Biology subjects

Chen, J.-H.

Publications and source records attributed to Chen, J.-H..

At least 19 recordsLinked to original sources

bioRxiv2026

Reverse engineering Lewy bodies suggests alpha-synuclein pathology as condensatopathy

Understanding how soluble monomeric alpha-synuclein transitions into pathological alpha-synuclein (Lewy bodies) has remained a central challenge in neurodegeneration. Existing models, largely based on treating cells and animals with pre-formed alpha-synuclein fibrils cannot account for the emergence of the first inclusion without pre-existing seeds. Here, we combine in vitro reconstitutions, computational modeling and a minimal cellular system to show that phase separation of alpha-synuclein with an interacting scaffold can recapitulate Lewy body-like structures. An unbiased proteomics screen, live-cell imaging, correlative light-electron microscopy, and soft X-ray tomography show that Lewy body-like structures are associated with redistribution of actin and membrane-bound organelles, with mitochondria accumulating at their periphery in an actin-dependent manner. Structural analyses further indicate that alpha-synuclein condensates provide local environments in which the insoluble beta-sheets can form. Together, these findings support a seed-independent trajectory from soluble alpha-synuclein through condensation and progressive immobilization to Lewy body-like assemblies and, subsequently, insoluble beta-sheet fibrils. This framework offers a new mechanistic route for understanding the disease progression and promises to guide the design of innovative therapeutic interventions.

neuroscience↗

bioRxiv2026

Paraspeckles translate microbial insult-induced inflammation into neurovascular remodeling by enhancing CYR61-FGF2 signaling via RBM14 sequestration

BackgroundSystemic inflammation triggered by microbial insults can disrupt endothelial homeostasis, impair blood-brain and blood-retinal barriers, leading to neurovascular remodeling in the central nervous system (CNS). Subnuclear condensates, paraspeckles, play a substantial role in stress-induced gene regulation, yet their contribution to the inflammatory relay from microbial insults to neurovascular remodeling remains unelucidated. ResultsOur comparative transcriptomic analysis followed by experimental validation identified a cross-species NEAT1_2/CYR61/FGF2 signature in the CNS positively associated with neurovascular remodeling across human disease cohorts and multiple mouse models. Notably, systemic inflammation triggered by microbial insults, including sepsis or gut dysbiosis, enhanced NEAT1_2 expression in the brain and retina with neurovascular remodeling. Microbial insults induced hyper-assembly of paraspeckles and the expression of CYR61 and FGF2 in vascular endothelial cells. Paraspeckle assembly and its required NEAT1_2 Domain C, rather than NEAT1_2 expression levels, play a pivotal role in endothelial homeostasis control and neurovascular remodeling by sequestering the RNA-binding protein RBM14 from the CYR61 promoter, thereby relieving its repression of CYR61 transcription. Moreover, secreted CYR61 enhanced FGF2-mediated endothelial remodeling signals in a paracrine manner. Disrupting paraspeckle assembly by targeting Domain C intercepts neurovascular remodeling, restoring endothelial homeostasis in vivo. ConclusionsOur results demonstrate an essential and conserved role for paraspeckles in the inflammatory relay from microbial insults to neurovascular remodeling by sequestering RBM14 to enhance CYR61-FGF2 signaling. Furthermore, our study underscores paraspeckle assembly as a promising therapeutic target for neurovascular remodeling and related diseases.

molecular biology↗

bioRxiv2025

Decoding acute neuroinflammatory states from the 3D architecture of in vitro microglia

Microglia, core brain immune defenders, rapidly polarize into pro- or anti-inflammatory states that shape neuronal survival during acute brain inflammation. Yet, how these inflammatory states are encoded at the native cellular level remains unclear. While microglial states associate with specific molecular and organelle markers, it is unknown whether their cellular architecture integrates robust metabolic and structural features. Here, we quantitatively decode inflammatory states from the 3D architecture of individual in vitro mouse microglia (BV-2). Using soft X-ray tomography on established BV-2 polarization, we identify coordinated intracellular organization distinguishing homeostatic, pro-inflammatory, and anti-inflammatory cells, including characteristic lipid droplet-endosome architectures. We further link lipid-endosome reorganization with mTORC1 pathway, by functionally implicating sestrin-2 in promoting anti-inflammation. Finally, we resolve the time-dependent remodeling of lipid droplet 3D profiles by their distinct lipidomic composition across inflammatory states. Overall, our work enables deciphering microglia states in disease-relevant models, with potential to ultimately understand brain immunity.

cell biology↗

bioRxiv2025

Sex-biased duplicates are rapidly generated during Drosophila tRNA repertoire evolution

Eukaryotic genomes encode hundreds of transfer RNA genes ostensibly to ensure efficient translation. How these seemingly redundant tRNA copies arise and are naturally selected in metazoan genomes and populations remains largely unexplored, owing to challenges in accurate sequencing of tRNA loci and their transcripts. We leveraged long-read genome assemblies of 24 Drosophilid species to infer the origination times of the entire Drosophila melanogaster tRNA gene repertoire and found that continuous gain and loss of tRNA duplicates throughout 60 million years of divergence has resulted in rapid taxonomic restriction of isodecoding and isoaccepting copies--even producing one isodecoder specific to D. melanogaster. Moreover, we identified patterns of global codon usage, especially in lineage-specific genes, incongruous with translational efficiency hypotheses. Through generation of tRNA sequencing in D. melanogaster we observed that recently duplicated, taxonomically restricted tRNA copies had sexually dimorphic patterns of expression, fragmentation, and nucleoside modification. Our work implicates the emergence of taxonomically restricted tRNA genes as sources of regulatory diversity and reveals that sexual and natural selection affect the evolutionary dynamics of the tRNA gene family in metazoan species.

evolutionary biology↗

bioRxiv2025

Interior soft x-ray tomography with sparse global sampling

Objective. To investigate the feasibility of interior imaging reconstruction in soft X-ray tomography to achieve higher spatial resolution cellular imaging, including whole-cell imaging. Approach. We develop an alignment and reconstruction algorithm that enables a combination of a low number of images from sparse whole-cell imaging with a high-resolution local interior scan. Based on numerical simulations, we demonstrate that combined reconstructions mitigate the depth of field limitation in high-resolution scans, enable radiation dose optimization, and yield quantitative X-ray absorption values with sparse sampling. Furthermore, we validate our numerical approach using experimental data from two different cell types and demonstrate that combined reconstruction is a reliable method for obtaining high and local spatial resolution within the volume of a whole cell. Significance. The developed sparse reconstruction algorithm provides a robust and faithful visualization of cellular organelles with soft X-ray tomography. A mesoscale imaging approach, such as an interior tomography scan, enables one to "scout" and zoom into the volumes of interest that contain organelles of interest. Utilizing sparse reconstructions, this increase in spatial resolution is achieved without sacrificing larger volume imaging, providing information on the relative position of all organelles within a cell.

cell biology↗

bioRxiv2025

Ultrahigh Resolution Structure of the Heat-stable Form-IAq RuBisCO from the Thermophilic Purple Sulfur Bacterium Thermochromatium tepidum

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the initial carbon fixation reaction in the Calvin-Benson-Bassham cycle in plants and most microbial autotrophs. Among the many forms of RuBisCOs, form-I--a protein complex containing 8 large and 8 small subunits--is the most common, representing over 90% of all known RuBisCOs. Although many form-I RuBisCO structures have been determined, no structure has been reported for a form-IAq RuBisCO. Here, and at ultrahigh (1.55 [A]) resolution, we detail the structure of the heat-stable form-IAq RuBisCO from the thermophilic and anaerobic purple bacterium Thermochromatium (Tch.) tepidum. The overall structure of the Tch. tepidum form-IAq RuBisCO resembles both a form-IAc RuBisCO from a chemolithotrophic sulfur bacterium and a synthetic form-I RuBisCO reconstructed from ancestral sequences. However, despite significant structural and sequence similarities with other form-I RuBisCOs, the Tch. tepidum form-IAq RuBisCO shows significantly greater interactions between adjacent small subunits through their extended N-terminal domains that contain a characteristic six-residue insertion unique to form-IAq RuBisCOs. Structural differences of Tch. tepidum RuBisCO from its mesophilic relative Allochromatium vinosum suggests the mechanisms of its enhanced thermostability. In addition to small subunit interactions, key substitutions on the hydrophilic surface of the small subunits likely contribute to its thermostability as well. It has been proposed that form-I RuBisCOs first appeared in anaerobic thermophiles. Our structure of the Tch. tepidum RuBisCO represents the first such structure of a form-IAq enzyme, providing fresh clues for unraveling the evolutionary history of RuBisCO and new details for how this key enzyme remains active at elevated temperatures.

plant biology↗

bioRxiv2025

Nucleus softens during herpesvirus infection

Nuclear mechanics is remodeled not only by extracellular forces but also by internal modifications, such as those induced by viral infections. During herpes simplex virus type 1 infection, the nuclear structures undergo drastic reorganization, but little is known about how nuclear mechanobiology changes as a result. We show that the nucleus softens dramatically during the infection. To understand the phenomenon, we used advanced microscopy and computational modeling. We discovered that the enlarged viral replication compartment had a low biomolecular density, partially explaining the observed nuclear softening. The mobility of the nuclear lamina decreased, which suggests increased rigidity and an inability to induce softening. However, computational modeling supported by experimental data showed that reduced outward forces, such as cytoskeletal pull and intranuclear osmotic pressure acting both on and within the nucleus, can explain the decreased nuclear stiffness. Our findings reveal that during infection, the nucleus is subject to changes in multiple mechanical forces, leading to decreased nuclear stiffness. Author SummaryDNA viruses take over the host cell nucleus, inducing dramatic structural modifications. There is currently very little knowledge of how the progression of viral infection modifies the mechanical properties of the nucleus, which are essential for various cellular processes, including gene expression and cell migration. Here, we show that the nucleus softens when herpesvirus infection progresses. We discovered that the viral replication compartment established in the central parts of the nucleus had a low biomolecular density, which may contribute to the nuclear softening. The shape and motion of the nuclear lamina suggested that it became more rigid, indicating that another mechanism was involved in the decreased elasticity. Our mechanical simulations and experiments showed that a reduction in outward forces, such as actin cytoskeleton pull or osmotic pressure, is the most likely factor in the nuclear softening. Our study provides new insights into the effects of DNA viruses on the mechanics of host cell nuclei, significantly expanding the knowledge of viral infection mechanobiology.

cell biology↗

bioRxiv2024

Soft X-ray tomography reveals variations in B.subtilis biofilm structure upon tasA deletion

Bacterial biofilms are complex communities of cells within a self-produced extracellular matrix. They play crucial roles in healthcare, nutrition, agriculture and environmental research, yet an analysis of their elaborate 3D architecture remains challenging. Understanding mechanisms of biofilm formation, particularly the effects of chemical, physical, and genetic influences or modifications, is crucial but requires structural information at subcellular resolution to enable a community-level analysis of biofilms. In this work, we developed a "biofilm-in-capillary" growth method compatible with full-rotation soft X-ray tomography, providing high-resolution 3D imaging of bacterial cells and their surrounding extracellular matrix during biofilm formation, without drying or fixation steps. This approach offers 50 nm isotropic spatial resolution, rapid imaging time, and quantitative native analysis of biofilm structure. We demonstrate the potential of our method using Bacillus subtilis biofilms, detecting coherent alignment and chaining of wild-type cells while they are travelling towards the oxygen-rich capillary tip region. In stark contrast, the genetic knock-out {Delta}tasA shows a loss of cellular orientation, including changes in the extracellular matrix in volume and chemical density. Notably, we show that the addition of TasA protein to a culture of a {Delta}tasA strain restores the extracellular matrix density and leads to a compaction of cell assemblies, yet no chaining is observed as for the wildtype. Our approach to imaging biofilms is scalable and transferable, opening new avenues for examining biofilm structure and function across various species, including mixed biofilms, and observing 3D reorganization in response to genetic and environmental factors.

microbiology↗

bioRxiv2024

Highly Dynamic and Sensitive NEMOer Calcium Indicators for Imaging ER Calcium Signals in Excitable Cells

Endoplasmic/sarcoplasmic reticulum (ER/SR) sits at the heart of the calcium (Ca2+) signaling machinery, yet current genetically encoded Ca2+ indicators (GECIs) lack the ability to detect elementary Ca2+ release events from ER/SR, particularly in muscle cells. Here we report a set of organellar GECIs, termed NEMOer, to efficiently capture ER Ca2+ dynamics with increased sensitivity and responsiveness. Compared to G-CEPIA1er, NEMOer indicators exhibit dynamic ranges that are an order of magnitude larger, which enables up to 5-fold more sensitive detection of Ca2+ oscillation in both non-excitable and excitable cells. The ratiometric version further allows super-resolution monitoring of local ER Ca2+ homeostasis and dynamics. Notably, the NEMOer-f variant enabled the inaugural detection of Ca2+ blinks, elementary Ca2+ releasing signals from the SR of cardiomyocytes, as well as in vivo spontaneous SR Ca2+ releases in zebrafish. In summary, the highly dynamic NEMOer sensors expand the repertoire of organellar Ca2+ sensors that allow real-time monitoring of intricate Ca2+ dynamics and homeostasis in live cells with high spatiotemporal resolution.

synthetic biology↗

bioRxiv2024

Multi-tissue transition of A-to-I RNA editing pattern and its regulatory relevance in transcription, splicing, and translation during development

Previous studies have shown that A-to-I RNA editing can occur in various organs and tissues under normal physiological conditions. However, the dynamics of RNA editing and its functional relevance in multiple tissues and organs during the embryo-to-adult transition in mammals remain to be elucidated. Here, we performed a comprehensive analysis of RNA-Seq and Ribo-Seq data from six mouse tissues at embryonic and adult stages to elucidate the A-to-I RNA editing transition during development and to validate it in additional mouse brain datasets spanning multiple developmental stages. Our results revealed a general transition of up-regulated A-to-I RNA editing activity across numerous tissue types during embryonic-adult development, indicated by significantly increased average RNA editing levels. Consistently, differential RNA editing (DRE) analysis showed more up-regulated than down-regulated RNA editing sites in all six tissue types. Furthermore, such RNA editing transitions during development could contribute to differential gene expression (DEG) at both transcriptional and translational levels, as well as differential alternative splicing (DAS) across multiple tissues. Differentially edited genes with DEG or DAS could be involved not only in tissue-specific biological functions but also in common routine biological processes during development. Notably, Adarb1 was found to be a more important player than Adar in the CNS (the brain and retina), with its expression levels increasing gradually and correlating with the A-to-I RNA editing activity. Our study demonstrates the potential role of A-to-I editing during development across multiple tissues, providing new insights into its regulatory relevance in both transcriptional and translational landscapes.

genomics↗

bioRxiv2023

Progression of herpesvirus infection remodels mitochondrial organization and metabolism

Viruses target mitochondria to promote their replication, and infection-induced stress during the progression of infection leads to the regulation of antiviral defenses and mitochondrial metabolism which are opposed by counteracting viral factors. The precise structural and functional changes that underlie how mitochondria react to the infection remain largely unclear. Here we show extensive transcriptional remodeling of protein-encoding host genes involved in the respiratory chain, apoptosis, and structural organization of mitochondria as herpes simplex virus type 1 lytic infection proceeds from early to late stages of infection. High-resolution microscopy and interaction analyses unveiled infection-induced emergence of rough, thin, and elongated mitochondria relocalized at the perinuclear area, a significant increase in the number and clustering of ER-mitochondria contact sites, and thickening and shortening of mitochondrial cristae. Finally, metabolic analyses demonstrated that reactivation of ATP production is accompanied by increased mitochondrial Ca2+ content and proton leakage as the infection proceeds. Overall, the significant structural and functional changes in the mitochondria triggered by the viral invasion are tightly connected to the progression of the virus infection.

microbiology↗

bioRxiv2023

Aberrant phase separation is a common killing strategy of positively charged peptides in biology and human disease

Positively charged repeat peptides are emerging as key players in neurodegenerative diseases. These peptides can perturb diverse cellular pathways but a unifying framework for how such promiscuous toxicity arises has remained elusive. We used mass-spectrometry-based proteomics to define the protein targets of these neurotoxic peptides and found that they all share similar sequence features that drive their aberrant condensation with these positively charged peptides. We trained a machine learning algorithm to detect such sequence features and unexpectedly discovered that this mode of toxicity is not limited to human repeat expansion disorders but has evolved countless times across the tree of life in the form of cationic antimicrobial and venom peptides. We demonstrate that an excess in positive charge is necessary and sufficient for this killer activity, which we name polycation poisoning. These findings reveal an ancient and conserved mechanism and inform ways to leverage its design rules for new generations of bioactive peptides.

cell biology↗

bioRxiv2022

An ESCRT-dependent pathway of Nuclear and Cytoplasmic Spatial PQC is coordinated at Nuclear Vacuolar Junctions

Effective Protein Quality Control (PQC), essential for cellular health, relies on spatial sequestration of misfolded proteins into defined inclusions. Here we elucidate the coordination of nuclear and cytoplasmic spatial PQC. While cytoplasmic misfolded proteins concentrate in a cytoplasmic, perinuclear Juxta Nuclear Quality control compartment (JUNQ), nuclear misfolded proteins sequester into a perinucleolar IntraNuclear Quality control compartment (INQ). Particle tracking reveals the INQ and JUNQ converge to face each other across the nuclear envelope at a site proximal to the Nuclear-Vacuolar Junction (NVJ) marked by perinuclear ESCRT-II/-III protein Chm7. Strikingly, this ESCRT-dependent convergence facilitates VPS4-dependent vacuolar clearance of misfolded cytoplasmic and nuclear proteins, the latter entailing extrusion of nuclear INQ into the vacuole. We propose perinuclear ESCRT coordinates spatial PQC at nuclear-vacuolar contacts to facilitate vacuolar clearance of nuclear and cytoplasmic misfolded proteins.

biochemistry↗

bioRxiv2022

Engineering of NEMO as calcium indicators with ultra-high dynamics and sensitivity

Genetically-encoded calcium indicators (GECI) are indispensable tools for real-time monitoring of intracellular calcium signals and cellular activities in living organisms. Current GECIs face the challenge of sub-optimal peak signal-to-baseline-ratio (SBR) with limited resolution for reporting subtle calcium transients. We report herein the development of a suite of calcium sensors, designated NEMO, with fast kinetics and wide dynamic ranges (>100-fold). NEMO indicators report Ca2+ transients with peak SBRs ~20-fold larger than the top-of-the-range GCaMP series. NEMO sensors further enable the quantification of absolution calcium concentration with ratiometric or photochromic imaging. Compared to GCaMPs, NEMOs could detect single action potentials in neurons with a peak SBR two times higher and a median peak SBR four times larger in vivo, thereby outperforming most existing state-of-the-art GECIs. Given their high sensitivity and resolution to report intracellular Ca2+ signals, NEMO sensors may find broad applications in monitoring neuronal activities and other Ca2+-modulated physiological processes in both mammals and plants.

synthetic biology↗

bioRxiv2022

Emergent Actin Flows Explain Diverse Parasite Gliding Modes

During host infection, single-celled apicomplexan parasites like Plasmodium and Toxoplasma use a motility mechanism called gliding, which differs fundamentally from other known mechanisms of eukaryotic cell motility. Gliding is thought to be powered by a thin layer of flowing filamentous (F)-actin1- 3 sandwiched between the plasma membrane and a myosin-coated4,5 inner membrane complex. How this surface actin layer drives the diverse apicomplexan gliding modes observed experimentally - helical, circular, and twirling6,7, and patch8, pendulum9, or rolling2 - presents a rich biophysical puzzle. Here, we use single-molecule imaging to track individual actin filaments and myosin complexes in live Toxoplasma gondii. Based on these data, we hypothesize that F-actin flows arise by self-organization, rather than following a microtubule-based template as previously believed. We develop a continuum model of emergent F-actin flow within the unusual confines provided by parasite geometry. In the presence of F-actin turnover, our model predicts the emergence of a steady-state mode in which actin transport is largely rearward. Removing actin turnover leads to actin patches that recirculate up and down the cell, a "cyclosis" that we observe experimentally for drug-stabilized actin bundles in live parasites. These findings provide a mechanism by which actin turnover governs a transition between distinct self-organized F-actin states, whose properties can account for the diverse gliding modes known to occur. More broadly, we illustrate how different forms of gliding motility can emerge as an intrinsic consequence of the self-organizing properties of F-actin flow in a confined geometry.

biophysics↗

bioRxiv2022

Extending of imaging volume in soft x-ray tomography

Soft x-ray tomography offers rapid whole single cell imaging with a few tens of nanometers spatial resolution without fixation or labelling. At the moment, this technique is limited to 10 {micro}m thick specimens, such that applications of soft x-ray tomography to large human cells or multicellular specimens are not possible. We have developed a theoretical and experimental framework for soft x-ray tomography to enable extension of imaging volume to 18 {micro}m thick specimens. This approach, based on long depth of field and half-acquisition tomography, is easily applicable to existing full-rotation based microscopes. This opens applications for imaging of large human cells, which are often observed in cancer research and cell to cell interactions.

cell biology↗

bioRxiv2020

Novel missense mutation E585K in retinitis pigmentosa leads to compromised RPGR splicing diversity

Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene, are the major cause of X-linked retinitis pigmentosa (RP). Herein we used whole-exome sequencing to screen possible novel RPGR mutations in RP patients, and identified a novel missense mutation E585K in a patient with early onset but slow disease progression, and a frameshift deletion E998Gfs*78 in a patient with RP sine pigmento and high myopia. Intriguingly, bioinformatic analysis indicated that E585K probably affected RPGR RNA splicing instead of the protein sequence directly. Mini-gene assays in 293T cells revealed that splicing events of the E585K mutant were found to be also exist in wildtype, but with a shifted pattern. In the E585K mini-gene usage of an upstream alternative 5' splice site (5' ss) of exon 14 was enhanced, and other splicing events were suppressed, including the canonical 5' ss of exon 14, skipping of exon 14/15 and retention of intron 14. As a result, RPGR splicing products of the E585K mini-gene were predominated by transcripts containing a 4-bp deletion, with a small fraction of in-frame transcripts containing a retended intron 14, which might explain the slow disease progression in the patient carrying the mutation. RNA-Seq analysis further confirmed existence of these splicing events in endogenous RPGR RNA in human retina, pointing to compromised splicing diversity in the E585K mutant. Our findings thus added to the understanding of genotype-phenotype correlation in RP, and suggested that compromised RPGR splicing diversity might play a role in molecular mechanism of the disease.

genetics↗

bioRxiv2020

Characterization of dynamic age-dependent changes and driver microbes in primate gut microbiota during host's development and healthy aging via captive crab-eating macaque model

Recent population studies have significantly advanced our understanding of how age shapes the gut microbiota. However, the actual role of age could be inevitably confounded due to varying environmental factors in human populations. A well-controlled environment is thus necessary to reduce undesirable cofounding effects, and recapitulate age-dependent taxonomic and functional changes in the healthy primate gut microbiota. Herein we performed 16S rRNA gene sequencing, characterized age-associated gut microbial profiles from infant to elderly crab-eating macaques reared in captivity, and systemically revealed lifelong dynamic changes of primate gut microbiota in the model. While the most significantly age-associated gut microbial taxa were mainly found in commensals such as Faecalibacterium, a set of suspicious pathogens such as Helicobacter were exclusively enriched in infants, pointing to their potential role in host development. Importantly, topology analysis indicated that the connectivity of gut microbial network was even more age-dependent than taxonomic diversity, with its tremendous decline probably linked to the hosts healthy aging. NetShift analysis identified Prevotella 9, Rikenellaceae RC9 gut group and Megasphaera as key drivers during gut microbiota maturation and development, actively involved in age-dependent changes in phenotypes and functions of the gut microbial community. The current study demonstrates lifelong age-dependent changes in healthy primate gut microbiota. Our findings indicate potential importance of appropriate exposure to suspicious pathogens in infant development. The age-associated baseline profiles and driver microbes of primate gut microbiota in the current study could provide new insight into its role in the hosts development and healthy aging.

microbiology↗