Biology subjects

Lim, C. T.

Publications and source records attributed to Lim, C. T..

At least 19 recordsLinked to original sources

bioRxiv2026

Theca cell mechanosensing and regulation of follicular extracellular matrix during ovarian follicle development

Mammalian folliculogenesis is essential for female hormonal regulation and successful reproduction. While the steroidogenic functions of theca cells (TCs) have been implicated in ovarian diseases and infertility, the physico-structural properties of TCs and their associated extracellular matrix (ECM), or theca matrix, remain poorly understood. Using murine ovaries, we show that a stiff basement membrane (BM) and theca matrix constitute a mechanically instructive niche that modulates TC proliferation and Yes-associated protein (YAP) signalling in secondary follicles. We identify hyaluronic acid (HA) as a key matrix component that is actively secreted by contractile TCs. The HA scaffold, in turn, regulates TC proliferation, YAP signalling and motility, and is required for overall follicle growth. We showed that stiffer substrates enhance YAP nuclear transport in TCs, while mechanical stretch, cell packing, and curvature affect TC proliferation. In addition, TCs exhibit directed migration towards regions of positive curvature. Together, this study reveals a mechanochemical feedback mechanism that establishes TC mechanics and HA as key regulators of theca matrix formation that is essential for mammalian folliculogenesis. SIGNIFICANCEThe structural properties and mechanical functions of the basement membrane and theca cell-matrix encapsulating mammalian ovarian follicles are poorly understood. Our findings reveal that during early stage of follicle development, the basement membrane remains thin and stiff, while the theca cells actively secrete hyaluronic acid in a contractility-dependent manner. The hyaluronic acid scaffold, in turn, regulates theca cell YAP signalling, proliferation and motility that are required for functional growth of follicles. We further showed that the theca cells are mechanosensitive and fine-tune their proliferative capacity and Hippo pathways in response to substrate stiffness, stretch and curvature. Together, our study uncovers mechanoregulatory feedback between theca cells and associated extracellular matrix, offering new insights into environmental control of folliculogenesis in female reproduction.

developmental biology↗

bioRxiv2025

Fibrillar adhesions are the primary integrin complexes shaped by matrix topography

Mechanisms of matrix topography recognition are poorly understood. Here, we show that 5{beta}1-integrin mediated fibrillar adhesions serve this function. While on planar substrates, their formation requires fibronectin secretion, tensins, and actomyosin contractility, these requirements are bypassed on nanotopographical features. While focal adhesions avoid these features, fibrillar adhesions rapidly align along pre-existing fibrous cell-derived matrix or electrospun nanofibers where they can then template fibronectin fibrils. Topography-induced fibrillar adhesions depend primarily on 5{beta}1-integrin clustering and disassemble upon cortical stiffening driven by myosin-II overactivation or increased membrane tension. We propose a generic theoretical model where the adhesion receptor favours the membrane and substrate planes to be tilted relative to each other. This model matches experimental observations of preferential 5{beta}1-integrin clustering along nanofibers and concave edges of large negative curvature along micro-ridges. These findings establish fibrillar adhesions as primary adhesion complexes that form independently of focal adhesions in response to matrix topography.

cell biology↗

bioRxiv2025

Graphene nanoedge electronics for monolithic and chronic recording of local microcircuits at neuronal density

High-density single-unit recording among closely spaced neurons over long durations is crucial for understanding the cellular-level functional architecture of the brain. Existing brain probes, however, either laterally sample neurons far more sparsely than neuronal density in the same cortical layer or suffer from long-term instability due to electrode modification, preventing precise neuron-to-neuron interrogation in local microcircuits. Here, we report a monolithic graphene-edge probe (NeuroEdge) that achieves single-unit recording at neuronal density (16 electrodes within 100 {micro}m diameter). We fabricate NeuroEdge using self-assembled reduced graphene oxide nanoflakes to form an electrochemically active tip consisting of exposed graphene nanoedges and electrolyte-filling nanotunnels, achieving an ultralow specific impedance of 20 M{Omega} {micro}m2. In vivo experiments over 5 months demonstrate recording at a high signal-to-noise ratio (>20 dB) and reliable interrogation of neighboring neurons. We also show that NeuroEdge can record from a single auditory cortical layer and reveal heterogeneities in the acoustic frequency response and dynamic connectivity among neighboring neurons. NeuroEdge provides a tool for precisely interrogating local microcircuits at the density of neurons in the brain.

bioengineering↗

bioRxiv2025

Chaperone AIP Couples mTORC1 Activation and Catabolic Metabolism During Neonatal Development

To grow and divide cells must tightly coordinate anabolic programs with the availability of nutrients and growth factors. This balance is especially critical during postnatal development, when biosynthetic and energetic demands are high, and nutrient supply and neonates have to adapt to periods of fasting. These conditions place acute stress on the proteostasis network, making autophagy essential for nutrient recycling. We found that the chaperone aryl hydrocarbon receptor-interacting protein (AIP) supports both arms of this metabolic balance: promoting anabolic PI3K-AKT signaling for mTORC1 activation and enabling catabolic processes such as proteasomal degradation and autophagy. Loss of AIP causes a severe neonatal metabolic disorder, where affected infants fail to thrive postnatally. Our findings establish AIP as a central regulator of neonatal metabolic adaptation and cellular homeostasis. One Sentence SummaryAIP integrates nutrient sensing and protein recycling to sustain neonatal survival.

developmental biology↗

bioRxiv2025

TRPM7 Annexin A1 Mechanosensitive Pathway Drives Capillary Infiltration by Circulating Tumor Cells

Successful metastatic dissemination requires tumor cells to overcome significant physical barriers. When circulating tumor cells (CTCs) become lodged in capillary beds, studies reveal that only a small subset can adapt to fluid shear stress (FSS) within the constricted vasculature. These rare, mechanically resilient cells may subsequently extravasate and form metastatic lesions, but details on this adaptation of the physical environment and its molecular basis are not well understood. Utilizing a microfluidics platform that mimics microcirculatory dynamics, we discover that only breast cancer cells with high metastatic potential maintain directional migration under physiologically relevant FSS conditions. This highlights the mechanical selection process of metastatic precursors during hematogenous dissemination. We identify the TRPM7-Annexin A1-actin signaling pathway as essential for overcoming physical barriers and regulating cell motility under capillary FSS. FSS prompts cytoskeletal reorganization and actin disassembly, restricting cell movement. Cancer cells respond to this by increasing mechanical loading, activating TRPM7 and triggering calcium influx, which then activates Annexin A1. This calcium-dependent protein, Annexin A1, interacts with the actin cortex to prevent FSS-induced actin disassembly, thus aiding migration. Experiments conducted in mouse liver capillaries validate the critical role of this pathway in cancer cell motility. Additionally, we propose a mechano-pharmacological strategy using FTY720 to target the TRPM7 pathway, highlighting its therapeutic potential to modify CTC receptor specificity and inhibit distant metastasis.

cancer biology↗

bioRxiv2025

Acute chromatin decompaction stiffens the nucleus as revealed by nanopillar-induced nuclear deformation in cells

Chromatin architecture is critical in determining nuclear mechanics. Most studies focus on the mechanical rigidity conferred by chromatin compaction from densely packed heterochromatin, but less is known on how transient changes in chromatin decompaction state impinge on nucleus stiffness. Here, we used an array of vertically aligned nanopillars to study nuclear deformability in situ after chromatin decompaction in cells. The nucleus significantly stiffened within 4 hours of chromatin decompaction but softened at longer timescales. This acute nucleus stiffening was predominantly underlied by an increase in nucleus volume, nuclear import and partially enhanced by lamin protein recruitment to the nuclear periphery. The coupling between nucleus stiffening and acute chromatin decompaction was observed in cancer cell lines with lower malignancy (e.g. MCF7, PEO1, A549) but weakened in those with higher metastatic potential (e.g. MDA-MB-231, HEYA8, HT1080), which was found to be associated with the capacity to efficiently compact heterochromatin into foci that sustains nucleus deformability required for confined migration. Our work signals how a rapid chromatin remodeling is a physiologically relevant pathway to modulate nucleus mechanics and cell migration behavior. STATEMENT OF SIGNIFICANCEMany cell processes such as wound healing, immune activation and DNA damage repair require a decompact and accessible chromatin structure. Whether such short-term remodeling of the chromatin impacts nucleus mechanics and function is poorly defined. Using nanopillars that allow interrogation of nucleus rigidity within intact cells, we showed that contrary to conventional knowledge the nucleus becomes less deformable and more rigid when chromatin is acutely decompacted due to enhanced nuclear import and swelling of the nucleus. In cancer cells, the coupling of transient chromatin decompaction to nucleus rigidity is weakened and appears to be countered by heterochromatin formation and compaction. We demonstrate here how short-term chromatin remodeling can impact nucleus and cellular properties in a time-dependent and non-genetic manner.

bioengineering↗

bioRxiv2024

Cell Cycle Regulation has Shaped Budding Yeast Replication Origin Structure and Function

Eukaryotic DNA replication initiates from multiple genomic loci known as origins. At budding yeast origins like ARS1, a double hexamer (DH) of the MCM replicative helicase is assembled by Origin Recognition Complex (ORC), Cdc6 and Cdt1 via sequential hexamer loading from two opposed ORC binding sites. Cyclin Dependent Kinase (CDK) inhibits DH assembly, which prevents re-replication by restricting helicase loading to G1 phase. Here we show that an intrinsically disordered region (IDR) in the Orc2 subunit promotes interaction between ORC and the first loaded, closed-ring MCM hexamer (the MO intermediate); CDK phosphorylation of this IDR blocks MO formation and DH assembly. We show that MO functions by stabilising ORC at the lower affinity binding sites required for second hexamer loading. Origins comprising two high affinity ORC sites can assemble DH efficiently without MO by independently loading single hexamers; these origins escape CDK inhibition in vitro and in vivo. Our work reveals mechanistic plasticity in MCM loading with implications for understanding how CDK regulation has shaped yeast origin evolution and how natural origins might escape cell cycle regulation. We also identify key steps common to loading pathways, with implications for understanding how MCM is loaded in other eukaryotes.

biochemistry↗

bioRxiv2023

Gel-Shrink Softlithography: Enabling 3D Tissue Culture Microstructure Miniaturization and Tacky Super-Soft Material Molding

In this study, we introduce an innovative hydrogel soft lithography technique that leverages the significant volume change of hydrogels during hydration. Through a systematic desiccation process applied to hydrogels containing microfeatures, we achieved a remarkable 2-fold reduction in feature dimensions. These shrunken hydrogel structures were then readily molded using standard soft lithography methods. Furthermore, we demonstrated the techniques utility in rectifying surface imperfections commonly found in 3D printed structures, offering promising prospects in fields like tissue engineering. Additionally, our study discovered a unique capability of hydrated hydrogel molds in molding super-soft, tacky elastomeric materials, exemplified by CY25-276 and low cross-linker ratio PDMS. This development opens doors to studying cellular forces through traction force microscopy to a broader spectrum of 3D structural contexts. In support of our efforts in microfabrication advancement, we explored multicellular responses to surface curvatures of diminishing dimensions. Our findings reveal a curvature-dependent alignment pattern: cells residing on convex surfaces align with the direction of least curvature, while those in concave regions align with the direction of the strongest curvature. These results underscore the critical influence of geometric complexity in biological systems and highlight the need for continuously improving methods to replicate geometric qualities relevant to these environments.

bioengineering↗

bioRxiv2022

Deleterious Mechanical Deformation Selects Mechanoresilient Cancer Cells with Enhanced Proliferation and Chemoresistance

Cancer cells derived from secondary tumors can form new distant metastases more efficiently as compared to their primary tumor counterparts. This is partially due to the unfavorable microenvironments encountered by metastasizing cancer cells that result in the survival of a more metastatic phenotype from the original population. However, it is unclear how cancer cells may acquire such metastatic competency after overcoming deleterious mechanical stresses. Here, by forcing cancer cells to flow through small capillary-sized constrictions, we demonstrate that mechanical deformation can select a tumor cell subpopulation that exhibits resilience to mechanical squeezing-induced cell death. Transcriptomic profiling reveals up-regulated proliferation and DNA damage response pathways in this subpopulation, which are further translated into a more proliferative and chemotherapy-resistant phenotype. Our results highlight a potential link between the microenvironmental physical barriers and the enhanced malignancy of metastasizing cancer cells which may potentially be utilized for novel therapeutic strategies in preventing the metastatic spread of cancer cells.

cancer biology↗

bioRxiv2022

Surface curvature and basal hydraulic stress induce spatial bias in cell extrusion

Epithelial cell extrusion is employed in maintaining a healthy epithelium. It remains unclear how environmental conditions specific to various epithelial tissues, such as geometry and fluid osmolarity, affect cell extrusions. We found that, over curved surfaces, epithelial monolayers exhibited higher extrusion rates in concave regions than convex ones. This difference, and overall extrusions, decreased when osmotically induced basal hydraulic stress was reduced by increasing media osmolarity or by culturing monolayers on hydrogels. Mechanistically, basal fluid accumulation antagonized cell-substrate adhesions and the subsequent FAK-Akt survival pathway, leading to apoptotic cell death. Convex surfaces induced cellular forces that acted against osmosis, thereby promoting adhesions and lowering apoptosis. This effect was reversed in concave regions, and together, resulted in a curvature induced spatial bias in cell extrusions.

cell biology↗

bioRxiv2021

Determination of nuclear position by the arrangement of actin filaments using deep generative networks

The cell nucleus is a dynamic structure that changes locales during cellular processes such as proliferation, differentiation, or migration1, and its mispositioning is a hallmark of several disorders2,3. As with most mechanobiological activities of adherent cells, the repositioning and anchoring of the nucleus are presumed to be associated with the organization of the cytoskeleton, the network of protein filaments providing structural integrity to the cells4. However, this correlation between cytoskeleton organization and nuclear position has not, to date, been demonstrated, as it would require the parameterization of the extraordinarily intricate cytoskeletal fiber arrangements. Here, we show that this parameterization and demonstration can be achieved outside the limits of human conceptualization, using generative network and raw microscope images, relying on machine-driven interpretation and selection of parameterizable features. The developed transformer-based architecture was able to generate high-quality, completed images of more than 8,000 cells, using only information on actin filaments, predicting the presence of a nucleus and its exact localization in more than 70 per cent of instances. Our results demonstrate one of the most basic principles of mechanobiology with a remarkable level of significance. They also highlight the role of deep learning as a powerful tool in biology beyond data augmentation and analysis, capable of interpreting--unconstrained by the principles of human reasoning--complex biological systems from qualitative data.

bioinformatics↗

bioRxiv2021

Biomimetic Vasculatures by 3D-Printed Porous Molds

Anatomically and biologically relevant vascular models are critical to progress our understanding of cardiovascular diseases (CVDs) that can lead to effective therapies. Despite advances in 3D bioprinting, recapitulating complex architectures (i.e., freestanding, branching, multilayered, perfusable) of a cell-laden vascular construct remains technically challenging, and the development of new techniques that can recapitulate both anatomical and biological features of blood vessels is of paramount importance. In this work, we introduce a unique, microfluidics-enabled molding technique that allows us to fabricate anatomically-relevant, cell-laden hydrogel vascular models. Our approach employed 3D-printed porous molds of poly(ethylene glycol) diacrylate (PEGDA) as templates to cast alginate-containing bioinks. Due to the porous and aqueous nature of the PEGDA mold, the calcium ion (Ca2+) was diffusively released to crosslink the bioinks to create hollow structures. Applying this technique, multiscale, multilayered vascular constructs that were freestanding and perfusable were readily fabricated using cell-compatible bioinks (i.e., alginate and gelatin methacryloyl (GelMA)). The bioinks were also readily customizable to either improve the compatibility with specific vascular cells or tune the mechanical modulus to mimic native blood vessels. Importantly, we successfully integrated smooth muscle cells and endothelial cells in a biomimetic organization within our vessel constructs and demonstrated a significant increase in monocyte adhesion upon stimulation with an inflammatory cytokine, tumor necrosis factor-alpha (TNF-). We also demonstrated that the fabricated vessels were amenable for testing percutaneous coronary interventions (i.e., drug-eluting balloons and stents) under physiologically-relevant mechanical states, such as vessel stretching and bending. Overall, we introduce a versatile fabrication technique with multi-faceted possibilities of generating biomimetic vascular models that can benefit future research in mechanistic understanding of CVD progression and the development of therapeutic interventions.

bioengineering↗

bioRxiv2021

Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp3 Papain-like Protease

The COVID-19 pandemic has emerged as the biggest life-threatening disease of this century. Whilst vaccination should provide a long-term solution, this is pitted against the constant threat of mutations in the virus rendering the current vaccines less effective. Consequently, small molecule antiviral agents would be extremely useful to complement the vaccination program. The causative agent of COVID-19 is a novel coronavirus, SARS-CoV-2, which encodes at least nine enzymatic activities that all have drug targeting potential. The papain-like protease (PLpro) contained in the nsp3 protein generates viral non-structural proteins from a polyprotein precursor, and cleaves ubiquitin and ISG protein conjugates. Here we describe the expression and purification of PLpro. We developed a protease assay that was used to screen a custom chemical library from which we identified Dihydrotanshinone I and Ro 08-2750 as compounds that inhibit PLpro in protease and isopeptidase assays and also inhibit viral replication in cell culture-based assays.

biochemistry↗

bioRxiv2021

Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of nsp5 Main Protease

The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5,000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.

biochemistry↗

bioRxiv2021

Identification of SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of the nsp14 RNA Cap Methyltransferase

The COVID-19 pandemic has presented itself as one of the most critical public health challenges of the century, with SARS-CoV-2 being the third member of the Coronaviridae family to cause fatal disease in humans. There is currently only one antiviral compound, remdesivir, that can be used for the treatment of COVID-19. In order to identify additional potential therapeutics, we investigated the enzymatic proteins encoded in the SARS-CoV-2 genome. In this study, we focussed on the viral RNA cap methyltransferases, which play a key role in enabling viral protein translation and facilitating viral escape from the immune system. We expressed and purified both the guanine-N7 methyltransferase nsp14, and the nsp16 2-O-methyltransferase with its activating cofactor, nsp10. We performed an in vitro high-throughput screen for inhibitors of nsp14 using a custom compound library of over 5,000 pharmaceutical compounds that have previously been characterised in either clinical or basic research. We identified 4 compounds as potential inhibitors of nsp14, all of which also show antiviral capacity in a cell based model of SARS-CoV-2 infection. Three of the 4 compounds also exhibited synergistic effects on viral replication with remdesivir.

bioengineering↗

bioRxiv2020

Adhesion-mediated heterogeneous actin organization governs apoptotic cell extrusion

Apoptotic extrusion is crucial in maintaining epithelial homeostasis and has implications in diseases of epithelial tissues. Current literature supports that epithelia respond to extrusion to maintain their integrity by the formation of a supracellular actomyosin ring (purse-string) in the neighbors that encompasses the dying cells. However, little is known about whether other types of actin structures could contribute to extrusion as well as how forces generated by mechanosensitive proteins in the cells are integrated. Here, we found that during extrusion, a heterogeneous actin network composed of lamellipodia protrusions and discontinuous actomyosin cables, was reorganized in the neighboring cells and was the main factor driving extrusion forwards. The early presence of basal lamellipodia protrusion participated both in basal sealing of the extrusion site and in orienting the actomyosin purse-string at the later stage of extrusion. These sequential events are essential in ensuring a successful extrusion in apicobasal direction. The co-existence of these two mechanisms is determined by the interplay between the cell-cell and cell-substrate adhesions. A theoretical model integrates the role of these cellular mechanosensitive components to explain why a dual-mode mechanism, which combined lamellipodia protrusion and purse-string contractility, leads to more efficient extrusion than a single-mode mechanism. We anticipate that our approach will be useful to provide mechanistic insight into epithelial homeostasis, morphogenetic events and tumorigenesis.

cell biology↗

bioRxiv2020

Cancer exosomes harbor diverse hypoxia-targeted mRNAs and contribute toward tumor angiogenesis

Exosomes are extracellular vesicles of endosome origin secreted by various cells. The exosomal cargo, especially proteins and microRNAs, have been extensively investigated for their roles in intercellular communication and as biomarkers for clinical applications. However, the understanding of types and functions of exosomal mRNAs remains limited. Here, we evaluated the mRNAs of 61 hypoxia-targeted genes in exosomes by quantitative reverse transcription PCR (RT-qPCR). Among these 61 mRNAs, 14.8% of them were detected in the MCF10A-derived exosomes, 42.6% in the MCF7-derived exosomes, and 49.2% in the MDA-MB-231-derived exosomes, many of which are differentially regulated in response to hypoxic stress in a cell-line dependent manner. Consequently, 30 exosomal mRNAs are identified as cancer related biomarkers as they are present in cancer cell-derived exosomes and absent in MCF10A-derived exosomes. Co-culture of MDA-MB-231 cells with HUVECs shows uptake of MDA-MB-231 secreted exosomes by the Human umbilical vein endothelial cells (HUVECs). Subsequently, the cancer exosomal VEGFA mRNAs were translated within the HUVECs into proteins that promoted VEGFR-dependent angiogenesis. This finding provides novel insights into how cancer cells can directly contribute towards angiogenesis. RNA-seq also shows that cancer exosomes can upregulate epithelial-mesenchymal transition-related and metabolism-related genes. The transcripts of these genes are found present in the cancer exosomes, suggesting that the uptake of exosomal mRNAs at least partially contributed to the upregulation of the corresponding mRNAs. This study shows that cancer exosomes harbor diverse mRNAs, some of which can act as promising biomarkers as well as contribute towards reprogramming of recipient cells.

cancer biology↗

bioRxiv2020

Endosomal spatio-temporal modulation of the cortical RhoA zone conditions epithelial cell organization

At the basis of cell shape and behavior, actomyosin organization and force-generating property are widely studied, however very little is known about the regulation of the contractile network in space and time. Here we study the role of the epithelial-specific protein EpCAM, a contractility modulator, in cell shape and motility, and we show that it is required for the maturation of stress fibers and frontrear polarity acquisition at the single cell level. There, EpCAM ensures the remodeling of a transient active RhoA zone in the cortex of spreading epithelial cells. GTP-RhoA follows the endosomal pathway mediated by Rab35 and EHD1, where it co-evolves together with EpCAM. In fact, EpCAM balances GTP-RhoA turnover in order to tune actomyosin remodeling for cell shape, polarity and mechanical property acquisition. Impairment of GTP-RhoA endosomal trafficking either by EpCAM silencing or Rab35 / EHD1 mutant expression prevents correct myosin-II activity, stress fiber formation, and ultimately cell polarization. Collectively, this work shows that the coupling of EpCAM/RhoA co-trafficking to actomyosin rearrangement is critical for spreading, and advances our understanding of how biochemical and mechanical properties can be coupled for cell plasticity.

cell biology↗