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You, C.

Publications and source records attributed to You, C..

17 recordsLinked to original sources

Sensory cortical disinhibition is required for long-term fear memory in humans

The transformation of transient threats into enduring memories is fundamental to survival, yet how the human brain constructs long-term fear memory remains elusive. Early sensory cortex, traditionally viewed as a passive feature analyzer, is increasingly recognized as an active participant in fear processing. Here, using a multimodal approach combining high-definition -frequency transcranial alternating current stimulation (HD -tACS) with simultaneous EEG-fMRI and transformer-based neural decoding, we tracked fear memory development across early formation, consolidation, and long-term retention stages (assessed at 15 min, 24 h, and 7 days post-conditioning). Spatiotemporal memory representations were evident at all three time points, beginning at an early sensory stage (40-60 ms post-stimulus). Conditioning potentiated occipital -oscillation desynchronization (a marker of visual cortical disinhibition), which predicted the strength of subsequent long-term memory traces. Critically, targeted -tACS over early visual cortex induced V1/V2 inhibition, abolished conditioning-potentiated desynchronization, and selectively disrupted fear-memory consolidation and retention while leaving initial acquisition intact. These findings establish sensory cortical disinhibition as a necessary mechanism for long-term fear memory in humans and identify early sensory cortex as a causal node for interventions targeting persistent maladaptive fear.

neuroscience↗

Metal-induced energy transfer uncovers activation-induced axial reorganization of signaling complexes inside cells

Transmembrane signaling mediated by cytokine receptors orchestrates key cellular processes such as proliferation, differentiation, and immune responses. While numerous high-resolution structures of cytokine receptor ectodomains are available, the structural organization of the largely disordered intracellular domain (ICD) has remained unclear. Here, we interrogate the axial organization of cytokine receptor signaling complexes at the plasma membrane by metal-induced energy transfer (MIET). For this purpose, we leveraged biofunctionalized nanodot arrays (bNDAs) to capture cell surface receptors at a defined distance from the substrate. Readout by fluorescence lifetime imaging microscopy enabled quantifying axial distances of proteins in the plasma membrane of cells at both ensemble and single-molecule levels with a resolution of [~]1 nm. Using the prototypic, biomedically relevant class I cytokine receptor GP130 as a model system, we uncover by MIET that the ICD extends randomly into the cytosol in the resting state, but surprisingly undergoes an axial compaction upon signal activation. These results demonstrate the potential of bNDA-supported MIET for resolving the axial architecture of signaling complexes within the cellular context.

biophysics↗

A Versatile Toolbox for Nanoscale Interrogation of Multiprotein Assemblies inside Living Cells

Quantitative analysis of protein interactions and the formation of higher-order assemblies in living cells remains a major challenge. Here, we introduce a versatile nanopatterning toolbox that employs capillary nanostamping of functionalized polymers to generate high contrast bio-functionalized nanodot arrays (bNDAs) with diameters below 500 nm. By leveraging orthogonal adaptor designs, we achieve robust immobilization of diverse fluorescent protein fusions, enabling simultaneous and selective recruitment of cytosolic and membrane-associated proteins into discrete nanodomains. This approach of forming cytosolic nanodot arrays (cNDAs) provides striking capabilities for dissecting cytosolic multiprotein complexes with molecular precision. Focusing on the assembly of the multimeric myddosome complex, we demonstrate density-dependent recruitment and co-localization of the core components MyD88, IRAK4, IRAK1, and TRAF6 within cNDAs. Super-resolution microscopy reveals distinct nanoscale clustering of MyD88 and IRAK4 and uncovers the ultrastructural architecture of IRAK4 oligomers. These analyses highlight the spatial organization and hierarchical assembly of the myddosome at the nanoscale in the native cellular context. Collectively, our findings establish cNDAs as a powerful platform for reconstituting and analyzing intricate multiprotein assemblies in live cells, offering new opportunities for elucidating the principles of complex protein networks.

biophysics↗

Multi-omic profiling identifies malignant subpopulations and a hypoxia-driven angiogenic axis as therapeutic vulnerabilities in sinonasal squamous cell carcinoma

Sinonasal squamous cell carcinoma (SNSCC) is a rare and aggressive malignancy with limited treatment options, necessitating comprehensive molecular characterization to uncover actionable therapeutic targets. Here, we integrate bulk and single-cell transcriptomics, epigenomics, and DNA methylation profiling to construct a detailed molecular and cellular landscape of SNSCC. Our analysis identifies five transcriptionally and epigenetically distinct malignant subpopulations, including hypoxic (TC1) and proliferative (TC2) clusters, with TC1 showing significant association with poor clinical outcomes. We uncover a hypoxia-driven angiogenic axis, wherein TC1 cells secrete adrenomedullin (ADM), MIF, and VEGFA to activate endothelial tip cells, fostering tumor angiogenesis. Epigenetic reprogramming, characterized by alterations in DNA methylation and chromatin accessibility, underpins these transcriptional programs, with AP-1 transcription factors emerging as key regulators. Notably, ADM expression is epigenetically controlled, correlates with advanced clinical stage, and predicts reduced survival in head and neck cancer patients. This multi-omic study highlights the tumor heterogeneity and microenvironmental interactions driving SNSCC progression, revealing epigenetically regulated pathways as promising therapeutic targets for future intervention.

cancer biology↗

Clonal analysis of murine HSC self-renewal and differentiation in native hematopoiesis

Hematopoietic stem cells (HSC) maintain lifelong hematopoiesis. However, in current clonal analyses with unbiased "barcoding" approaches, steady-state hematopoietic clones in young and middle-aged adults rarely have detectable HSCs, which precludes comprehensive interrogation of HSC clonal behaviors. In the current study, we used the previously described Sleeping Beauty transposon model to investigate HSC self-renewal and differentiation at a clonal level following a lifelong chase that significantly enriched HSC-derived clones. From seventeen mice, we detected over seventy thousand clones in native hematopoiesis that reflected the known HSC differentiation biases observed in transplantation. Our data indicated an intimate connection between megakaryocytic-restricted differentiation and HSC self-renewal expansion. By comparing the differentiation patterns of clones derived from transplanted HSCs, we further demonstrated the abilities of HSCs to preserve their cell fates towards self-renewal or multilineage differentiation. Unlike HSCs, clonal expansion in multipotent progenitors was associated with either a differentiation-active or differentiation-indolent state. Moreover, the clonal expansion events in the more differentiated stem and progenitor cells, but not the most primitive HSCs, drove clonal expansion in the megakaryocyte and myeloid cell lineages. Our study provided a comprehensive portrait of native hematopoiesis at a clonal level and revealed the general patterns in which HSCs maintained self-renewal and multi-lineage differentiation.

developmental biology↗

Long-term single molecule localization microscopy uncovers dynamic co-assembly of Lrp6 and Ror2 into Wnt-signalosomes

The conserved Wnt signaling has been classified as two categories of canonical and noncanonical Wnt signaling. With a high promiscuity of Wnt signaling, how receptors from the two distinct pathways re-arrange in multi-protein signalosomes remains elusive. We here developed single-molecule tracking and localization microscopy based on labeling with reversibly binding nanobodies (rbTALM) for imaging receptor dynamics in the plasma membrane for extended time periods. To this end, we engineered nanobody-tag pairs with fine-tuned binding stabilities ensuring single-molecule tracking with high fidelity, yet continuous exchange of photobleached labels. Multicolor rbTALM imaging enabled simultaneous tracking and super-resolution imaging of three different Wnt co-receptors in the same cell for more than one hour at video rate. Time-lapse correlation analyses uncovered cooperative association of canonical and noncanonical Wnt co-receptors into a common, hybrid Wnt signalosome, demonstrating the exciting possibilities of rbTALM imaging for exploring nanoscale dynamics across millisecond to hour timescales.

biophysics↗

Rescuing missing data in connectome-based predictive modeling

Recent evidence suggests brain-behavior predictions may require very large sample sizes. However, as the sample size increases, the amount of missing data also increases. Conventional methods, like complete-case analysis, discard useful information and shrink the sample size. To address the missing data problem, we investigated rescuing these missing data through imputation. Imputation is the substitution of estimated values for missing data to be used in downstream analyses. We integrated imputation methods into the Connectome-based Predictive Modeling (CPM) framework. Utilizing four open-source datasets--the Human Connectome Project, the Philadelphia Neurodevelopmental Cohort, the UCLA Consortium for Neuropsychiatric Phenomics, and the Healthy Brain Network (HBN)--we validated and compared our framework with different imputation methods against complete-case analysis for both missing connectomes and missing phenotypic measures scenarios. Imputing connectomes exhibited superior prediction performance on real and simulated missing data as compared to complete-case analysis. In addition, we found that imputation accuracy was a good indicator for choosing an imputation method for missing phenotypic measures but not informative for missing connectomes. In a real-world example predicting cognition using the HBN, we rescued 628 individuals through imputation, doubling the complete case sample size and increasing explained variance by 45%. Together, our results suggest that rescuing data with imputation, as opposed to discarding subjects with missing information, improves prediction performance.

neuroscience↗

Single-cell transcriptome analysis reveals the cellular atlas of human intracranial aneurysm and highlights inflammation features associated with aneurysm rupture

Intracranial aneurysm (IA) is pouch-like pathological dilations of cerebral arteries, which often affects middle-aged people and culminates in life-threatening hemorrhagic stroke. A deeper knowledge of the cellular and gene expression perturbations in human IA tissue deepens our understanding of disease mechanisms and facilitates developing pharmacological targets for unruptured IA. In this study, 21,332 qualified cells were obtained from cell-sparse ruptured and unruptured human IA tissues and a detailed cellular profile was determined, including conventional endothelial cells, smooth muscle cells (SMC), fibroblasts and the newly identified pericytes. Notably, striking proportion of immune cells were identified in IA tissue, with the number of monocyte/macrophages and neutrophils being remarkably higher in ruptured IA. By leveraging external datasets and machine learning algorithms, a subset of macrophages characterized by high expression of CCL3 and CXCL3, and transcriptional activation of NF-{kappa}B and HIVEP2 was identified as the cell most associated with IA rupture. Further, the interactome of CCL3/CXCL3 macrophages disclosed their role in regulating vascular cell survival and orchestrating inflammation. In summary, this study illustrated the profile and interactions of vascular and immune cells in human IA tissue and the opportunities for targeting local chronic inflammation.

cell biology↗

Arabidopsis AAR2, a conserved splicing factor in eukaryotes, acts in microRNA biogenesis

MicroRNAs (miRNAs) play an essential role in plant growth and development, and as such, their biogenesis is fine-tuned via regulation of the core microprocessor components. Here, we report that Arabidopsis AAR2, a homolog of a U5 snRNP assembly factor in yeast and humans, not only acts in splicing but also promotes miRNA biogenesis. AAR2 interacts with the microprocessor component HYPONASTIC LEAVES1 (HYL1) in the cytoplasm, nucleus and dicing bodies. In aar2 mutants, abundance of nonphosphorylated HYL1, the active form of HYL1, and the number of HYL1-labeled dicing bodies are reduced. Primary miRNA (pri-miRNA) accumulation is compromised despite normal promoter activities of MIR genes in aar2 mutants. RNA decay assays show that the aar2-1 mutation leads to faster degradation of pri-miRNAs in a HYL1-dependent manner, which reveals a previously unknown and negative role of HYL1 in miRNA biogenesis. Taken together, our findings reveal a dual role of AAR2 in miRNA biogenesis and pre-mRNA splicing. SignificanceIn yeast and humans, AAR2 is involved in pre-mRNA splicing through regulating U5 snRNP assembly. This study shows that Arabidopsis AAR2 promotes microRNA (miRNA) accumulation in addition to its conserved role in pre-mRNA splicing. AAR2 is associated with the microprocessor component HYL1 and promotes its dephosphorylation to produce the active form in miRNA biogenesis. The study also reveals a previously unknown role of HYL1 in causing the degradation of the primary precursors to miRNAs (pri-miRNAs) and a role of AAR2 in protecting pri-miRNAs from HYL1-depedent degradation. Taken together, our findings provide new insights into the role of a conserved splicing factor in miRNA biogenesis in plants.

plant biology↗

Live cell nanodot arrays uncover synergistic co-condensation of Wnt signalodroplets

Qualitative and quantitative analysis of transient signaling platforms in the plasma membrane has remained a key experimental challenge. Here, we have developed biofunctional nanodot arrays (bNDAs) to spatially control dimerization and clustering of cell surface receptors at nanoscale. High-contrast bNDAs with spot diameters of [~]300 nm were obtained by capillary nanostamping of BSA bioconjugates, which were subsequently biofunctionalized by reaction with tandem anti- GFP clamp fusions. We achieved spatially controlled assembly of active Wnt signalosomes at the nanoscale in the plasma membrane of live cells by capturing the co-receptor Lrp6 into bNDAs via an extracellular GFP tag. Strikingly, we observed co-recruitment of co-receptor Frizzled-8 as well as the cytosolic scaffold proteins Axin-1 and Disheveled-2 into Lrp6 nanodots in the absence of ligand. Density variation and the high dynamics of effector proteins uncover highly cooperative liquid-liquid phase separation (LLPS)-driven assembly of Wnt "signalodroplets" at the plasma membrane, pinpointing the synergistic effects of LLPS for Wnt signaling amplification. These insights highlight the potential of bNDAs for systematically interrogating nanoscale signaling platforms and condensation at the plasma membrane of live cells.

biophysics↗

Parallel analysis of RNA ends reveals global microRNA-mediated target RNA cleavage in maize

MicroRNAs (miRNAs) are endogenous 20-to 24-nucleotide (nt) noncoding RNAs that play important regulatory roles in many biological processes in eukaryotes. miRNAs modulate the expression of target genes at the post-transcriptional level by transcript cleavage or translational inhibition. Identification of miRNA target genes have been extensively investigated in Arabidopsis and rice, but an in-depth global analysis of miRNA-mediated target regulation is still lacking in maize. Here, we report a transcriptome-wide identification of miRNA targets by analyzing Parallel Analysis of RNA Ends (PARE) datasets derived from nine different tissues at five developmental stages of the maize (Zea mays L.) B73 cultivar. 246 targets corresponding to 60 miRNAs from 25 families were identified, including transcription factors and other genes. In addition, PARE analysis revealed that miRNAs guide specific target transcript cleavage in a tissue-preferential manner. Interestingly, primary transcripts of MIR159c and MIR169e were found to be cleaved by mature miR159 and miR169, respectively, indicating a positive-feedback regulatory mechanism in miRNA biogenesis. Moreover, several new miRNA-target gene pairs involved in seed germination were identified and experimentally validated. Our PARE analyses generated a wide and detailed miRNA-target interaction atlas, which provides a valuable resource for investigating the roles of miRNAs and their targets in maize.

plant biology↗

Physiological and molecular mechanisms regulated mesophyll conductance under severe drought in water-saving drought-resistant rice

Water-saving and drought-resistant rice (WDR) is a new type of rice varieties. It plays an important role in responding to drought with high yield and has been widely planted in central China at present. High photosynthetic production potential caused by high mesophyll conductance (gm) is the main factor promoted high yield formation in drought for WDR. But little is known about physiological and molecular mechanisms regulated gm in drought for WDR. Therefore, WDR cultivar HY73 and drought-sensitive cultivar HLY898 were used for comparative studies with three irrigation regimes before applying severe drought treatment at heading to create different differential individuals of photosynthetic potential and gm. The results showed that cultivar HY73 had lower up-regulation different expression genes (DEGs) than cultivar HLY898 in drought at transcriptional level. Conversely, DEGs of down-regulation was higher in cultivar HY73 than cultivar HLY898. In addition, 3071 DEGs were clustered in 3 modules named Midnightblue (734 DEGs), Blue (921 DEGs), and Turquoise (1416 DEGs) in severe drought merged three irrigation regimes and both cultivars, which the modules had significant correlational relationship with gm based on weighted gene co-expression network analysis (P<0.05). Only DEGs in midnightblue module were enriched in photosynthesis process and positively regulated gm (P<0.05). The main biological process were photosynthesis (GO:0015979), light harvesting in photosystem I (GO:0009768), reductive pentose-phosphate cycle (GO:0019253), protein-chromophore linkage (GO:0018298), photosynthetic electron transport in photosystem I (GO:0009773), and photosystem II repair (GO:0010206). These results indicate that gm and energy distribution in PSI and PSII systems could synergistic effect photosynthetic production potential in severe drought for rice plants. In the modules, the 18 most highly connected hub genes were screened using co-expression networks method. RT-PCR analysis indicated that CSP41B, PGLP1A, LHCA5, and GSTU6 genes had a similar variation trend with gm among treatments for both cultivar. LHCA5 and CSP41B genes were significantly up-regulated in HY73 compared with HLY898 in drought (P<0.05). And the both genes locates in thylakoid membrane in photosystems. Therefore, LHCA5 and CSP41B genes could be key genes to synergistically manage gm and energy distribution in photosystems. Our results provide some new physiological and molecular mechanisms regulated gm in severe drought for WDR.

physiology↗

Structural insights into the peptide selectivity and activation of human neuromedin U receptors

Neuromedin U receptors (NMURs), including NMUR1 and NMUR2, are a group of Gq/11-coupled G protein-coupled receptors (GPCRs) related to pleiotropic physiological functions. Upon stimulation by two endogenous neuropeptides, neuromedin U and S (NMU and NMS) with similar binding affinities, NMUR1 and NMUR2 primarily display distinct peripheral tissue and central nervous system (CNS) functions, respectively, due to their distinct tissue distributions. These NMU receptors have triggered extensive attention as drug targets for obesity and immune inflammation. Specifically, selective agonists for NMUR1 in peripheral tissue show promising long-term anti-obesity effects with fewer CNS-related side effects. However, the mechanisms of peptide binding specificity and receptor activation remain elusive due to the lack of NMU receptor structures, which hamper drug design targeting NMU receptors. Here, we report four cryo-electron microscopy structures of Gq chimera-coupled NMUR1 and NMUR2 bound with NMU and NMS. These structures present the conserved overall peptide-binding mode and reveal the mechanism of peptide selectivity for specific NMURs, as well as the common activation mechanism of the NMUR subfamily. Together, these findings provide insights into the molecular basis of the peptide recognition selectivity and offer a new opportunity for designing selective drugs targeting NMURs.

biophysics↗

Structural basis for recognition of anti-migraine drug lasmiditan by the serotonin receptor 5-HT1F-G protein complex

Migraine headache has become global pandemics and is the number one reason of work day loss. The most common drugs for anti-migraine are the triptan class of drugs that are agonists for serotonin receptors 5-HT1B and 5-HT1D. However, these drugs have side effects related to vasoconstriction that could have fatal consequences of ischemic heart disease and myocardial infarction. Lasmiditan is a new generation of anti-migraine drug that selectively binds to the serotonin receptor 5-HT1F due to its advantage over the tripan class of anti-migraine drugs. Here we report the cryo-EM structure of the 5-HT1F in complex with Lasmiditan and the inhibitory G protein heterotrimer. The structure reveals the mechanism of 5-HT1F-selective activation by Lasmiditan and provides a template for rational design of anti-migraine drugs.

biophysics↗

TIN2 facilitates TRF1-mediated trans- and cis-interactions on physiologically relevant long telomeric DNA

The shelterin complex consisting of TRF1, TRF2, RAP1, TIN2, TPP1, and POT1, functions to prevent false recognition of telomeres as double-strand DNA breaks, and to regulate telomerase and DNA repair protein access. TIN2 is a core component linking double-stranded telomeric DNA binding proteins (TRF1 and TRF2) and proteins at the 3 overhang (TPP1-POT1). Since knockdown of TIN2 also removes TRF1 and TRF2 from telomeres, determining TIN2s unique mechanistic function has been elusive. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 using complementary single-molecule imaging platforms, including atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that TIN2S and TIN2L isoforms facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (6 TTAGGG repeats), the majority of TRF1 mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG), TIN2 forms multi-protein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last for at least minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 significantly reduces TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protects the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Our study provides evidence that TIN2 functions to promote TRF1 mediated trans-interactions of telomeric DNA, leading to new mechanistic insight into sister telomere cohesion.

biophysics↗

Estrogen Receptor α Regulates Ethanol Excitation of Ventral Tegmental Area Neurons and Binge Drinking in Female Mice

Elevations in estrogen (17{beta}-estradiol, E2) are associated with increased alcohol drinking by women and experimentally in rodents. E2 alters the activity of the dopamine system, including the ventral tegmental area (VTA) and its projection targets, which plays an important role in binge drinking. A previous study demonstrated that during high E2 states, VTA dopamine neurons in female mice are more sensitive to ethanol excitation. However, the mechanisms responsible for the ability of E2 to enhance ethanol sensitivity of VTA dopamine neurons have not been investigated. In this study, we used selective agonists and antagonists to examine the role of estrogen receptor subtypes (ER and ER{beta}) in regulating the ethanol sensitivity of VTA dopamine neurons and found that ER promotes the enhanced ethanol response of VTA dopamine neurons. We also demonstrated that the E2-induced increase in ethanol excitation requires the activity of the metabotropic glutamate receptor, mGluR1, which is known to couple with ER at the plasma membrane. To investigate the behavioral relevance of these findings, we administered lentivirus expressing short hairpin RNAs targeting either ER or ER{beta} into the VTA and found that knockdown of each receptor in the VTA reduced binge-like ethanol drinking in female, but not male, mice. Reducing ER in the VTA had a more dramatic effect on binge-like drinking than reducing ER{beta}, consistent with the ability of ER to alter ethanol sensitivity of dopamine neurons. These results provide important insight into sex-specific mechanisms that drive excessive alcohol drinking.\n\nSignificance StatementEstrogen has potent effects on the dopamine system and increases the vulnerability of females to develop addiction to substances such as cocaine and alcohol. We investigated the mechanisms by which estrogen increases the response of dopamine neurons in the ventral tegmental area to ethanol. We found that activation of the estrogen receptor, ER, increased the ethanol-induced excitation of dopamine neurons and that this required the metabotropic glutamate receptor mGluR1. We also demonstrated that estrogen receptors in the ventral tegmental area regulate binge-like alcohol drinking by female, but not male, mice. The influence of estrogen receptors on binge drinking in female mice suggests that treatments for alcohol use disorder in women may need to account for this sex difference.

neuroscience↗

NuSeT: A Deep Learning Tool for Reliably Separating and Analyzing Crowded Cells

Segmenting cell nuclei within microscopy images is a ubiquitous task in biological research and clinical applications. Unfortunately, segmenting low-contrast overlapping objects that may be tightly packed is a major bottleneck in standard deep learning-based models. We report a Nuclear Segmentation Tool (NuSeT) based on deep learning that accurately segments nuclei across multiple types of fluorescence imaging data. Using a hybrid network consisting of U-Net and Region Proposal Networks (RPN), followed by a watershed step, we have achieved superior performance in detecting and delineating nuclear boundaries in 2D and 3D images of varying complexities. By using foreground normalization and additional training on synthetic images containing non-cellular artifacts, NuSeT improves nuclear detection and reduces false positives. NuSeT addresses common challenges in nuclear segmentation such as variability in nuclear signal and shape, limited training sample size, and sample preparation artifacts. Compared to other segmentation models, NuSeT consistently fares better in generating accurate segmentation masks and assigning boundaries for touching nuclei.

bioinformatics↗