Biology subjects

Yi, J.

Publications and source records attributed to Yi, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Compact lens-based dual-channel adaptive optics scanning laser ophthalmoscopy for in-vivo three-dimensional retinal imaging in mice

Adaptive optics (AO) enables cellular-resolution retinal imaging, yet mirror-based AOSLO systems are constrained by off-axis aberrations that restrict high-quality imaging to narrow fields of view, limiting in vivo studies of dynamic, large-scale retinal processes. Clinical translation of regenerative cell therapy to neurologic disease is hampered by attrition of donor neurons following transplantation. We hypothesized that early innate neuroinflammatory responses to retinal ganglion cell (RGC) transplantation underlie early death of donor cells and that next-generation imaging technologies would provide evidence for microglial attack of grafted neurons. We present a compact refractive lens-based AOSLO system that achieves two-color fluorescence imaging across up to a 16{degrees} field of view in mouse retina. Dual-wavelength excitation (488 nm and 552 nm) enables visualization of two fluorescence labels, while AO correction improves axial resolution and depth fidelity, allowing robust separation of structures through anatomical layers in retina. Using this platform, we performed 3D time-lapse imaging of microglia and longitudinal imaging in an optic nerve crush model, revealing layer-dependent differences in microglial motility, early activation signatures, and large-scale redistribution longitudinally. The system enabled widefield visualization of injury-associated vascular changes and spatial coupling between microglia and vasculature. Finally, depth-resolved two-color imaging captured immune responses to intravitreally transplanted RGCs, including host-cell recruitment, rapid neurite retraction following local immune-cell contact, and microglial phagocytosis of donor RGCs. Together, these results demonstrate that refractive AOSLO enables in vivo observations of microvascular organization, neuroimmune dynamics, injury responses, and transplanted-cell behavior with spatiotemporal resolution. Our data also suggests that modulation of microglial reactivity may improve outcomes of RGC transplantation. Significance StatementWide-field, depth-resolved imaging is essential for understanding how different cell types interact across a large retinal area, yet existing AOSLO systems retain limited imaging fields due to a conventional optical design using reflective spherical mirrors. Our refractive large-FOV AOSLO overcomes this limitation, enabling simultaneous two-color, diffraction-limited 3D imaging across an up to 16{degrees} field in vivo. This platform reveals previously inaccessible biological phenomena--including layer-specific microglial dynamics after optic nerve injury, microvascular remodeling, and rapid microglial rejection of transplanted RGCs--providing critical insight into neuroimmune behavior and retinal repair mechanisms at single-cell and subcellular resolution.

bioengineering↗

bioRxiv2025

Human sensory neurons exhibit cell-type-specific, pain-associated differences in intrinsic excitability and expression of SCN9A and SCN10A

Despite major advances in pain science, the approval of novel therapeutics has been slow. A major cause for the lack of new analgesics may be fundamental biological differences between humans and model organisms used in preclinical research. Large-scale transcriptional profiling efforts on human dorsal root ganglia (hDRG) have now identified at least 22 distinct neuronal subtypes; however, a significant knowledge gap exists in ascribing functional phenotypes to these diverse neuronal populations. In this study, we use Patch-seq recordings in hDRG to link electrical properties to transcriptionally defined cell types. First, through unbiased clustering of electrophysiological properties from 228 hDRG neurons, we identify three electrophysiological subtypes (E-types). Next, we show that E-types can be mapped onto specific transcriptional classes (T-types) of hDRG neurons. We find that donors pain history is associated with E-type-specific differences in electrical properties, some of which may be associated with higher expression of voltage-gated sodium channels, NaV1.7 and NaV1.8. These results highlight the importance of using multimodal profiling to better understand human sensory neuron biology and may help reveal novel therapeutic targets driving chronic pain. TeaserMapping electrical properties to transcriptional profiles of human sensory neurons offers new insights into pain neurobiology

neuroscience↗

bioRxiv2025

RNA-triggered fluorescence controlled by RNA switches for real-time RNA expression tracking in living plants

Real-time visualization of RNA dynamics across spatial scales in living plants is desirable yet remains challenging, hindered by the absence of imaging tools that simultaneously offer high sensitivity and deployability in whole living plants. Here, we engineered a modular, RNA-triggered fluorescence (RTF) reporter system de novo for spatiotemporal imaging of RNA from cellular to whole-plant scales in vivo. This system integrates three functional modules: a target-specific allosteric RNA switch, a degradable adapter for background suppression, and a fluorescent reporter. All components are stably expressed from a single vector and assemble in vivo, enabling uniform deployment throughout entire plants. The modified RTF (RTFst) achieves single-molecule sensitivity (signal-to-noise ratio >60) for dynamic tracking of developmentally regulated, tissue-specific, circadian, and stress-responsive mRNAs. Crucially, it enables the first direct, real-time observation of graft-transmissible mRNA trafficking across tissue scales and cross-kingdom transfer of the aphid-secreted long non-coding RNA Ya1 to plant cells. RTF establishes a versatile platform for studying gene regulation, signaling, and transcript mobility in plants, with broad applicability in synthetic biology and crop improvement.

molecular biology↗

bioRxiv2025

Expression of Fibroblast Growth Factor Receptor 3 (FGFR3) in the Human Peripheral Nervous System: Implications for the Putative Pathogenic Role of FGFR3 Autoantibodies in Neuropathy

IntroductionAutoantibodies to the Fibroblast Growth Factor Receptor 3 (FGFR3-AAbs) have been associated with idiopathic sensory-predominant neuropathy. The pathogenicity of FGFR3-AAbs in this disorder is unknown. Pathogenic mechanisms of autoantibodies in other dysimmune neuropathies commonly involve their direct binding to antigens on either neurons or glia. The expression of FGFR3 in the human peripheral nervous system is unknown. Therefore, as an initial step toward clarifying the pathogenicity of FGFR3-AAbs, we characterized the expression of FGFR3 in nerve (hNerve), dorsal root ganglia (hDRG) and spinal cord (hSC) in human. MethodsFGFR3 mRNA was assayed via in situ hybridization (ISH) on post-mortem sections of hNerve, hDRG and hSC, and by re-analysis of RNA-sequencing data from hDRG. FGFR3 protein was assayed in these tissues using capillary electrophoretic immunoassays (CEIA) with several validated anti-FGFR3 antibodies. ResultsFGFR3 mRNA was not detected in hNerve or hDRG but was abundant in hSC by ISH. FGFR3 protein was absent from hNerve and hDRG by CEIA but was moderately expressed in hSC. DiscussionA direct pathogenic mechanism of FGFR3-AAbs in sensory neuropathy would require the expression of FGFR3 in either neurons or non-neuronal cells in nerve or DRG. Using multiple methods, we did not detect FGFR3 expression at the mRNA or protein levels in these tissues. Given the absence of FGFR3 from hNerve and hDRG, it is improbable that FGFR3-AAbs cause direct damage to the neural components involved in neuropathy and thus are unlikely to be pathogenic, although indirect mechanisms via non-neural cells cannot be excluded.

neuroscience↗

bioRxiv2025

Brucella proline racemase protein A targets Tpl2 to promote IL-10 secretion for establishment of chronic infection

IL-10, an anti-inflammatory cytokine, plays a crucial role in limiting immune responses to pathogens, preventing host damage. However, the mechanisms underlying Brucella-mediated IL- 10 production remain incompletely understood. In this study, we demonstrate that the proline racemase protein A (PrpA) of Brucella melitensis M5-90 induces macrophages to secrete IL-10 by activating the Tpl2-ERK signaling pathway, thereby promoting chronic infection. Moreover, Tpl2 deletion impairs macrophage bactericidal ability, accompanied by reduced TNF- and IL-1{beta} but unaffected IL-10 levels. Additionally, Trp309, Glu103, and Glu129 of PrpA participate in interaction with Tpl2, but these residues do not influence PrpA-mediated IL-10 production in macrophages. PrpA deletion enhances IFN-{gamma} levels, specific anti-Brucella IgG, and CD4+ and CD8+ T cell numbers in mice. Furthermore, the Brucella melitensis M5-90 prpA mutant provides higher protection than the parental strain against virulent Brucella melitensis M28 infection in mice. Our findings suggest that Brucella PrpA promotes IL-10 secretion by macrophages through Tpl2 activation for bacterial survival and persistent infection, making the Brucella melitensis M5-90 prpA mutant a promising vaccine for enhanced protection. Author SummaryIL-10, an anti-inflammatory cytokine, is exploited by Brucella for survival. We identified Brucella PrpA as a potent IL-10 inducer, activating the Tpl2-ERK signaling pathway. Tpl2 deletion increased Brucella survival in macrophages, accompanied by reduced TNF- and IL-1{beta} but unaffected IL-10 secretion. Residues (Trp309, Glu103, and Glu129) of PrpA were crucial for interaction with Tpl2, yet did not impact PrpA-mediated IL-10 production. The B. melitensis M5-90 prpA mutant induced higher anti-Brucella IgG, IFN-{gamma}, and CD4+ and CD8+ T cell numbers in mice, providing better protection than B. melitensis M5-90. These findings unveil PrpAs role in IL-10 production and highlight the potential of the B. melitensis M5-90 prpA mutant as a promising vaccine for further evaluation.

microbiology↗

bioRxiv2025

Mouse Single-Cell Long-Read Splicing Atlas by Ouro-Seq

Alternative splicing creates cellular and phenotypic diversity; thus, characterizing splicing patterns across diverse cell types is essential for a comprehensive understanding of these cell types. However, existing full-length single-cell RNA-sequencing methods suffer from multiple technical limitations, such as the pervasive presence of reverse transcription artifacts across genes. Here, we introduce Ouro-Seq, a novel single-cell long-read sequencing framework with a versatile artifact removal mechanism for characterizing full-length mRNA species expressed by individual cells. We also developed a computational pipeline that identifies genuine full-length cDNAs and normalizes full-length mRNA size distributions for integration. Using Ouro-Seq, we constructed the Mouse Single-Cell Long-Read Splicing Atlas by collecting full-length transcriptomes of 103,304 nuclei or cells from 12 major organs and tissues of adult mice. Our atlas comprehensively characterizes alternative splicing, alternative promoter usage, and alternative polyadenylation events across cell types, revealing previously unappreciated functional heterogeneity among individual cells. Different cell types exhibit largely distinct expression patterns of various isoforms, corresponding to their specific morphologies and biological functions. Our splicing atlas and generalized long-read frameworks will empower researchers to fully interpret biological variation across full-length transcriptomes of single cells.

genomics↗

bioRxiv2025

Preclinical and Toxicology Assessment of ISFP10, an Inhibitor of Fungal Phosphoglucomutase (PGM)

Background and ObjectivePreviously, the novel small molecule ISFP10 has been shown to inhibit fungal phosphoglucomutase (PGM) activity in Aspergillus fumigatus and Pneumocystis spp. With 50-fold selectivity over the human PGM molecule due to the presence of a unique yet conserved cysteine residue present in a number pathogenic fungal PGMs, use of this compound may provide a novel broad-spectrum approach to treating fungal infections. Accordingly, we sought to determine the tolerability in test animals receiving this compound, as well as the potential antifungal activity of ISFP10 on cultures of the common fungal pathogens Candida albicans and Candida glabrata. MethodsC57BL6 mice received once daily intraperitoneal (IP) injections of 100 mL of vehicle control (DMSO) or ISFP10 at a concentration of 10 mg/kg. Body weights were recorded daily for 7 days of treatment. On the final day, mice were weighed and euthanized. Postmortem blood collection was conducted via cardiac puncture and distributed to EDTA and lithium heparin tubes for complete blood count (CBC) and comprehensive blood chemistry panels, respectively. Liver, kidney, and lung tissue were also harvested and placed in 10% formalin for H&E staining and blinded histopathologic scoring. Lung samples were further analyzed for proinflammatory cytokines using enzyme-linked immunosorbent assays (ELISA) and quantitative PCR (qPCR). Furthermore, ISPF10 was tested for antifungal activity via 8-hour growth curve analysis in a concentration-dependent fashion against Candida albicans and Candida glabrata. ResultsThere was no significant difference in the daily or final body weights of the mice receiving 10 mg/kg of ISFP10 compared to those of the vehicle control group. Extracellular matrix (ECM) transcripts for IL-6 and TNF were statistically similar via qPCR. ELISA results of proinflammatory cytokines for IL-6 was not significant whereas TNF levels in lung tissue from the ISFP10 treatment group were significantly reduced, indicating a potential anti-inflammatory effect of ISFP10 at this dosage. Overall, blood chemistry and CBC analysis revealed no overall significant differences between the two groups, except for increased neutrophil counts and decreased potassium levels in samples collected from ISFP10 treated animals compared to the vehicle control group. These laboratory abnormalities were not of clinical significance to the test animals. Blinded histopathological examination revealed no abnormalities or evidence of critical organ toxicity from all groups. Inhibition of C. albicans and C. glabrata culture growth by ISFP10 was concentration-dependent in YPD liquid media containing the ISFP10 compared to vehicle control. ConclusionsOur preliminary testing of ISFP10 revealed no inherent safety or toxicology concerns within the observed parameters. These data further support significant culture suppressive activity against C. albicans and C. glabrata. Taken together, these observations of ISFP10 further indicate that targeting PGM might be a novel and viable therapeutic strategy for serious fungal infections. Key pointsAn inhibitor specific to fungal PGM enzymes, termed ISFP10, was generally well tolerated when administered via intraperitoneal (IP) injection in mice. ISFP10 displays antifungal activity in a concentration-dependent manner against C. albicans and C. glabrata.

pharmacology and toxicology↗

bioRxiv2024

T505A variant of ETV5 promotes proliferation of precursor B cells in a mouse model of acute lymphoblastic leukemia

Precursor B cell acute lymphoblastic leukemia (pre-B-ALL) arises as a result of precursor B cells acquiring driver mutations that lead to arrested differentiation and increased proliferation. Identification of driver mutations and understanding their biological function is critical to understanding pre-B-ALL development and advancing disease treatment. Using a mouse model of pre-B-ALL driven by deletion of genes encoding the related E26-transformation-specific (ETS) transcription factors PU.1 and Spi-B, we performed whole exome sequencing to identify secondary driver mutations. We identified recurrent variants in E26 transformation-specific transcription variant 5 (ETV5) resulting in R392P, V444I, and T505A amino acid changes. We found that the R392P and V444I variants altered the ability of ETV5 to bind to DNA using electrophoretic mobility shift assay. R392P and V444I variants did not activate a Dual-Specificity-Phosphatase 6 (DUSP6) reporter. In contrast, T505A ETV5 could interact with DNA and activate the DUSP6 promoter. To determine biological function, we forced expression of wild type, R392P, V444I, or T505A ETV5 in an interleukin-7-dependent pre-B cell line. Proliferation and apoptosis assays showed that T505A ETV5 conferred a proliferative advantage to pre-B cells. RNA sequencing showed that expression of ETV5 variants significantly altered gene expression in cultured cells. Through gene set enrichment analysis, T505A was suggested to downregulate the p53 pathway and the anti-proliferative protein, B cell translocation gene 2 (encoded by Btg2). In summary, these data suggest that ETV5 mutations play a role in pre-B-ALL by affecting proliferation and cell survival.

immunology↗

bioRxiv2024

ALDH3A1-mediated detoxification of reactive aldehydes contributes to distinct muscle responses to denervation and Amyotrophic Lateral Sclerosis progression

Different muscles exhibit varied susceptibility to degeneration in Amyotrophic Lateral Sclerosis (ALS), a fatal neuromuscular disorder. Extraocular muscles (EOMs) are particularly resistant to ALS progression, and exploring the underlying molecular nature may offer significant therapeutic value. Reactive aldehyde 4-hydroxynonenal (HNE) is implicated in ALS pathogenesis, and Aldh3a1 is an inactivation-resistant intracellular aldehyde dehydrogenase that detoxifies 4-HNE to protect eyes against UV-induced oxidative stress. We detected prominently higher levels of Aldh3a1 in mouse EOMs compared to other muscles under normal physiological conditions. In an ALS mouse model (hSOD1G93A) reaching end-stage, Aldh3a1 expression was maintained high in EOMs, substantially elevated in soleus and diaphragm, but only moderately increased in extensor digitorum longus (EDL) muscle, which endured the most severe pathological remodeling, as demonstrated by unparalleled upregulation of a denervation marker Ankrd1. Importantly, sciatic nerve transection in wildtype mice further confirmed induced Aldh3a1 and Ankrd1 expression in an inverse manner across muscle types in response to denervation. Mechanistically, whole-muscle RNA-Seq and pharmacological tests indicate that higher basal levels of lipid oxidation in soleus and diaphragm muscles may predispose them to stronger Nrf2 antioxidant responses under pathological stress compared to EDL, leading to more prominent Aldh3a1 upregulation. Additionally, the identification of the myoblast fusion marker Mymk as an EOM signature gene suggests that the spontaneous activation of satellite cells contributes to high levels of Aldh3a1 in EOMs. Functionally, adeno-associated virus-mediated overexpression of Aldh3a1 protected myotubes from 4-HNE-induced DNA fragmentation and plasma membrane leakage. It also restored MG53-mediated membrane repair, highlighting its potential for clinical applications.

cell biology↗

bioRxiv2024

Secretory trafficking maintains the organelle band for spindle separation during Arabidopsis meiosis

Ploidy reduction is a critical aspect of meiosis, essential for the successful development of haploid germline cells. Notably, cellular processes during male meiosis vary between the major groups of flowering plants, with eudicots exhibiting simultaneous cytokinesis without an interkinesis phase, unlike monocots. Recent studies on the Arabidopsis jason (jas) mutant revealed that an organelle band plays a key role in spindle separation during simultaneous cytokinesis, though the mechanism is not fully understood. Here we identify a novel jas suppressor mutant, peleus (pele), which restores haploid pollen production in the jas background. PELE encodes a vacuole-localized protein that contributes to spindle separation by balancing the vacuolar pathway and secretory trafficking to the organelle band. JAS, PELE, and the ubiquitin-like protein UBQL coordinate the delivery of proteins and membrane components to the organelle band. Specifically, PELE and UBQL antagonistically fine-tune trafficking to the vacuole, while JAS adjusts the flow of secretory trafficking towards the organelle band. Our findings underscore the critical role of secretory trafficking in maintaining organelle band integrity and in ensuring precise spindle positioning during meiosis II. Our work provides novel insights into the regulation of the organelle band and spindle separation, enhancing our understanding of polyploidy generation in plants.

plant biology↗

bioRxiv2024

The tetrapod fauna of the upper Permian Naobaogou Formation of China: a new mid-sized pareiasaur Yinshanosaurus angustus gen. et sp. nov. and its implication for the phylogenetic relationship of pareiasaurs

Pareiasauria is a specialized clade of herbivorous tetrapods that existed throughout Pangaea during the mid to late Permian period. The phylogenetic relationships of Chinese pareiasaur species remain controversy for several decades, primarily due to the poor preservation of known specimens. Until the report of Shihtienfenia completus in 2019, no complete skull had been documented for Chinese pareiasaurs. The present study describes a mid-sized pareiasaur, Yinshanosaurus angustus gen. et sp. nov., based on a nearly complete skull and an articulated partial postcranial skeleton collected from the Naobaogou Formation in 2018. It presents several significant new morphological features such as high and narrow skull, with the length of the skull table exceeding the width between the two quadratojugals; snout dimension as high as wide; long frontal with a length-to-width ratio of [~]3.0; U-shaped paraoccipital process in occipital view; and maxillary teeth oriented vertically, with only 7-9 cusps. Although the phylogenetic framework of pareiasaurs still requires further refinement, the current analysis yields distinct phylogenetic positions for Chinese pareiasaurs and establishes a new monophyletic clade that includes S. completus and Y. angustus.

paleontology↗

bioRxiv2024

Comparative spatiotemporal single cell transcriptomes reveal rewiring of pre-existing regulations during emergence of Kranz anatomy in C4 grasses

Many of the worlds most productive food and bioenergy crops use C4 photosynthesis, which have high photosynthetic efficiency due to a Kranz anatomy-based CO2 concentrating mechanism. Here, we took a comparative transcriptomics approach using single cell spatial transcriptomes of leaf primordia for maize (Zea mays) and single cell RNA-seq (scRNA-seq) atlases of corresponding leaf tissues for three C4 species (Zea mays, Sorghum bicolor, Setaria viridis) and one C3 rice (Oryza sativa) to study regulatory networks involved in the development and evolution of Kranz anatomy. We show that the formation of Kranz anatomy involves extensive recruitment and modification of pre-existing regulatory modules, especially the SHR-SCR module and the auxin signaling pathway. Members of the INDETERMINATE DOMAIN (IDD) family transcription factors, including IDD7 and IDDP1, contribute to the modification of the involved modules. These extensive recruitment and modification of existing genetic regulatory programs underlie the repeated emergence of C4 photosynthesis during evolution.

plant biology↗

bioRxiv2024

Novel regulator Fpr2 controls immune homeostasis via CCL4-dependent neutrophils

Polymorphonuclear leukocytes (PMNs) in Fpr2-deficient mice have significantly impaired bactericidal activity, yet the precise mechanism remains elusive. Our research unveiled the cellular heterogeneity of peripheral blood PMNs through single-cell RNA-sequencing (scRNA-seq) and identified two new neutrophil clusters related to bacterial infection, including a sterilization cluster named Neu_CSTA and a regulation cluster named Neu_CCL4. The lack of Fpr2 leads to an immune regulation imbalance of Neu_CCL4 on other clusters in uninfected conditions, resulting in persistent instability of PMNs in peripheral blood, elevated inflammatory factors, and increased NOX2 expression. Furthermore, our findings corroborate that the regulatory function of Neu_CCL4 on PMNs immune balance is closely tied to its high expression of the anti-inflammatory ligand Anxa1. Regulon analysis inferred by scRNA-seq and ChIP-seq data suggested that a specific transcription factor Elf4, could modulate the expression of Anxa1, thereby facilitating its regulatory impact on other clusters. In the infection state, there was a noted increase in intercellular interactions across different clusters, leading to a general improvement in the bactericidal activity of PMNs. In summary, our research revealed distinct functional cellular sub-populations of PMNs, discovered the potential regulatory mechanisms, and elucidated the essential roles of Fpr2 in their ability to resist infection.

immunology↗

bioRxiv2024

Awe is characterized as an ambivalent experience in the human behavior and cortex: integrated virtual reality-electroencephalogram study

Ambivalent feelings are a defining feature of awe, which has been understood as a possible source of its psychosocial benefits. However, due to the conventional unidimensional model of affective valence, behavior and neural representation of ambivalent feelings during awe remain elusive. To address this gap, we combined awe-inducing virtual reality clips, electroencephalogram, and a deep learning-based dimensionality reduction technique (N = 43). Behaviorally, awe ratings were precisely predicted by the duration and intensity of ambivalent feelings, not by single valence-related metrics. In the electrophysiological analysis, we identified latent neural space for each participant sharing valence representation structures across individuals and stimuli. In these spaces, ambivalent feelings during awe were distinctly represented from positive and negative ones, and the variability in their distinctiveness specifically predicted awe ratings. Additionally, frontal delta oscillations mainly engaged in differentiating valence representations. Our findings demonstrate that awe is fundamentally an ambivalent experience reflected in both behavior and electrophysiological activities. This work provides a new framework for understanding complex emotions and their neural underpinnings, with potential implications for affective neuroscience and relevant fields.

neuroscience↗

bioRxiv2024

Single-cell resolved differentiation of pre-Kranz anatomy in maize leaf primordia

Typical C4 plants such as maize possess highly optimized Kranz-type leaf anatomy, whereby concentric wreaths of mesophyll and bundle sheath cells surround closely spaced veins. The veins and the cells that surround them are derived from the middle ground meristem (mGM) through processes that are as yet undefined. Here we distinguished the active zone of vascular development within early leaf primordia, and used comparative transcriptomics of sub-sectioned maize and rice primordia to identify cohorts of genes likely involved in early Kranz development. Leveraging single-nucleus RNA sequencing (snRNA-seq) we then explored the cell heterogeneity and developmental trajectories within single maize leaf primordia. Assisted by in situ hybridization, cell clusters of mGM and procambium were identified, with candidate marker genes showing different yet inter-related expression patterns. Localization of the vascular marker ZmSHR1 was preceded by that of ZmEREB161 and ZmEREB114 in terms of procambium initiation. Potential subclusters of bundle sheath cells and different layer of mesophyll cells were depicted from developing cells toward the tip of sub-sectioned maize primordia. Collectively our results identify potential mGM derived or procambium localized Kranz regulators and provide resources for investigating leaf vein development in maize and rice, at sub-primordium and single-cell resolution.

plant biology↗

bioRxiv2024

CD28 Shapes T Cell Receptor Signaling by Regulating ZAP70 Activation and Lck Dynamics

T cell activation requires T cell receptor (TCR) engagement, which initiates a series of proximal events including tyrosine phosphorylation of the CD3 and TCR{zeta} chains, recruitment, and activation of the protein tyrosine kinases Lck and ZAP70, followed by recruitment of adapter and signaling proteins. CD28 co-stimulation is also required to generate a functional immune response. Currently we lack a full understanding of the molecular mechanism of CD28 activation. TCR microclusters (MC) are submicron-sized molecular condensates and basic signaling units that form immediately after TCR ligation. Our results show that CD28 co-stimulation specifically accelerated recruitment of ZAP70 to the TCR{zeta} chain in MCs and increased ZAP70 activation. This CD28-mediated acceleration of ZAP70 recruitment was driven by enhanced Lck recruitment to the MCs. A greater spatial separation between active and inactive species of Lck was also observed in the MCs as a consequence of CD28 co-stimulation. These results suggest that CD28 co-stimulation may lower the TCR activation threshold by enhancing the activated form of Lck in the TCR MCs.

immunology↗

bioRxiv2024

Functional architecture of intracellular oscillations in hippocampal dendrites

Fast electrical signaling in dendrites is central to neural computations that support adaptive behaviors. Conventional techniques lack temporal and spatial resolution and the ability to track underlying membrane potential dynamics present across the complex three-dimensional dendritic arbor in vivo. Here, we perform fast two-photon imaging of dendritic and somatic membrane potential dynamics in single pyramidal cells in the CA1 region of the mouse hippocampus during awake behavior. We study the dynamics of subthreshold membrane potential and suprathreshold dendritic events throughout the dendritic arbor in vivo by combining voltage imaging with simultaneous local field potential recording, post hoc morphological reconstruction, and a spatial navigation task. We systematically quantify the modulation of local event rates by locomotion in distinct dendritic regions and report an advancing gradient of dendritic theta phase along the basal-tuft axis, then describe a pre-dominant hyperpolarization of the dendritic arbor during sharp-wave ripples. Finally, we find spatial tuning of dendritic representations dynamically reorganizes following place field formation. Our data reveal how the organization of electrical signaling in dendrites maps onto the anatomy of the dendritic tree across behavior, oscillatory network, and functional cell states.

neuroscience↗

bioRxiv2023

RFC1 AAGGG pentanucleotide repeats form parallel G-quadruplex: structural implications for aberrant molecular cascades in CANVAS

A pentanucleotide repeat expansion (PRE) of (AAGGG)n in the replication factor C subunit 1 (RFC1) gene is recently identified as the genetic cause of cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS), and also linked to several other neurodegenerative disorders including the Parkinsons disease. However, the molecular mechanism by which RFC1 PRE drives pathology remains poorly understood. Here, for the first time we discovered and determined the high-resolution structures of parallel G-quadruplex formed by AAGGG repeats within the pathogenic RFC1 PRE, revealing an intriguing conformational plasticity at the 3-termi that allows stacking of multiple G4s. We further identify a molecular mechanism by which the DNA G4 in RFC1 PRE impedes polymerase processivity leading to replication stalling and transcription inhibition in vitro in a repeat-length-dependent manner, and the transcription inhibition could partially contribute to a reduced gene expression in cells. Our results demonstrate that the DNA G-quadruplex of RFC1 PRE participate in aberrant molecular cascades, and provide an unprecedented high-resolution structural target to discover helicases and ligands that resolve the pathogenic G4 for therapeutic intervention.

biochemistry↗