Biology subjects

Chen, K. H.

Publications and source records attributed to Chen, K. H..

11 recordsLinked to original sources

bioRxiv2025

Puromycin-sensitive aminopeptidase acts as an inhibitory auxiliary subunit of volume-regulated anion channels

Volume-regulated anion channels (VRACs) are large-pore channels present in nearly all vertebrate cells, playing key roles in cell volume regulation and autocrine/paracrine signaling. Here, we identify the ubiquitously expressed puromycin-sensitive aminopeptidase (PSA) as a binding partner of the obligatory VRAC subunit SWELL1 (also known as LRRC8A) and report the cryo-electron microscopy structure of the SWELL1-PSA complex. Three PSA molecules associate with a single SWELL1 hexamer, coupling adjacent leucine-rich repeat (LRR) domains into local dimers. Functionally, PSA overexpression suppresses VRAC activation, whereas its deletion results in elevated basal channel activity. Notably, PSAs regulatory role on VRACs is independent of its aminopeptidase activity. Our findings identify PSA as the first auxiliary subunit of VRACs, highlight the role of intracellular LRR domains in allosteric channel gating, and propose a new strategy for modulating VRAC function in diverse physiological contexts, including cGAMP transport and STING signaling.

molecular biology↗

bioRxiv2025

The proton-activated chloride channel inhibits SARS-CoV-2 spike protein-mediated viral entry through the endosomal pathway.

SARS-CoV-2 binds to its obligatory receptor, angiotensin-converting enzyme 2 (ACE2) and capitalizes on decreasing endosomal acidity and cathepsin-mediated spike protein cleavage to enter cells. Endosomal acidification is driven by V-ATPase which pumps protons (H+) into the lumen. The driving force for H+ is maintained by the import of chloride (Cl-) which is mediated by intracellular CLC transporters. We have recently identified the Proton-Activated Chloride (PAC) channel as a negative regulator of endosomal acidification. PAC responds to low pH and releases Cl- from the lumen to prevent endosomal hyperacidification. However, its role in SARS-CoV-2 viral entry remains unexplored. Here, we show that overexpressing the PAC channel in ACE2 expressing HEK 293T cells markedly inhibited the SARS-CoV-2 spike-mediated viral entry. Several lines of evidence suggest that this effect was due to the suppression of the endosomal entry pathway. First, the abilities of PAC to regulate endosomal acidification and inhibit pseudoviral entry were both dependent on its endosomal localization and channel activity. Second, the inhibitory effect on viral entry was similar to the suppression mediated by E64-d, a cathepsin inhibitor, while no major additive effect for both treatments was observed. Third, this inhibition was also attenuated in cells expressing TMPRSS2, which provides the alternative entry pathway through cell surface. Importantly, PAC overexpression also inhibited the number and size of plaques formed by two live SARS-CoV-2 isolates (B.1 and Omicron XBB.1.16) in Vero E6 cells. Altogether, our data indicates that PAC plays a vital role in inhibiting SARS-CoV-2 viral entry and identifies this endosomal channel as a potential novel target against the infection of SARS-CoV-2 and other viruses, which rely on the endosomal pathway.

physiology↗

bioRxiv2025

Phagosome-mediated anti-bacterial immunity is governed by the proton-activated chloride channel in peritoneal macrophages

The success of phagosome degradation relies on the ability of phagocytes to regulate the maturation of phagosomes. However, its underlying molecular mechanisms remain poorly understood. Here, we identify the proton-activated chloride (PAC) channel as a key negative regulator of phagosome maturation. PAC deletion enhanced phagosomal acidification and protease activities, leading to augmented bacterial killing in large peritoneal macrophages (LPMs) upon Escherichia coli infection in mice. Surprisingly, phagosome degradation also stimulated STING-IRF3-interferon responses and inflammasome activation in LPMs, both of which are enhanced upon PAC deletion. The increased inflammasome activation induced the release of cleaved gasdermin D, which localized to the surface of bacteria in the peritoneum and further contributed to their killing. Finally, enhanced bacterial clearance by PAC-deficient LPMs reduced proinflammatory immune cell infiltration and peritoneal inflammation, resulting in improved survival in mice. Our study thus provides new insights into the molecular mechanism of phagosome maturation and the dynamics of host defense response following phagosome-mediated bacterial degradation in peritoneal macrophages. SummaryThe PAC channel mediates phagosome maturation during bacterial infection in macrophages. PAC deletion promotes phagosome-mediated STING-interferon signaling and inflammasome-mediated gasdermin D secretion during bacterial infection in peritoneal macrophages.

immunology↗

bioRxiv2025

Guidestar: a spike-in approach to improve RNA detection accuracy in imaging-based spatial transcriptomics

Imaging-based spatial transcriptomics technologies, such as MERFISH and seq-FISH, use combinatorial barcoding and imaging to simultaneously detect individual RNA molecules from 10s to 10,000s of genes. These technologies require the decoding of individual RNA molecules location and gene identity from stacks of images. However, beyond using blank code-words as negative controls, there is a lack of ground truth information embedded within the assay to experimentally measure the accuracy and sensitivity of the decoding algorithm. We introduce Guidestar, a system of spike-in controls integrated within a combinatorial FISH assay, that labels a subset of RNA transcripts with additional probes. These probes are imaged separately as guide bits, which serve as ground-truth data to assess decoding accuracy at the level of individual RNA molecules. Using Guidestar to evaluate accuracy of an existing decoding method suggested alternative parameter settings that increased sensitivity with minimal impact on accuracy. We also used the Guidestar dataset to train a machine-learning based classifier to distinguish true from false RNA calls, yielding 9% and 40% higher F1 scores across cell line and tissue samples, respectively.

genomics↗

bioRxiv2024

Spatial Perturb-Seq: Single-Cell Functional Genomics within Intact Tissue Architecture

We develop Spatial Perturb-Seq, an in vivo CRISPR technology that interrogates multiple genes within single cells of intact tissues. We apply Spatial Perturb-Seq to screen risk genes for neurodegenerative diseases in the mouse brain, uncovering cell autonomous and cell-cell microenvironmental effects within the spatially intact tissue. Spatial Perturb-Seq functionally screens multiple genes in situ and in vivo, identifying candidate genes underlying dysregulated neuronal intercellular communication pathways in Lrp1 signalling and ephrin-Eph receptor interactions.

cell biology↗

bioRxiv2024

Spatial mRNA profiling using Rapid Amplified Multiplexed-FISH (RAM-FISH)

Spatial localization of multiple mRNAs in intact tissues provides vital insights into development and function, yet routine multiplexed imaging remains constrained by complex workflows, specialized equipment costs, or sample preparation requirements. Here, we present Rapid Amplified Multiplexed FISH (RAMFISH), an accessible, modular, benchtop workflow for targeted spatial mRNA profiling in intact tissues and whole-mount specimens. RAMFISH achieves reliable multiplexing of over 30 transcripts through iterative cycles of standard hybridization either manually or via open-source automation and imaging in shared confocal systems. To streamline analysis, we introduce an integrated, open-source pipeline automating signal normalization, rigid and non-rigid alignment, Laplacian of Gaussian spot calling, and 30-channel composite merging. We validate the platform by mapping multiple transcripts in developing Bicyclus anynana butterfly wings and intact 14 days-post-fertilization Danio rerio larvae, resolving both historically inferred domains and newly characterized gene expression. Overall, RAMFISH methodology offers an end-to-end, completely open-source ecosystem for robust, semi-quantitative, spatial mRNA mapping on a variety of sample types. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/627193v4_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@14dc9a3org.highwire.dtl.DTLVardef@1f11e3dorg.highwire.dtl.DTLVardef@f85483org.highwire.dtl.DTLVardef@1746701_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2024

KAT6A deficiency impairs cognitive functions through suppressing RSPO2/Wnt signaling in hippocampal CA3

Intellectual disability (ID) affects [~]2% of the general population and is often genetic in origin. ID-associated genes are enriched for epigenetic factors, including those encoding the largest family of histone lysine acetyltransferases (KAT5-KAT8). Among them is KAT6A, whose de novo heterozygous mutations cause KAT6A Syndrome (or Arboleda-Tham Syndrome), with ID as a common clinical feature. However, the underlying molecular mechanisms remain elusive. Here, we show that haploinsufficiency of Kat6a impairs learning and memory in mice, and specific deletion of Kat6a in excitatory neurons recapitulates the hippocampus-dependent memory deficits. Unexpectedly, KAT6A deficiency results in impaired synaptic structure and plasticity in hippocampal CA3, but not in CA1 region. Combining single-nucleus RNA-sequencing and chromatin analysis, we identify a CA3-enriched gene Rspo2, encoding a Wnt activator R-spondin 2, as a key transcriptional target of KAT6A. Moreover, deletion of Rspo2 in excitatory neurons phenocopies the loss of Kat6a, resulting in defective Wnt/{beta}-catenin signaling and synaptic plasticity in CA3, and abnormal cognitive behaviors in mice. Importantly, restoring RSPO2 expression in CA3 pyramidal neurons rescues the deficits in Wnt signaling and learning-associated behaviors in Kat6a mutant mice. Collectively, our results demonstrate that KAT6A plays a critical role in regulating synaptic plasticity and memory formation through RSPO2-mediated Wnt signaling in hippocampal CA3, shedding new light on the fundamental mechanisms of ID and providing potential therapeutic targets for the treatment of KAT6A Syndrome and related neurodevelopmental diseases.

neuroscience↗

bioRxiv2023

Highly sensitive spatial transcriptomics using FISHnCHIPs of multiple co-expressed genes

High-dimensional, spatially resolved analysis of intact tissue samples promises to transform biomedical research and diagnostics, but existing spatial omics technologies are costly and labor-intensive. We present FISHnCHIPs for highly sensitive in situ profiling of cell types and gene expression programs. FISHnCHIPs achieves this by simultaneously imaging [~]2-35 co-expressed genes that are spatially co-localized in tissues, resulting in similar spatial information as single-gene FISH, but at [~]2-20-fold higher sensitivity. Using FISHnCHIPs, we imaged up to 53 gene modules from the mouse kidney and mouse brain, and demonstrated high-speed, large field-of-view profiling of a whole tissue section. FISHnCHIPS also revealed spatially restricted localizations of cancer-associated fibroblasts in a human colorectal cancer biopsy. Overall, FISHnCHIPs enables robust and scalable spatial transcriptomics analysis of tissues with normal physiology or undergoing pathogenesis.

genomics↗

bioRxiv2022

BANKSY: A Spatial Omics Algorithm that Unifies Cell Type Clustering and Tissue Domain Segmentation

Each cell type in a solid tissue has a characteristic transcriptome and spatial arrangement, both of which are observable using modern spatial omics assays. However, the common practice is still to ignore spatial information when clustering cells to identify cell types. In fact, spatial location is typically considered only when solving the related, but distinct, problem of demarcating tissue domains (which could include multiple cell types). We present BANKSY, an algorithm that unifies cell type clustering and domain segmentation by constructing a product space of cell and neighbourhood transcriptomes, representing cell state and microenvironment, respectively. BANKSYs spatial kernel-based feature augmentation strategy improves per-formance and scalability on both tasks when tested on FISH-based and sequencing-based spatial omics data. Uniquely, BANKSY identified hitherto undetected niche-dependent cell states in two mouse brain regions. Lastly, we show that quality control of spatial omics data can be formulated as a domain identification problem and solved using BANKSY. BANKSY represents a biologically motivated, scalable, and versatile framework for analyzing spatial omics data.

bioinformatics↗

bioRxiv2018

Tfap2a is a novel gatekeeper of differentiation in renal progenitors during kidney development

Renal functional units known as nephrons undergo patterning events during development that create a segmental array of cellular populations with discrete physiological tasks. Knowledge about the terminal differentiation programs of each nephron segment has central importance for understanding kidney disease and to advance regenerative medicine, as mammalian nephrons grown in organoid cultures from pluripotent cells fail to terminally differentiate. Here, from a novel forward genetic screen using zebrafish we report the discovery that transcription factor AP-2 alpha (tfap2a) coordinates a gene regulatory network that controls the progression of nephron distal segment progenitors into the differentiated state. Overexpression of tfap2a rescued differentiation in mutants and caused ectopic expression of distal segment markers in wild-type nephrons, indicating tfap2a is sufficient to instigate the distal segment differentiation program. tfap2a/2b deficiency exacerbated distal nephron segment differentiation defects, revealing functional redundancy where tfap2a has a dominant role upstream of its family member. With further genetic studies, we assembled a blueprint of the tfap2a gene regulatory network during nephrogenesis. We demonstrate that tfap2a acts downstream of Iroquois homeobox 3b, a conserved distal lineage transcription factor. tfap2a controls a circuit consisting of irx1a, tfap2b, and genes encoding solute transporters that dictate the specialized metabolic functions of the distal nephron segments, and we show for the first time that this regulatory node is distinct from the pathway circuits controlling aspects such as apical-basal polarity and ciliogenesis during the differentiation process. Thus, our studies reveal new insights into the genetic control of differentiation, where tfap2a regulates the suite of segment transporter traits. These findings have relevance for understanding renal birth defects, as well as efforts to recapitulate nephrogenesis in vivo to make functional units that can facilitate organoid applications such as drug discovery and regenerative therapies.\n\nSummary StatementHere, we report for the first time that transcription factor AP-2 alpha (tfap2a) controls the progression from nephron progenitor into the fully differentiated state. This fundamentally deepens our knowledge about the genetic control of kidney development.

developmental biology↗

bioRxiv2018

Small non-coding RNA expression in developing mouse nephron progenitor cells

MicroRNAs (miRNAs) are small non-coding RNAs that are essential for the regulation of gene expression and play critical roles in human health and disease. Here we present comprehensive miRNA profiling data for mouse nephron progenitors, which give rise to most of the cell-types of the nephron, the functional units of the kidney. We describe a miRNA expression profile for nephron progenitors, with 162 miRNAs differentially expressed in nephron progenitors when compared to whole kidney. We also annotated 52, and experimentally validated 4 novel miRNAs, which are expressed in developing kidney. Our data is available as a public resource, so that it can be integrated into future studies and analyzed in the context of other functional and epigenomic data in kidney development. Specifically, it will be useful in the effort to shed light on molecular mechanisms underlying processes essential for normal kidney development, such as nephron progenitor specification, self-renewal and differentiation.

developmental biology↗