Biology subjects

Ji, W.

Publications and source records attributed to Ji, W..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

Tex2 is required for lysosomal functions at TMEM55-dependent ER membrane contact sites

ER tubules form and maintain membrane contact sites (MCSs) with late endosomes/lysosomes (LE/lys). The molecular composition and cellular functions of these MCSs are poorly understood. Here we find that Tex2, an SMP domain-containing lipid transfer protein conserved in metazoen and yeast, is a tubular ER protein, and enriches at ER-LE/lys MCSs dependent on TMEM55, phosphatases that convert PI(4,5)P2 to PI5P on LE/lys. We show that the Tex2-TMEM55 interaction occurs between a N-terminal region of Tex2 and a catalytic motif in PTase domain of TMEM55. The Tex2-TMEM55 interaction can be regulated by endosome-resident type 2 PI4K activities. Functionally, Tex2 knockout results in severe defects in lysosomal digestive capacity and blocked autophagic flow, as well as an aberrant accumulation of PI3P on the LE/lys membranes. These defects can be substantially rescued by wild type Tex2 other than a lipid transfer-defective Tex2 mutant, indicating an important role of lipid transfer in these processes. Together our data identify Tex2 as a tubular ER protein that resides at TMEM55-depedent ER-LE/lys MCSs required for lysosomal functions.

cell biology↗

bioRxiv2022

DNA packaging via hierarchical chromatin structures revealed by live-cell 3D imaging

Single-molecule localization microscopy is a powerful superresolution imaging technique to study biological questions by visualizing subcellular fine structures with nanometer-scale precision. However, its application in live-cell imaging studies has been impeded by the paucity of self-blinking organic fluorophores that enable high spatiotemporal resolution and labeling/localization density at a moderate laser intensity. Herein, we report a self-blinking Si-rhodamine dye 6-HESiR with a suitably increased "ON" fraction and a fluorogenic self-blinking dsDNA probe 6-HoeHESiR as a powerful tool for 3D superresolution imaging of native chromatin in eukaryotes without the use of photoswitching buffer and high laser intensity. With the probe 6-HoeHESiR, 3D superresolution imaging of in vitro reconstituted nucleosomal arrays and chromatin fibers yielded results consistent with EM analysis. Similar euchromatin and heterochromatin structures were visualized in fixed and live cells with high spatiotemporal resolution and labeling density, providing the first live-cell evidence for a hierarchical model of chromatin organization. 3D imaging results obtained in the presence of selective inhibitors of histone deacetylases also corroborate chromatin fiber decompaction upon hyperacetylation of histones.

cell biology↗

bioRxiv2022

Covalent Disruptor of YAP-TEAD Association Suppresses Defective Hippo Signaling

The transcription factor TEAD, together with its coactivator YAP/TAZ, is a key transcriptional modulator of the Hippo pathway. Activation of TEAD transcription by YAP has been implicated in a number of malignancies, and this complex represents a promising target for drug discovery. However, both YAP and its extensive binding interfaces to TEAD have been difficult to address using small molecules, mainly due to a lack of druggable pockets. TEAD is post-translationally modified by palmitoylation that targets a conserved cysteine at a central pocket, which provides an opportunity to develop cysteine-directed covalent small molecules for TEAD inhibition. Here, we employed covalent fragment screening approach followed by structure-based design to develop an irreversible TEAD inhibitor MYF-03-69. Using a range of in vitro and cell-based assays we demonstrated that through a covalent binding with TEAD palmitate pocket, MYF-03-69 disrupts YAP-TEAD association, suppresses TEAD transcriptional activity and inhibits cell growth of Hippo signaling defective malignant pleural mesothelioma (MPM). Further, a cell viability screening with a panel of 903 cancer cell lines indicated a high correlation between TEAD-YAP dependency and the sensitivity to MYF-03-69. Transcription profiling identified the upregulation of proapoptotic BMF gene in cancer cells that are sensitive to TEAD inhibition. Further optimization of MYF-03-69 led to an in vivo compatible compound MYF-03-176, which shows strong antitumor efficacy in MPM mouse xenograft model via oral administration. Taken together, we disclosed a story of the development of covalent TEAD inhibitors and its high therapeutic potential for clinic treatment for the cancers that are driven by TEAD-YAP alteration.

cancer biology↗

bioRxiv2021

Genome-wide identification and expression analysis of the invertase gene family in common wheat

BackgroundThe degradation of sucrose plays an important role in the process of crop biomass allocation and yield formation. Invertase (INV) irreversibly catalyzes the conversion of sucrose into glucose and fructose, which doomed its important role in plant development and stress tolerance. However, the functions of INV genes in wheat, one of the most important crops, were less studied due to the polyploidy. ResultsHere, we systematically analyzed the INV gene family based on the latest published wheat reference genomic information. A total of 126 TaINV genes were identified and classified into three classes based on the phylogenetic relationship and their gene structure. Of which, 11 and 83 gene pairs were identified as tandem and segmental duplication genes respectively, while the Ka/Ks ratios of tandem and segmental duplication TaINV genes were less than 1. Expression profile analysis shows that 18 TaINV genes have tissue-specific expression, and 54 TaINV genes were involved in stress response. Furthermore, RNA-seq showed that 35 genes are differentially expressed in grain weight NILs N0910-81L/N0910-81S, in which 9 TaINVs were stably detected by qRT-PCR at three time-points, 4, 7 and 10 DPA. Four of them (TaCWI47, TaCWI48, TaCWI50 and TaVI27) different expressed between the NILs resided in 4 QTL segments (QTGW.nwafu-5DL.1, QTGW.nwafu-5DL.2, QTGW.nwafu-7AS.1 and QTGW.nwafu-7AS.2). These findings facilitate function investigations of the wheat INV gene family and provide new insights into the grain development mechanism in wheat. ConclusionsOur results showed that allopolyploid events were the main reason for the expansion of the TaINV gene family in hexaploid wheat, and duplication genes might undergo purifying selection. The expression profiling of TaINV genes implied that they are likely to play an important role in wheat growth and development and adaption to stressful environments. And TaCWI47, TaCWI48, TaCWI50 and TaVI27 may have more important roles in grain developments. Our study lay a base for further dissecting the functional characterization of TaINV family members.

genomics↗

bioRxiv2021

Wall-associated Receptor Kinase and The Expression Profiles in Wheat Responding to Fungal Stress

Cell wall-associated kinases (WAKs), which are encoded by conserved gene families in plants, are crucial for development and responses to diverse stresses. However, the wheat (Triticum aestivum L.) WAKs have not been systematically classified, especially those involved in protecting plants from disease. Here, we classified 129 WAK proteins (encoded by 232 genes) and 75 WAK-Like proteins (WAKLs; encoded by 109 genes) into four groups, via a phylogenetic analysis. An examination of protein sequence alignment revealed diversity in the GUB-domain of WAKs structural organization, but it was usually characterized by a PYPFG motif followed by CxGxGCC motifs, while the EGF-domain was usually initiated with a YAC motif, and eight cysteine residues were spliced by GNPY motif. The expression profiles of WAK-encoding homologous genes varied in response to Blumeria graminis f. sp. tritici (Bgt), Puccinia striiformis f. sp. tritici (Pst) and Puccinia triticina (Pt) stress. A quantitative real-time polymerase chain reaction (qRT-PCR) analysis proved that TaWAK75 and TaWAK76b were involved in wheat resistance to Bgt. This study revealed the structure of the WAK-encoding genes in wheat, which may be useful for future functional elucidation of wheat WAKs responses to fungal infections.

plant biology↗

bioRxiv2020

Trans-Golgi network-lipid droplet contacts maintain the TGN integrity and function via lipid transfer activities of VPS13B

While the physical interactions between the Golgi apparatus (Golgi) and lipid droplets (LDs) have been suggested through system-level imaging, the bona fide and functional Golgi-LD membrane contact sites (MCSs) remain largely uncharacterized. Here, we demonstrate that vacuolar protein sorting-associated protein 13B (VPS13B) mediates trans-Golgi network (TGN)-LD interactions. VPS13B is specifically accumulated at TGN-LD MCSs with its C-terminal region targeting LDs via an amphipathic helix while a putative WD40 module and a C-terminal Pleckstrin homology (PH) domain independently recognizing TGN via directly binding to Rab6. A putative lipid transfer domain (LTD) at the N-terminal portion of VPS13B binds glycerophospholipids in vitro. VPS13B suppression results in severe fragmentation of the TGN, an effect that can be almost completely rescued by the expression of VPS13B-LTD. Collectively, our findings demonstrate that VPS13B mediates lipid transfer at TGN-LD MCSs to maintain TGN integrity and function.

cell biology↗

bioRxiv2020

A novel high-accuracy genome assembly method utilizing a high-throughput workflow

Across domains of biological research using genome sequence data, high-quality reference genome sequences are essential for characterizing genetic variation and understanding the genetic basis of phenotypes. However, the construction of genome assemblies for various species is often hampered by complexities of genome organization, especially repetitive and complex sequences, leading to mis-assembly and missing regions. Here, we describe a high-throughput gold standard genome assembly workflow using a large-scale bacterial artificial chromosome (BAC) library with a refined two-step pooling strategy and the Lamp assembler algorithm. This strategy minimizes the laborious processes of physical map construction and clone-by-clone sequencing, enabling inexpensive sequencing of several thousand BAC clones. By applying this strategy with a minimum tiling path BAC clone library for the short arm of chromosome 2D (2DS) of bread wheat, 98% of BAC sequences, covering 92.7% of the 2DS chromosome, were assembled correctly for this species with a highly complex and repetitive genome. We also identified 48 large mis-assemblies in the reference wheat genome assembly (IWGSC RefSeq v1.0) and corrected these large mis-assemblies in addition to filling 92.2% of the gaps in RefSeq v1.0. Our 2DS assembly represents a new benchmark for the assembly of complex genomes with both high accuracy and efficiency.

genomics↗

bioRxiv2020

On the role of photoreceptor identity in controlling accurate wiring of the Drosophila visual circuit

During development, neurons extend in search of synaptic partners. Precise control of axon extension velocity can therefore be crucial to ensuring proper circuit formation. How velocity is regulated - particularly by the extending axons themselves - remains poorly understood. Here, we investigate this question in the Drosophila visual system, where photoreceptors make precise connections with a specific set of synaptic partners that together create a circuit underpinning neural superposition (NSP). We used a combination of genetic perturbations and quantitative image analysis to investigate the influence of cell identity on growth cone velocity and subsequent spatial-temporal coincidence of presynaptic and postsynaptic neurons. Our study provides a case study of how cell autonomous properties of presynaptic axons play a pivotal role in controlling the dynamics of growing axons and determining the formation of a precise neuronal circuit.

bioinformatics↗

bioRxiv2020

Drone-derived canopy height predicts biomass across non-forest ecosystems globally

Non-forest ecosystems, dominated by shrubs, grasses and herbaceous plants, provide ecosystem services including carbon sequestration and forage for grazing, yet are highly sensitive to climatic changes. Yet these ecosystems are poorly represented in remotely-sensed biomass products and are undersampled by in-situ monitoring. Current global change threats emphasise the need for new tools to capture biomass change in non-forest ecosystems at appropriate scales. Here we assess whether canopy height inferred from drone photogrammetry allows the estimation of aboveground biomass (AGB) across low-stature plant species sampled through a global site network. We found mean canopy height is strongly predictive of AGB across species, demonstrating standardised photogrammetric approaches are generalisable across growth forms and environmental settings. Biomass per-unit-of-height was similar within, but different among, plant functional types. We find drone-based photogrammetry allows for monitoring of AGB across large spatial extents and can advance understanding of understudied and vulnerable non-forested ecosystems across the globe.

ecology↗

bioRxiv2020

Essential amnion signals for primate primitive streak formation resolved by scRNA map

Essential genes for murine embryonic development can demonstrate a disparate phenotype in human cohorts. By generating a transcriptional atlas containing >30,000 cells from postimplantation non-human primate embryos, we discovered that ISL1, a gene with a well-established role in cardiogenesis, controls a gene regulatory network in primate amnion. CRISPR/Cas9-targeting of ISL1 resulted in non-human primate embryos which did not yield viable offspring, demonstrating that ISL1 is critically required in primate embryogenesis. On a cellular level, mutant ISL1 embryos displayed a failure in mesoderm formation due to reduced BMP4 signaling from the amnion. Via loss of function and rescue studies in human embryonic stem cells we confirmed a similar role of ISL1 in human in vitro derived amnion. This study highlights the importance of the amnion as a signaling center during primate mesoderm formation and demonstrates the potential of in vitro primate model systems to dissect the genetics of early human embryonic development.

developmental biology↗

bioRxiv2020

Single-cell regulatory landscape and disease vulnerability map of adult Macaque cortex

Single cell approaches have increased our knowledge about the cell type composition of the non-human primate (NHP), but a detailed characterization of area-specific regulatory features remains outstanding. We generated single-cell chromatin accessibility (single-cell ATAC) and transcriptomic data of 358,237 cells from prefrontal cortex (PFC), primary motor cortex (M1) and primary visual cortex (V1) of adult cynomolgus monkey brain, and integrated this dataset with Stereo-seq (Spatio-Temporal Enhanced REsolution Omics-sequencing) of the corresponding cortical areas to assign topographic information to molecular states. We identified area-specific chromatin accessible sites and their targeted genes, including the cell type-specific transcriptional regulatory network associated with excitatory neurons heterogeneity. We reveal calcium ion transport and axon guidance genes related to specialized functions of PFC and M1, identified the similarities and differences between adult macaque and human oligodendrocyte trajectories, and mapped the genetic variants and gene perturbations of human diseases to NHP cortical cells. This resource establishes a transcriptomic and chromatin accessibility combinatory regulatory landscape at a single-cell and spatially resolved resolution in NHP cortex.

neuroscience↗

bioRxiv2020

Single-cell atlas of a non-human primate reveals new pathogenic mechanisms of COVID-19

Stopping COVID-19 is a priority worldwide. Understanding which cell types are targeted by SARS-CoV-2 virus, whether interspecies differences exist, and how variations in cell state influence viral entry is fundamental for accelerating therapeutic and preventative approaches. In this endeavor, we profiled the transcriptome of nine tissues from a Macaca fascicularis monkey at single-cell resolution. The distribution of SARS-CoV-2 facilitators, ACE2 and TMRPSS2, in different cell subtypes showed substantial heterogeneity across lung, kidney, and liver. Through co-expression analysis, we identified immunomodulatory proteins such as IDO2 and ANPEP as potential SARS-CoV-2 targets responsible for immune cell exhaustion. Furthermore, single-cell chromatin accessibility analysis of the kidney unveiled a plausible link between IL6-mediated innate immune responses aiming to protect tissue and enhanced ACE2 expression that could promote viral entry. Our work constitutes a unique resource for understanding the physiology and pathophysiology of two phylogenetically close species, which might guide in the development of therapeutic approaches in humans. Bullet pointsO_LIWe generated a single-cell transcriptome atlas of 9 monkey tissues to study COVID-19. C_LIO_LIACE2+TMPRSS2+ epithelial cells of lung, kidney and liver are targets for SARS-CoV-2. C_LIO_LIACE2 correlation analysis shows IDO2 and ANPEP as potential therapeutic opportunities. C_LIO_LIWe unveil a link between IL6, STAT transcription factors and boosted SARS-CoV-2 entry. C_LI

cell biology↗

bioRxiv2020

Canonical and noncanonical features of the mouse visual cortical hierarchy

Neocortical circuit computations underlying active vision are performed by a distributed network of reciprocally connected, functionally specialized areas. Mouse visual cortex is a dense, hierarchically organized network, comprising subnetworks that form preferentially interconnected processing streams. To determine the detailed layout of the mouse visual hierarchy, laminar patterns formed by interareal axonal projections, originating in each of ten visual areas were analyzed. Reciprocally connected pairs of areas, and shared targets of pairs of source areas, exhibited structural features consistent with a hierarchical organization. Beta regression analyses, which estimated a continuous measure of hierarchical distance, indicated that the network comprises multiple hierarchies embedded within overlapping processing levels. Single unit recordings showed that within each processing stream, receptive field sizes typically increased with increasing hierarchical level; however, ventral stream areas showed overall larger receptive field diameters. Together, the results reveal canonical and noncanonical hierarchical network motifs in mouse visual cortex.

neuroscience↗

bioRxiv2020

Identification and Expression Analysis of Heat Shock Proteins in Wheat Infected with Powdery Mildew and Stripe Rust

Heat shock proteins (HSPs), which are encoded by conserved gene families in plants, are crucial for development and responses to diverse stresses. However, the wheat (Triticum aestivum) HSPs have not been systematically classified, especially those involved in protecting plants from disease. Here, we classified 119 DnaJ (Hsp40) proteins (TaDnaJs; encoded by 313 genes) and 41 Hsp70 proteins (TaHsp70s; encoded by 95 genes) into six and four groups, respectively, via a phylogenetic analysis. An examination of protein structures and a multiple sequence alignment revealed diversity in the TaDnaJ structural organization, but a highly conserved J-domain, which was usually characterized by an HPD motif followed by DRD or DED motifs. The expression profiles of these HSP-encoding homologous genes varied in response to Blumeria graminis f. sp. tritici and Puccinia striiformis f. sp. tritici. A quantitative real-time PCR analysis indicated a lack of similarity in the expression of DnaJ70b, Hsp70-30b, and Hsp90-4b in wheat infected by B. graminis f. sp. tritici, although the expression levels of these genes were abnormal in the infected resistant and susceptible lines. Furthermore, a direct interaction between DnaJ70 and TaHsp70-30 was not detected in a yeast two-hybrid assay. This study revealed the structure and expression profiles of the HSP-encoding genes in wheat. The resulting data may be useful for future functional analyses and for further elucidating the roles of wheat HSPs during responses to fungal infections.

plant biology↗

bioRxiv2019

Novel truncating mutations in CTNND1 cause a dominant craniofacial and cardiac syndrome

AbstractCTNND1 encodes the p120-catenin (p120) protein, which has a wide range of functions, including the maintenance of cell-cell junctions, regulation of the epithelial-mesenchymal transition and transcriptional signaling. Due to advances in next generation sequencing, CTNND1 has been implicated in human diseases including cleft palate and blepharocheilodontic syndrome (BCD) albeit only recently. In this study, we identify eight novel protein-truncating variants, six de novo, in thirteen participants presenting with craniofacial dysmorphisms including cleft palate and hypodontia, as well as congenital cardiac anomalies, limb dysmorphologies and neurodevelopmental disorders. Using conditional deletions in mice as well as CRISPR/Cas9 approaches to target CTNND1 in Xenopus, we identified a subset of phenotypes that can be linked to p120-catenin in epithelial integrity and turnover, and additional phenotypes that suggest mesenchymal roles of CTNND1. We propose that CTNND1 variants have a wider developmental role than previously described, and that variations in this gene underlie not only cleft palate and BCD but may be expanded to a broader velocardiofacial-like syndrome.

genetics↗

bioRxiv2019

Two distinct actin filament populations have effects on mitochondria, with differences in stimuli and assembly factors

Recent studies show that mitochondria and actin filaments work together in two contexts: 1) increased cytoplasmic calcium induces cytoplasmic actin polymerization that stimulates mitochondrial fission, and 2) mitochondrial depolarization causes actin assembly around mitochondria, with roles in mitophagy. It is unclear whether these two processes utilize similar actin assembly mechanisms. Here, we show that these are distinct actin assembly mechanisms in the acute phase after treatment (<10 min). Calcium-induced actin assembly is INF2-dependent and Arp2/3 complex-independent, whereas depolarization-induced actin assembly is Arp2/3 complex-dependent and INF2-independent. The two types of actin polymerization are morphologically distinct, with calcium-induced filaments throughout the cytosol and depolarization-induced filaments as \"clouds\" around depolarized mitochondria. We have previously shown that calcium-induced actin stimulates increases in both mitochondrial calcium and recruitment of the dynamin GTPase Drp1. In contrast, depolarization-induced actin is temporally-associated with extensive mitochondrial dynamics that do not result in mitochondrial fission, but in circularization of the inner mitochondrial membrane (IMM). These dynamics are dependent upon the protease Oma1 and independent of Drp1. Actin cloud inhibition causes increased IMM circularization, suggesting that actin clouds limit these dynamics.\n\nSummary statementMitochondrial depolarization induces Arp2/3 complex-dependent actin clouds that restrain mitochondrial shape changes induced by Oma1 on the inner mitochondrial membrane. A distinct actin network stimulates mitochondrial fission in response to calcium.

cell biology↗

bioRxiv2019

Spatial clustering of inhibition in mouse primary visual cortex

Whether mouse visual cortex contains orderly feature maps is debated. The overlapping pattern of geniculocortical (dLGN) inputs with M2 muscarinic acetylcholine receptor-rich patches in layer 1 (L1) suggests a non-random architecture. Here, we found that L1 inputs from the lateral posterior thalamus (LP) avoid patches and target interpatches. Channelrhodopsin-assisted mapping of EPSCs in L2/3 shows that the relative excitation of parvalbumin-expressing interneurons (PVs) and pyramidal neurons (PNs) by dLGN, LP and cortical feedback are distinct and depend on whether the neurons reside in clusters aligned with patches or interpatches. Paired recordings from PVs and PNs shows that unitary IPSCs are larger in interpatches than patches. The spatial clustering of inhibition is matched by dense clustering of PV-terminals in interpatches. The results show that the excitation/inhibition balance across V1 is organized into patch and interpatch subnetworks which receive distinct long-range inputs and are specialized for the processing of distinct spatiotemporal features.

neuroscience↗

bioRxiv2019

Extensive adaptive immune response of AAVs and Cas proteins in non-human primates

The CRISPR-mediated Cas system is the most widely used tool in gene editing and gene therapy for its convenience and efficiency. Delivery of the CRISPR system by adeno-associated viruses (AAVs) is currently the most promising approach to gene therapy. However, pre-existing adaptive immune responses against CRISPR nuclease (PAIR-C) and AAVs has been found in human serum, indicating that immune response is a problem that cannot be ignored, especially for in vivo gene correction. Non-human primates (NHPs) share many genetic and physiological traits with human, and are considered as the bridge for translational medicine. However, whether NHPs have same PAIR-C status with human is still unknown. Here, macaques (rhesus and cynomolgus), including normal housed and CRISPR-SpCas9 or TALENs edited individuals, were used to detect PAIR-C which covered SaCas9, SpCas9, AsCas12a and LbCas12a. Dogs and mice were also detected to expand the range of species. In addition, pre-existing adaptive antibodies to AAV8 and AAV9 were performed against macaques of different ages. The results showed that adaptive immunity was pre-existing in the macaques regardless of Cas proteins and AAVs. These findings indicate that the pre-existing adaptive immune of AAV-delivered CRISPR construction and correction system should be concerned for in vivo experiments.

immunology↗