Biology subjects

Lee, C. F.

Publications and source records attributed to Lee, C. F..

11 recordsLinked to original sources

bioRxiv2026

Intravital microscopy and statistical image analysis of early engraftment of haematopoietic stem cells reveal dynamic and evolving cellular behaviours linked to progressive differentiation.

Haematopoietic stem cells (HSCs) have long been used in the clinic for bone marrow transplantation applications, critical for the survival of an increasingly wide range of patients with haematological, oncological and immunological pathologies. Despite this, little is known about the mechanisms through which relatively few stem cells are able to regenerate the entire haematopoietic tissue of transplant recipients. To gain insights on this biological process, we used intravital microscopy of calvarium bone marrow, collecting tissue-wide images and a total of 850 hours of tracks of engrafting haematopoietic stem and progenitor cells from 24 hours to 8 days following injection in lethally irradiated recipients. Analysis of the data revealed that regenerating HSCs and their immediate progeny are highly dynamic, migrating through the parenchyma at both the microscopic and near-macroscopic scales, i.e. within and in-and-out of fields of view. HSC-derived cell clusters, expanded locally between day 2 and day 4 post-transplant, leading to patches of densely populated bone marrow by day 8. Single cell level analysis highlighted increasingly heterogenous cellular behaviours over time. Track clustering based on migration and niche interaction parameters combined with post-tracking whole mount immunostaining revealed that persistence in the vicinity of nestin-GFP perivascular cells and relatively slow movement correlated with stemness, while more heterogeneous niche interactions coupled with slowest or faster migration correlated with differentiation, validated through flow cytometry analysis and functional studies. The findings presented here shed light on the fundamental principles driving haematopoietic regeneration in transplantation settings.

developmental biology↗

bioRxiv2026

PFKFB2 Gates a Relationship Between Cardiac Glycolytic Regulation and Electrophysiological Function

BackgroundThe cardiac isoform of phosphofructokinase-2/fructose 2,6-bisphosphatase (PFKFB2) is the hearts strongest glycolytic regulator but is degraded in the absence of insulin signaling. This makes PFKFB2 loss critical to understand in metabolic heart disease, of which impaired insulin signaling is a hallmark. Prolongation of the QT interval, risk of arrhythmia, and sudden cardiac death are also augmented in metabolic heart disease, raising a question as to whether potential crosstalk between glycolytic dysregulation and electrophysiological dysfunction exists. MethodsWe therefore assessed the impact of PFKFB2 loss on cardiac electrophysiology using a cardiomyocyte-specific PFKFB2 knockout mouse model (cKO) and litter-matched controls (CON). To do so, we employed electrocardiography in the fed state and following 12 hours of fasting, examining physiology both at baseline and in the presence of an acute stimulant stress. To further investigate the arrhythmia mechanism, we used patch-clamp electrophysiology and IonOptix Ca2+ transient measurements in ventricular cardiomyocytes isolated from CON and cKO hearts. ResultsThe hearts of cKO mice exhibited prolonged repolarization, marked by QT and action potential duration prolongations. This occurred with impaired Ca2+ reuptake and increased spontaneous Ca2+ release events in ventricular cardiomyocytes. Ultimately, these changes culminated in ventricular tachyarrhythmia in cKO mice, which was enhanced in the fed relative to the fasted state. ConclusionThese data suggest that in the presence of sufficient glucose availability, cardiac glycolytic dysregulation at the phosphofructokinase nexus is sufficient to promote cardiac electrophysiological instability. Clinical PerspectiveO_ST_ABSWhat is KnownC_ST_ABSO_LIMetabolic heart diseases, such as heart failure with preserved ejection fraction and diabetic cardiomyopathy, are associated with heightened risks of arrhythmogenesis and sudden cardiac death. C_LI What the Study AddsO_LIHere, we show for the first time that PFKFB2 is decreased in human hearts with heart failure with preserved ejection fraction. C_LIO_LIFurthermore, we show that loss of cardiac PFKFB2 is sufficient to promote impaired ventricular repolarization at baseline and ventricular tachyarrhythmia upon stress test. C_LIO_LIThis identifies PFKFB2 stabilization and activation as key potential targets in conferring electrophysiological stability in metabolic heart disease. C_LI

physiology↗

bioRxiv2026

Viscosity-mediated signal amplification enables robust left-right symmetry breaking in the mouse node

Left-right (LR) symmetry breaking in vertebrates depends on a directional fluid flow generated in the embryonic LR organizer, yet how this flow is sensed remains unresolved. Mechanosensing and chemosensing models each capture part of the process but face key limitations when considered independently. Here, we introduce a minimal theoretical model of viscosity-mediated signal amplification that unifies these perspectives. In this framework, macromolecules secreted by organizer cells locally increase the near-surface viscosity, creating another viscous fluid layer that is entrained by the leftward nodal flow. This layer can significantly amplifies the drag and torque exerted on immotile perinodal cilia, enabling robust discrimination of flow direction even when flow magnitudes on the left and right are nearly identical. The model naturally incorporates macromolecule secretion, clarifies the complementary roles of motile and immotile cilia, and resolves the major shortcomings of previous proposals. Together, these results provide a simple and physically grounded mechanism for LR determination in the mouse node.

biophysics↗

bioRxiv2025

Optimizing scheduling in dual-pulse nucleoside labeling experiments for cell cycle analysis

All eukaryotic cells go though a universal sequence of phases during their division cycle, where the phase timings vary according to cell type and state. Dual-pulse nucleoside labeling (DPNL) is a standard, widely applicable experimental technique to probe cell cycle kinetics at the population level, including in living organisms. In such an experimental protocol, a key scheduling parameter is the choice of waiting time between the two labeling pulses. Here, we use simulation to show that the waiting time can be optimized with regards to the signal-to-noise ratio of the cycle parameters as inferred from experimental outputs, which is of particular importance in DPNL experiments with small numbers of cells and repeats. We further discuss the procedure to perform such a task in an experimentally relevant setting.

biophysics↗

bioRxiv2024

The synaptonemal complex assembles between meiotic chromosomes by wetting

AbstractDuring meiosis, the parental chromosomes are drawn together to enable exchange of genetic information. Chromosomes are aligned through the assembly of a conserved interface, the synaptonemal complex, composed of a central region that forms between two parallel chromosomal backbones called axes. Here we identify the axis-central region interface in C. elegans, containing a conserved positive patch on the axis component HIM-3 and the C-terminus of the central region protein SYP-5. Crucially, the canonical ultrastructure of the synaptonemal complex is altered upon weakening this interface. We developed a thermodynamic model that recapitulates our experimental observations, indicating that the liquid-like central region can assemble by wetting the axes without active energy consumption. More broadly, our data show that condensation drives tightly regulated nuclear reorganization during sexual reproduction.

cell biology↗

bioRxiv2023

Late-life Rapamycin Treatment Enhances Cardiomyocyte Relaxation Kinetics and Reduces Myocardial Stiffness

Diastolic dysfunction is a key feature of the aging heart. We have shown that late-life treatment with mTOR inhibitor, rapamycin, reverses age-related diastolic dysfunction in mice but the molecular mechanisms of the reversal remain unclear. To dissect the mechanisms by which rapamycin improves diastolic function in old mice, we examined the effects of rapamycin treatment at the levels of single cardiomyocyte, myofibril and multicellular cardiac muscle. Compared to young cardiomyocytes, isolated cardiomyocytes from old control mice exhibited prolonged time to 90% relaxation (RT90) and time to 90% Ca2+ transient decay (DT90), indicating slower relaxation kinetics and calcium reuptake with age. Late-life rapamycin treatment for 10 weeks completely normalized RT90 and partially normalized DT90, suggesting improved Ca2+ handling contributes partially to the rapamycin-induced improved cardiomyocyte relaxation. In addition, rapamycin treatment in old mice enhanced the kinetics of sarcomere shortening and Ca2+ transient increase in old control cardiomyocytes. Myofibrils from old rapamycin-treated mice displayed increased rate of the fast, exponential decay phase of relaxation compared to old controls. The improved myofibrillar kinetics were accompanied by an increase in MyBP-C phosphorylation at S282 following rapamycin treatment. We also showed that late-life rapamycin treatment normalized the age-related increase in passive stiffness of demembranated cardiac trabeculae through a mechanism independent of titin isoform shift. In summary, our results showed that rapamycin treatment normalizes the age-related impairments in cardiomyocyte relaxation, which works conjointly with reduced myocardial stiffness to reverse age-related diastolic dysfunction.

physiology↗

bioRxiv2022

Genetic Architecture of Heart Mitochondrial Proteome influencing Cardiac Hypertrophy

Mitochondria play a key role in the normal function of the heart as well as in the pathogenesis of diseases. We report analysis of common genetic variations contributing to mitochondrial and heart functions using an integrative proteomics approach in a panel of inbred mouse strains called the Hybrid Mouse Diversity Panel (HMDP). We performed a whole heart proteomic analysis in the HMDP (72 strains, n=2-3 mice) and retrieved 840 mitochondrial proteins (quantified in [≥]50 strains). High-resolution association mapping on their respective abundance levels identified three trans-acting genetic loci, located on chromosome (chr) 7, chr13 and chr17, that control distinct classes of mitochondrial proteins as well as heart hypertrophy. Follow-up high resolution regional mapping identified NDUFS4, LRPPRC and COQ7 as the candidate genes for chr13, chr17 and chr7 loci, respectively, and both experimental and statistical analyses supported their causal roles. Variations of all three were associated with heart mass in two independent heart stress models, namely, isoproterenol (ISO)-induced heart failure and diet-induced obesity (DIO) models. To identify the aspects of mitochondrial metabolism regulated by these loci, we constructed co-expression protein networks using weighted gene co-expression network analysis (WGCNA). DAVID enrichment analyses of genes regulated by each of the loci revealed that the chr13 locus was highly enriched for complex-I proteins (24 proteins, P = 2.2E-61), the chr17 locus for mitochondrial ribonucleoprotein complex (17 proteins, P = 3.1E-25) and the chr7 locus for ubiquinone biosynthesis (3 proteins, P = 6.9E-05). These results indicate that common variations of certain mitochondrial proteins can act in trans to influence mitochondrial functions and contribute to heart hypertrophy, elucidating mechanisms that may underlie genetic susceptibility to heart failure in human populations.

genetics↗

bioRxiv2022

Model of inverse bleb growth explains giant vacuole dynamics during cell mechanoadaptation

Cells can withstand hostile environmental conditions manifest as large mechanical forces such as pressure gradients and/or shear stresses by dynamically changing their shape. Such conditions are realized in the Schlemms canal of the eye where endothelial cells that cover the inner vessel wall are subjected to the hydrodynamic pressure gradients exerted by the aqueous humor outflow. These cells form fluid-filled dynamic outpouchings of their basal membrane called giant vacuoles. The inverse of giant vacuoles are reminiscent of cellular blebs, extracellular cytoplasmic protrusions triggered by local temporary disruption of the contractile actomyosin cortex. Inverse blebbing has been first observed experimentally during sprouting angiogenesis, but its underlying physical mechanisms are poorly understood. Here, we identify giant vacuole formation as inverse blebbing and formulate a biophysical model of this process. Our model elucidates how cell membrane mechanical properties affect the morphology and dynamics of giant vacuoles and predicts coarsening akin to Ostwald ripening between multiple invaginating vacuoles. Our results are in qualitative agreement with observations from the formation of giant vacuoles during perfusion experiments. Our model not only elucidates the biophysical mechanisms driving inverse blebbing and giant vacuole dynamics, but also identifies universal features of the cellular response to pressure loads that are relevant to many experimental contexts. Significance statementHuman Schlemms canal endothelial cells in physiological conditions are subjected to a pressure gradient caused by the flow of aqueous humor in the basal-to-apical direction across the endothelium leading to the formation of cellular outpouchings called giant vacuoles. The physical mechanisms regulating giant vacuole formation are unknown. By describing giant vacuoles as inward blebs, we formulate a model of their growth and collapse that captures the characteristic features observed experimentally. Our theory reveals that the abrupt increase in surface tension caused by membrane stretching, which is required to accommodate the large areal strains locally induced by inward blebbing, limits giant vacuole growth. The model also predicts a competition between multiple invaginating vacuoles in which big vacuoles win over small vacuoles.

biophysics↗

bioRxiv2021

Heterogeneous run-and-tumble motion accounts for transient non-Gaussian super-diffusion in haematopoietic multi-potent progenitor cells

Multi-potent progenitor (MPP) cells act as a key intermediary step between haematopoietic stem cells and the entirety of the mature blood cell system. Their eventual fate determination is thought to be achieved through migration in and out of spatially distinct niches. Here we first analyze statistically MPP cell trajectory data obtained from a series of long time-course 3D in-vivo imaging experiments on irradiated mouse calvaria, and report that MPPs display transient super-diffusion with apparent non-Gaussian displacement distributions. Second, we explain these experimental findings using a run-and-tumble model of cell motion which incorporates the observed dynamical heterogeneity of the MPPs. Third, we use our model to extrapolate the dynamics to time-periods currently inaccessible experimentally, which enables us to quantitatively estimate the time and length scales at which super-diffusion transitions to Fickian diffusion. Our work sheds light on the potential importance of motility in early haematopoietic progenitor function.

biophysics↗

bioRxiv2021

Pickering stabilization of a dynamic intracellular emulsion

Biomolecular condensates are cellular compartments that form by phase separation in the absence of limiting membranes. Studying the P granules of C. elegans, we find that condensate dynamics are regulated by protein clusters that adsorb to the condensate interface. Using in vitro reconstitution, live observations and theory, we demonstrate that localized assembly of P granules is controlled by MEG-3, an intrinsically disordered protein that forms low dynamic assemblies on P granules. Following classic Pickering emulsion theory, MEG-3 clusters lower surface tension and slow down coarsening. During zygote polarization, MEG-3 recruits DYRK/MBK-2 kinase to accelerate localized growth of the P granule emulsion. By tuning condensate-cytoplasm exchange, interfacial clusters regulate the structural integrity of biomolecular condensates, reminiscent of the role of lipid bilayers in membrane-bound organelles. One Sentence SummaryBiomolecular condensates are stabilized by interfacial nanoscale protein clusters.

molecular biology↗

bioRxiv2020

NAD+ Redox Imbalance in the Heart Exacerbates Diabetic Cardiomyopathy

BackgroundDiabetes is a risk factor of heart failure and promotes cardiac dysfunction. Diabetic tissues are associated with NAD+ redox imbalance; however, the hypothesis that NAD+ redox imbalance leads to dysfunction of diabetic hearts has not been tested. In this study, we employed mouse models with altered NAD+ redox balance to test the hypothesis. Methods and ResultsDiabetes was induced in C57BL/6 mice by streptozotocin injections, and diabetic cardiomyopathy (DCM) was allowed to develop for 16 weeks. Diabetic stress led to cardiac dysfunction and lowered NAD+/NADH ratio. This diabetogenic regimen was administered to cardiac-specific knockout mice of complex I subunit Ndufs4 (cKO), a model with lowered cardiac NAD+/NADH ratio without baseline dysfunction. Cardiac NAD+ redox imbalance in cKO hearts exacerbated systolic and diastolic dysfunction of diabetic mice in both sexes. Collagen levels and transcript analyses of fibrosis and extracellular matrix-dependent pathways did not show change in diabetic cKO hearts, suggesting that the exacerbated cardiac dysfunction was likely due to cardiomyocyte dysfunction. We found that cardiac NAD+ redox imbalance promoted superoxide dismutase 2 (SOD2) acetylation, protein oxidation, induced troponin I S150 phosphorylation and impaired energetics in diabetic cKO hearts. Importantly, elevation of cardiac NAD+ levels by nicotinamide phosphoribosyltransferase (NAMPT) normalized NAD+ redox balance, over-expression alleviated cardiac dysfunction and reversed pathogenic mechanisms in diabetic mice. ConclusionOur results show that NAD+ redox imbalance to regulate protein acetylation and phosphorylation is a critical mediator of the progression of DCM, and suggest the therapeutic potential of harnessing NAD+ metabolism in DCM.

physiology↗