methyl transfer reaction of METTL3 can be useful for understanding the mechanisms of other RNA MTases and for the design of transition state analogues as their inhibitors.

Eukaryotic gene expression is linked to chromatin structure and nucleosome positioning by ATP-dependent chromatin remodelers that establish and maintain nucleosome-depleted regions (NDRs) near transcription start sites. Conserved yeast RSC and ISW2 remodelers exert antagonistic effects on nucleosomes flanking NDRs, but the temporal dynamics of remodeler search, engagement, and directional nucleosome mobilization for promoter accessibility are unknown. Using optical tweezers and two-color single-particle imaging, we investigated the Brownian diffusion of RSC and ISW2 on free DNA and sparse nucleosome arrays. RSC and ISW2 rapidly scan DNA by one-dimensional hopping and sliding, respectively, with dynamic collisions between remodelers followed by recoil or apparent co-diffusion. Static nucleosomes block remodeler diffusion resulting in remodeler recoil or sequestration. Remarkably, both RSC and ISW2 use ATP hydrolysis to translocate mono-nucleosomes processively at ~30 bp/s on extended linear DNA under tension. Processivity and opposing push-pull directionalities of nucleosome translocation shown by RSC and ISW2 shape the distinctive landscape of promoter chromatin.

Habitat loss and fragmentation per se have been shown to be a major threat to global biodiversity and ecosystem function. However, little is known about how habitat loss and fragmentation per se alters the relationship between biodiversity and ecosystem function (BEF relationship) in the natural landscape context. Based on 130 landscapes identified by a stratified random sampling in the agro-pastoral ecotone of northern China, we investigated the effects of landscape context (habitat loss and fragmentation per se) on plant richness, above-ground biomass, and the relationship between them in grassland communities using a structural equation model. We found that habitat loss directly decreased plant richness and hence decreased above-ground biomass, while fragmentation per se directly increased plant richness and hence increased above-ground biomass. Fragmentation per se also directly decreased soil water content and hence decreased above-ground biomass. Meanwhile, habitat loss decreased the magnitude of the positive relationship between plant richness and above-ground biomass by reducing the percentage of grassland specialists in the community, while fragmentation per se had no significant modulating effect on this relationship. These results demonstrate that habitat loss and fragmentation per se have inconsistent effects on BEF, with the BEF relationship being modulated by landscape context. Our findings emphasise that habitat loss rather than fragmentation per se can weaken the positive BEF relationship by decreasing the degree of habitat specialisation of the community.

Down syndrome (DS) is characterized by skeletal and brain structural malformations, cognitive impairment, altered hippocampal metabolite concentration and gene expression imbalance. These alterations were usually investigated separately, and the potential rescuing effects of green tea extracts enriched in epigallocatechin-3-gallate (GTE-EGCG) provided disparate results due to different experimental conditions. We overcame these limitations by conducting the first longitudinal controlled experiment evaluating genotype and GTE-EGCG prenatal chronic treatment effects before and after treatment discontinuation. Our findings revealed that the Ts65Dn mouse model reflected the pleiotropic nature of DS, exhibiting brachycephalic skull, ventriculomegaly, neurodevelopmental delay, hyperactivity, and impaired memory robustness with altered hippocampal metabolite concentration and gene expression. GTE-EGCG treatment modulated most systems simultaneously but did not rescue DS phenotypes. On the contrary, the treatment exacerbated trisomic phenotypes including body weight, tibia microarchitecture, neurodevelopment, adult cognition, and metabolite concentration, not supporting the therapeutic use of GTE-EGCG as a prenatal chronic treatment. Our results highlight the importance of longitudinal experiments assessing the co-modulation of multiple systems throughout development when characterizing preclinical models in complex disorders and evaluating the pleiotropic effects and general safety of pharmacological treatments.

Current models of scene processing in the human brain include three scene-selective areas: the parahippocampal place area (or the temporal place areas), the restrosplenial cortex (or the medial place area), and the transverse occipital sulcus (or the occipital place area). Here, we challenged this model by showing that at least one other scene-selective site can also be detected within the human posterior intraparietal gyrus. Despite the smaller size of this site compared to the other scene-selective areas, the posterior intraparietal gyrus scene-selective (PIGS) site was detected consistently in a large pool of subjects (n = 59; 33 females). The reproducibility of this finding was tested based on multiple criteria, including comparing the results across sessions, utilizing different scanners (3T and 7T) and stimulus sets. Furthermore, we found that this site (but not the other three scene-selective areas) is significantly sensitive to ego-motion in scenes, thus distinguishing the role of PIGS in scene perception relative to other scene-selective areas. These results highlight the importance of including finer scale scene-selective sites in models of scene processing - a crucial step toward a more comprehensive understanding of how scenes are encoded under dynamic conditions.

The physical basis of phase separation is thought to consist of the same types of bonds that specify conventional macromolecular interactions yet is unsatisfyingly often referred to as 'fuzzy'. Gaining clarity on the biogenesis of membraneless cellular compartments is one of the most demanding challenges in biology. Here, we focus on the chromosome passenger complex (CPC), that forms a chromatin body that regulates chromosome segregation in mitosis. Within the three regulatory subunits of the CPC implicated in phase separation - a heterotrimer of INCENP, Survivin, and Borealin - we identify the contact regions formed upon droplet formation using hydrogen/deuterium exchange mass spectrometry (HXMS). These contact regions correspond to some of the interfaces seen between individual heterotrimers within the crystal lattice they form. A major contribution comes from specific electrostatic interactions that can be broken and reversed through initial and compensatory mutagenesis, respectively. Our findings reveal structural insight for interactions driving liquid-liquid demixing of the CPC. Moreover, we establish HXMS as an approach to define the structural basis for phase separation.

Synapse is the fundamental structure for neurons to transmit information between cells. The proper synapse formation is crucial for developing neural circuits and cognitive functions of the brain. The aberrant synapse formation has been proved to cause many neurological disorders, including autism spectrum disorders and intellectual disability. Synaptic cell adhesion molecules (CAMs) are thought to play a major role in achieving mechanistic cell-cell recognition and initiating synapse formation via trans-synaptic interactions. Due to the diversity of synapses in different brain areas, circuits and neurons, although many synaptic CAMs, such as Neurexins (NRXNs), Neuroligins (NLGNs), Synaptic cell adhesion molecules (SynCAMs), Leucine-rich-repeat transmembrane neuronal proteins (LRRTMs), and SLIT and NTRK-like protein (SLITRKs) have been identified as synaptogenic molecules, how these molecules determine specific synapse formation and whether other molecules driving synapse formation remain undiscovered are unclear. Here, to provide a tool for synapse labeling and synaptic CAMs screening by artificial synapse formation (ASF) assay, we generated synaptotagmin-1-tdTomato (Syt1-tdTomato) transgenic mice by inserting the tdTomato-fused synaptotagmin-1 coding sequence into the genome of C57BL/6J mice. In the brain of Syt1-tdTomato transgenic mice, the tdTomato-fused synaptotagmin-1 (SYT1-tdTomato) signals were widely observed in different areas and overlapped with synapsin-1, a widely-used synaptic marker. In the olfactory bulb, the SYT1-tdTomato signals are highly enriched in the glomerulus. In the cultured hippocampal neurons, the SYT1-tdTomato signals showed colocalization with several synaptic markers. Compared to the wild-type (WT) mouse neurons, cultured hippocampal neurons from Syt1-tdTomato transgenic mice presented normal synaptic neurotransmission. In ASF assays, neurons from Syt1-tdTomato transgenic mice could form synaptic connections with HEK293T cells expressing NLGN2, LRRTM2, and SLITRK2 without immunostaining. Therefore, our work suggested that the Syt1-tdTomato transgenic mice with the ability to label synapses by tdTomato, and it will be a convenient tool for screening synaptogenic molecules.

Voltage-gated sodium channels (Naᵥ) are membrane proteins which open to facilitate the inward flux of sodium ions into excitable cells. In response to stimuli, Naᵥ channels transition from the resting, closed state to an open, conductive state, before rapidly inactivating. Dysregulation of this functional cycle due to mutations causes diseases including epilepsy, pain conditions, and cardiac disorders, making Naᵥ channels a significant pharmacological target. Phosphoinositides are important lipid cofactors for ion channel function. The phosphoinositide PI(4,5)P2 decreases Naᵥ1.4 activity by increasing the difficulty of channel opening, accelerating fast inactivation and slowing recovery from fast inactivation. Using multiscale molecular dynamics simulations, we show that PI(4,5)P2 binds stably to inactivated Naᵥ at a conserved site within the DIV S4-S5 linker, which couples the voltage-sensing domain (VSD) to the pore. As the Naᵥ C-terminal domain is proposed to also bind here during recovery from inactivation, we hypothesize that PI(4,5)P2 prolongs inactivation by competitively binding to this site. In atomistic simulations, PI(4,5)P2 reduces the mobility of both the DIV S4-S5 linker and the DIII-IV linker, responsible for fast inactivation, slowing the conformational changes required for the channel to recover to the resting state. We further show that in a resting state Naᵥ model, phosphoinositides bind to VSD gating charges, which may anchor them and impede VSD activation. Our results provide a mechanism by which phosphoinositides alter the voltage dependence of activation and the rate of recovery from inactivation, an important step for the development of novel therapies to treat Naᵥ-related diseases.

Plant pathogens secrete proteins, known as effectors, that function in the apoplast or inside plant cells to promote virulence. Effector recognition by cell-surface or cytosolic receptors results in the activation of defence pathways and plant immunity. Despite their importance, our general understanding of fungal effector function and recognition by immunity receptors remains poor. One complication often associated with effectors is their high sequence diversity and lack of identifiable sequence motifs precluding prediction of structure or function. In recent years, several studies have demonstrated that fungal effectors can be grouped into structural classes, despite significant sequence variation and existence across taxonomic groups. Using protein X-ray crystallography, we identify a new structural class of effectors hidden within the secreted in xylem (SIX) effectors from Fusarium oxysporum f. sp. lycopersici (Fol). The recognised effectors Avr1 (SIX4) and Avr3 (SIX1) represent the founding members of the Fol dual-domain (FOLD) effector class, with members containing two distinct domains. Using AlphaFold2, we predicted the full SIX effector repertoire of Fol and show that SIX6 and SIX13 are also FOLD effectors, which we validated experimentally for SIX6. Based on structural prediction and comparisons, we show that FOLD effectors are present within three divisions of fungi and are expanded in pathogens and symbionts. Further structural comparisons demonstrate that Fol secretes effectors that adopt a limited number of structural folds during infection of tomato. This analysis also revealed a structural relationship between transcriptionally co-regulated effector pairs. We make use of the Avr1 structure to understand its recognition by the I receptor, which leads to disease resistance in tomato. This study represents an important advance in our understanding of Fol-tomato, and by extension plant-fungal interactions, which will assist in the development of novel control and engineering strategies to combat plant pathogens.