Biology subjects

Lin, W.-H.

Publications and source records attributed to Lin, W.-H..

At least 19 recordsLinked to original sources

bioRxiv2026

Plekha7 promotes retina organization and inhibits inflammation and photoreceptor loss

Plekha7 associates with p120 catenin and localizes at apical cell-cell junctions where it has been implicated in junction stabilization. However, its in vivo function and underlying mechanisms of action remain unclear. Here, we show that constitutive Plekha7 loss in mice disrupts retinal organization, reduces retinal thickness, and induces RPE multinucleation, microglia infiltration, and photoreceptor loss. Plekha7 deficiency reduces the accumulation of cadherin-catenin complexes at apical junctions without altering overall cadherin levels, indicating a defect in junctional maintenance. Mechanistically, PLEKHA7 regulates cadherin trafficking by limiting levels of endocytosed E-cadherin, thereby maintaining junctional cadherin pools. Notably, these effects are largely independent of p120 catenin. In addition, further Plekha7 roles in the regulation of centrosome, cilia, microtubule dynamics, and cytokinesis, likely contribute to the phenotypic changes induced by Plekha7 knockout. These findings establish Plekha7 as a key regulator of cadherin trafficking and epithelial organization with relevance to retinal disease.

cell biology↗

bioRxiv2026

Functional genomics identifies therapeutic options, biomarkers, and resistance mechanisms for high-grade gliomas

High-grade gliomas (HGGs) are aggressive tumors with poor outcomes and limited treatment options. Here, we combined genomic and transcriptomic tumor profiling with drug testing in a patient-derived 3-dimensional culture model to identify individualized treatments and predictive biomarkers. Activity of single agents targeting frequently dysregulated glioma pathways was relatively poor ex vivo and generally reflected historical patient data. However, compounds targeting PI3K, epigenetic, and survival/senescence signaling were effective in some cases. Drug sensitivity correlated with transcriptional rather than genomic features and suggested heterogeneity as a resistance mechanism. Bromodomain and extraterminal domain inhibition was particularly effective in tumors enriched in the mesenchymal transcriptional subtype, promoted proneural transition, and was overcome by upregulated PI3K signaling. Notably, combinations were largely effective, with 6 strategies exhibiting stronger efficacy than corresponding single agents in most cases (58-77%). This study identifies HGG vulnerabilities and associated biomarkers, resistance mechanisms, and effective combination strategies that warrant further clinical validation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=171 SRC="FIGDIR/small/701806v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@cdb75corg.highwire.dtl.DTLVardef@1c133e6org.highwire.dtl.DTLVardef@1365153org.highwire.dtl.DTLVardef@11543a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Time-resolved phenotyping at subcellular resolution reveals shared principles and key trade-offs across antimicrobial peptide activities

Cationic antimicrobial peptides are a broad family of host defense molecules that neutralize bacteria by permeabilizing one or more membranes and/or inhibiting intracellular targets. Here, we present a time-resolved single-cell pipeline for quantifying these effects in Escherichia coli. Applying this pipeline to 18 diverse natural peptides and synthetic peptidomimetics reveals shared core activities, but with different kinetics, defining two classes with opposite trade-offs. Class I peptides cause abrupt growth arrest, predominantly coupled with inner membrane permeabilization and ribosome/DNA reorganization, conferring fast, multipronged action. However, rapid intracellular absorption by the first permeabilized cells depletes the extracellular pool, rendering them ineffective against dense populations, including biofilms. Class II peptides act more gradually, with delayed or absent inner membrane permeabilization, limiting their speed of action. However, this results in slower intracellular absorption and greater efficacy at high cell densities and against biofilms. These opposing functional trade-offs point to important immunological and therapeutic implications.

microbiology↗

bioRxiv2025

Macrophages promote collagen deposition through circadian regulation of fibroblasts

Collagen deposition in fibroblasts, the primary collagen-producing cells, is regulated by both macrophages and the circadian rhythm, although how these regulatory processes interact with one another is unknown. Here, we reveal that macrophage-fibroblast interaction enhances collagen deposition which requires a functional circadian rhythm. This response is also dependent on macrophage polarisation status, where M0 (naive) and M1 (pro-inflammatory) macrophages require direct cell-cell contact; whereas M2 (anti-inflammatory) macrophages utilise an additional mechanism through secreted soluble factors that strengthen circadian rhythms and significantly increase collagen deposition in fibroblasts, independent of cell-cell contact. Using mass spectrometry proteomics analysis, we identified PDGFA as a key factor in induction of circadian amplitude in fibroblasts. Crucially, macrophages lacking PDGFA showed diminished ability to modulate fibroblast circadian rhythms and collagen production. Further, in fibroblasts with impaired circadian rhythms, neither macrophages nor macrophage-secreted factors are capable of inducing collagen deposition. These results confirmed that the collagen secretory pathway is also under circadian clock control in lung fibroblasts, and demonstrated that macrophages influence collagen deposition via circadian clock mediated mechanisms. In conclusion, our findings highlight M2-derived PDGFA as a key macrophage-derived factor in extracellular matrix remodelling.

cell biology↗

bioRxiv2024

Fluorescence based live cell imaging identifies exon 14 skipped hepatocyte growth factor receptor (MET) degraders

Despite ongoing efforts to employ structure-based methods to discover targeted protein degraders (TPD), the prevailing strategy continues to be the synthesis of a focused set of heterobifunctional compounds and screen them for target protein degradation. Here we used a fluorescence based live cell imaging screen to identify degraders that target exon 14 skipped hepatocyte growth factor receptor (MET). MET is a known oncogenic driver. MET exon 14 skipping mutations (METex14{Delta}) are found in lung cancers and result in the loss of a degron that is required for E3-ligase recognition and subsequent ubiquitination, prolonging the half-life and oncogenicity of MET. Since proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that promote target degradation by the proteosome, we sought to restore degradation of MET lost with METex14{Delta} using a MET-targeting PROTAC. We generated a library of sixty PROTACs of which 37 used the MET inhibitor capmatinib as the protein of interest targeting ligand. We screened this PROTAC library for targeted degradation of METex14{Delta}-GFP using live cell imaging. We benchmarked out MET-targeting PROTACs to that of a previously reported MET-targeting PROTAC, SJF8240. Curve fitting live cell imaging data affords determination of time required to degrade 50% of the target protein (DT50), which was used in determining structure activity relationships. A promising candidate, 48-284, identified from the screen, exhibited classic PROTAC characteristics, was > 15-fold more potent than SJF8240, had fewer off targets compared to SJF8240, and degraded MET in multiple cell lines.

cancer biology↗

bioRxiv2024

SCAD Delivery Platform: A Novel Approach for Efficient CNS and Extrahepatic Oligonucleotide Therapeutics

Oligonucleotide therapeutics, including antisense oligonucleotides (ASOs) and duplex RNAs like siRNA, saRNA, and miRNA, hold immense potential for treating genetic and acquired diseases by modulating gene expression in a target-specific manner. However, effective delivery to extrahepatic tissues, particularly the central nervous system (CNS), remains a significant challenge. While GalNAc conjugation has enabled liver-specific delivery, leading to several approved siRNA drugs for hepatic targets, CNS delivery lags. ASOs, on the other hand, can self-deliver to the CNS when administered locally, as seen with nusinersen and tofersen. To address this disparity, weve developed the Smart Chemistry Aided Delivery (SCAD) platform which enables duplex RNA delivery by conjugating it to an accessory oligonucleotide (ACO), which acts as an aptamer to mediate protein binding and facilitate cellular uptake. Through extensive screening, we identified an optimal SCAD architecture that demonstrates enhanced cell-free protein binding and in vitro activity. In rodent models, local administration of SCAD-siRNA conjugates resulted in broad biodistribution throughout the CNS and sustained mRNA knockdown for over five months, with a favorable safety profile. The SCAD platform also exhibited efficient delivery to other tissues, including the eye, the lung and the joint. These features support its potential for broader clinical applications, as evidenced by an ongoing trial targeting amyotrophic lateral sclerosis (ALS) associated with mutations in the SOD1 gene. The modular design of SCAD allows it to easily adapt to any duplex RNA, making it a powerful tool for advancing oligonucleotide therapeutics.

neuroscience↗

bioRxiv2024

DNA/polysome phase separation and cell width confinement couple nucleoid segregation to cell growth in Escherichia coli

Chromosome segregation is essential for cellular proliferation. Unlike eukaryotes, bacteria lack cytoskeleton-based machinery to segregate their chromosomal DNA (nucleoid). The bacterial ParABS system segregates the duplicated chromosomal regions near the origin of replication. However, this function does not explain how bacterial cells partition the rest (bulk) of the chromosomal material. Furthermore, some bacteria, including Escherichia coli, lack a ParABS system. Yet, E. coli faithfully segregates nucleoids across various growth rates. Here, we provide theoretical and experimental evidence that polysome production during chromosomal gene expression helps compact, split, segregate, and position nucleoids in E. coli through out-of-equilibrium dynamics and polysome exclusion from the DNA meshwork, inherently coupling these processes to biomass growth across nutritional conditions. Halting chromosomal gene expression and thus polysome production immediately stops sister nucleoid migration while ensuing polysome depletion gradually reverses nucleoid segregation. Redirecting gene expression away from the chromosome and toward plasmids causes ectopic polysome accumulations that are sufficient to drive aberrant nucleoid dynamics. Cell width enlargement suggest that the proximity of the DNA to the membrane along the radial axis is important to limit the exchange of polysomes across DNA-free regions, ensuring nucleoid segregation along the cell length. Our findings suggest a self-organizing mechanism for coupling nucleoid segregation to cell growth.

microbiology↗

bioRxiv2024

Functional impact of the hyperduplication genomophenotype in high copy number endometrial cancer

High copy number endometrial cancers (HCNEC) are dominated by excessive duplications scattered across the genome, termed here as the HyperDuplication GenomoPhenotype (HDGP). Although correlated with cancer progression, its biological significance and implications for therapy have not yet been established. We identified locations and sizes of duplications in 171 endometrial cancer cases and designated 71 HCNEC cases as HDGP. We also investigated the response to the pan-ERBB inhibitor afatinib in a subset of HDGP-EC cases with ERBB2/ERBB3 duplications using a patient-derived three-dimensional culture model. Our analysis demonstrates that beyond tandem duplications there is a more general pattern involving coordinated duplication of multiple distant regions of the genome, demonstrating preferential selectivity to over-expressed potential oncogenes within a broad network. This suggests that HDGP increases tumor fitness and resistance to therapy by perturbing important gene networks in concert rather than only driver genes, suggesting a mechanistic basis for the ineffectiveness of targeted drugs in these patients and highlighting the need for combination therapies in these highly aggressive cases.

genomics↗

bioRxiv2024

Coupling of cell growth modulation to asymmetric division and cell cycle regulation in Caulobacter crescentus

In proliferating bacteria, growth rate is often assumed to be similar between daughter cells. However, most of our knowledge of cell growth derives from studies on symmetrically dividing bacteria. In many -proteobacteria, asymmetric division is a normal part of the life cycle, with each division producing daughter cells with different sizes and fates. Here, we demonstrate that the functionally distinct swarmer and stalked daughter cells produced by the model -proteobacterium Caulobacter crescentus have different average growth rates despite sharing an identical genome and environment. The discrepancy in growth rate is due to a growth slowdown during the cell cycle stage preceding DNA replication (the G1 phase), which is predominantly associated with swarmer cell functionality. We also provide evidence that the second messenger (p)ppGpp extends the timing of the G1/swarmer cell stage by slowing growth specifically during the beginning of the cell cycle. Our data further show that cells factor the amount and rate of their growth to control the G1/S transition, allowing them to adjust the time they spend with ecologically important G1-specific traits. Significance statementBacterial growth rate modulation is generally associated with changes in genetic make-up or environmental condition. This study demonstrates that the rate of cell growth can also vary between daughter cells and across cell cycle stages under invariant and unstressed environmental conditions. This is illustrated by the asymmetrically dividing -proteobacterium Caulobacter crescentus, which, at each division, produces two functionally distinct daughter cells that differ in average growth rate. This growth rate difference arises from a G1 phase-specific growth slowdown mediated, in part, by the (p)ppGpp alarmone. Altogether, this study showcases the coupling of cell growth modulation to asymmetric division and cell cycle regulation, which may have implications for other -proteobacteria given their cell cycle similarities with C. crescentus.

microbiology↗

bioRxiv2024

Genome concentration limits cell growth and modulates proteome composition in Escherichia coli

Defining the cellular factors that drive growth rate and proteome composition is essential for understanding and manipulating cellular systems. In bacteria, ribosome concentration is known to be a constraining factor of cell growth rate, while gene concentration is usually assumed not to be limiting. Here, using single-molecule tracking, quantitative single-cell microscopy, and modeling, we show that genome dilution in Escherichia coli cells arrested for DNA replication limits total RNA polymerase activity within physiological cell sizes across tested nutrient conditions. This rapid-onset limitation on bulk transcription results in sub-linear scaling of total active ribosomes with cell size and sub-exponential growth. Such downstream effects on bulk translation and cell growth are near-immediately detectable in a nutrient-rich medium, but delayed in nutrient-poor conditions, presumably due to cellular buffering activities. RNA sequencing and tandem-mass-tag mass spectrometry experiments further reveal that genome dilution remodels the relative abundance of mRNAs and proteins with cell size at a global level. Altogether, our findings indicate that chromosome concentration is a limiting factor of transcription and a global modulator of the transcriptome and proteome composition in E. coli. Experiments in Caulobacter crescentus and comparison with eukaryotic cell studies identify broadly conserved DNA concentration-dependent scaling principles of gene expression.

cell biology↗

bioRxiv2023

Intact reinforcement learning in healthy ageing

What does age in ageing? Results in reinforcement learning (RL) are mixed. Some studies found deteriorated performance in older participants compared to younger controls whereas other studies did not. Daniel et al. (2020) suggested that task demand can explain these differences, with less demanding tasks showing no effect of age. Here, we increased the task demand of previous studies turning them into a classic navigation task. We extracted 4 behavioral parameters and 2 parameters (learning and exploration rates) of a classic Q-learning model. Except for one specific parameter, all other parameters showed no group differences, i.e., RL turned out to be intact in older individuals also with higher task demands. It is important to publish such null results to avoid the stigmatizing impression of an overall performance deficit among older people.

neuroscience↗

bioRxiv2022

Structural analyses of the plant PRT6-UBR box in the Cys-Arg/N-degron pathway and insights into the plant submergence response

The submergence response in higher plants is highly dependent on the protein stability of group VII ethylene response factors, which are primarily degraded through the oxygen-dependent Cys-Arg branch of the N-degron pathway of targeted proteolysis. Knockout of PRT6, an E3 ligase and a vital component of the N-degron pathway, improves submergence tolerance in Arabidopsis and barley but is associated with side effects such as germination deficiency. In this study, we determined structures of rice and Arabidopsis PRT6-UBR box in complex with various Arg/N-degron related peptides. We identified two highly conserved motifs in the plant PRT6-UBR box, which is responsible for Cys-Arg/N-degron recognition. Structural and mutagenesis studies revealed the importance of two conserved motifs for Cys-Arg/N-degron recognition. The phenotype of Arabidopsis seedlings with PRT6-UBR mutants in these newly identified conserved motifs showed superior submergence survival suggesting that rational manipulation of the PRT6-UBR box can improve flood tolerance. Our results provide an engineering platform for generating crops with improved submergence tolerance.

biochemistry↗

bioRxiv2022

The curse of optimism: a persistent distraction by novelty

Humans successfully explore their environment to find extrinsic rewards, even when exploration requires several intermediate reward-free decisions. It has been hypothesized that intrinsic rewards such as novelty guide this reward-free exploration. However, different intrinsic rewards lead to different exploration strategies, some prone to suboptimal attraction to irrelevant stochastic stimuli, sometimes called the noisy TV problem. Here, we ask whether humans show a similar attraction to reward-free stochasticity and, if so, which type of intrinsic reward guides their exploration. We design a multi-step decision-making paradigm where human participants search for rewarding states in an environment with a highly stochastic but reward-free sub-region. We show that (i) participants persistently explore the stochastic sub-region and (ii) their decisions are best explained by algorithms driven by novelty but not by optimal algorithms driven by information gain. Our results suggest that humans use suboptimal but computationally cheap strategies for exploration in complex environments.

neuroscience↗

bioRxiv2022

Connecting single-cell ATP dynamics to overflow metabolism, cell growth and the cell cycle in Escherichia coli

Adenosine triphosphate (ATP) is a universal energy-carrying molecule that cells consume and regenerate in vast amounts to support growth. Despite this high turnover, bacterial cultures maintain a similar average concentration of ATP even when the carbon source conditions lead to large differences in population growth rate. What happens in individual bacterial cells is, however, less clear. Here, we use the QUEEN-2m biosensor to quantify ATP dynamics in single Escherichia coli cells in relation to their growth rate, metabolism, cell cycle, and cell lineage. We find that ATP dynamics are more complex than expected from population studies and are associated with growth rate variability. Under a nutrient-rich condition, cells can display large fluctuations in ATP level that are partially coordinated with the cell cycle. Abrogation of aerobic acetate fermentation (overflow metabolism) through genetic deletion considerably reduces both the amplitude of ATP level fluctuations and the cell cycle trend. Similarly, growth in media in which acetate fermentation is lower or absent results in reduction of ATP level fluctuation and cell cycle trend. This suggests that overflow metabolism exhibits temporal dynamics, which contributes to fluctuating ATP levels during growth. Remarkably, at the single-cell level, growth rate negatively correlates with the amplitude of ATP fluctuation for each tested condition, linking ATP dynamics to growth rate heterogeneity in clonal populations. Our work highlights the importance of single-cell analysis in studying cellular energetics and its implication to phenotypic diversity and cell growth.

cell biology↗

bioRxiv2022

Targeting neuronal homeostasis to prevent seizures

Manipulating neuronal homeostasis, which enables neurons to regulate their intrinsic excitability, offers an attractive opportunity to prevent seizures. However, no anticonvulsant compounds have yet been reported that directly manipulate neuronal homeostasis. Here, we describe a novel class of anticonvulsant compounds, based on 4-tert-butyl-benzaldehyde (4-TBB), with a mode-of-action that includes increased expression of the homeostatic regulator Pumilio (Pum). In Drosophila and mouse we use a pentylenetetrazole (PTZ) induced seizure model, and an electrically induced seizure model for refractory seizures to evaluate anticonvulsant efficacy. The pyrazole analogue (RAB216) demonstrates best efficacy, protecting 50% of mice from PTZ-induced seizure. Knock-down of Pum, in Drosophila, blocks anticonvulsive effects, whilst analysis of validated Pum targets show significant reductions following exposure of mouse brain to 4-TBB. This study provides proof-of-principle that anticonvulsant effects can be achieved through regulation of neuronal homeostasis and identifies a chemical lead compound for future development.

neuroscience↗

bioRxiv2021

A Syx-RhoA-Dia1 signaling axis regulates cell cycle progression, DNA damage, and therapy resistance in glioblastoma

Glioblastomas (GBM) are aggressive tumors that lack effective treatments. Here, we show that the Rho family guanine nucleotide exchange factor Syx promotes GBM cell growth both in vitro and in orthotopic GBM patient-derived xenografts. Growth defects upon Syx depletion are attributed to prolonged mitosis, increased DNA damage, G2/M cell cycle arrest, and cell apoptosis, mediated by altered mRNA and protein expression of various cell cycle regulators. These effects are phenocopied by depletion of the Rho downstream effector Dia1 and are due at least in part to increased cytoplasmic retention and reduced activity of the YAP/TAZ transcriptional coactivators. Further, targeting Syx signaling cooperates with radiation treatment and temozolomide (TMZ) to decrease viability in GBM cells irrespective of their inherent response to TMZ. Taken together, the data indicate that a Syx-RhoA-Dia1-YAP/TAZ signaling axis regulates cell cycle progression, DNA damage, and therapy resistance in GBM and argue for its targeting for cancer treatment. One Sentence SummarySyx promotes growth and therapy resistance in glioblastoma.

cancer biology↗

bioRxiv2021

Characterization and computational simulation of human Syx, a RhoGEF implicated in glioblastoma

Structural discovery of guanine nucleotide exchange factor (GEF) protein complexes is likely to become increasingly relevant with the development of new therapeutics targeting small GTPases and development of new classes of small molecules that inhibit protein-protein interactions. Syx (also known as PLEKHG5 in humans) is a RhoA GEF implicated in the pathology of glioblastoma (GBM). Here we investigated protein expression and purification of ten different human Syx constructs and performed biophysical characterizations and computational studies that provide insights into why expression of this protein was previously intractable. We show that human Syx can be expressed and isolated and Syx is folded as observed by circular dichroism (CD) spectroscopy and actively binds to RhoA as determined by co-elution during size exclusion chromatography (SEC). This characterization may provide critical insights into the expression and purification of other recalcitrant members of the large class of oncogenic -- Diffuse B-cell lymphoma (Dbl) homology GEF proteins. In addition, we performed detailed homology modeling and molecular dynamics simulations on the surface of a physiologically realistic membrane. These simulations reveal novel insights into GEF activity and allosteric modulation by the plekstrin homology (PH) domain. These newly revealed interactions between the GEF PH domain and the membrane embedded region of RhoA support previously unexplained experimental findings regarding the allosteric effects of the PH domain from numerous activity studies of Dbl homology GEF proteins. This work establishes new hypotheses for structural interactivity and allosteric signal modulation in Dbl homology RhoGEFs.

biochemistry↗

bioRxiv2021

Evolution of the open-access CIViC knowledgebase is driven by the needsof the cancer variant interpretation community

CIViC (Clinical Interpretation of Variants in Cancer; civicdb.org) is a crowd-sourced, public domain knowledgebase composed of literature-derived evidence characterizing the clinical utility of cancer variants. As clinical sequencing becomes more prevalent in cancer management, the need for cancer variant interpretation has grown beyond the capability of any single institution. With nearly 300 contributors, CIViC contains peer-reviewed, published literature curated and expert-moderated into structured data units (Evidence Items) that can be accessed globally and in real time, reducing barriers to clinical variant knowledge sharing. We have extended CIViCs functionality to support emergent variant interpretation guidelines, increase interoperability with other variant resources, and promote widespread dissemination of structured curated data. To support the full breadth of variant interpretation from basic to translational, including integration of somatic and germline variant knowledge and inference of drug response, we have enabled curation of three new evidence types (predisposing, oncogenic and functional). The growing CIViC knowledgebase distributes clinically-relevant cancer variant data currently representing >2500 variants in >400 genes from >2800 publications.

genomics↗