Revealing the Biophysics of Lamina-Associated Domain Formation by Integrating Theoretical Modeling and High-Resolution Imaging.

The interactions between chromatin and the nuclear lamina orchestrate cell type-specific gene activity by forming lamina-associated domains (LADs) which preserve cellular characteristics through gene repression. However, unlike the interactions between chromatin segments, the strength of chromatin-lamina interactions and their dependence on cellular environment are not well understood. Here, we develop a theory to predict the size and shape of peripheral heterochromatin domains by considering the energetics of chromatin-chromatin interactions, the affinity between chromatin and the nuclear lamina and the kinetics of methylation and acetylation9in human mesenchymal stem cells (hMSCs). Through the analysis of super-resolution images of peripheral heterochromatin domains using this theoretical framework, we determine the nuclear lamina-wide distribution of chromatin-lamina affinities. We find that the extracted affinity is highly spatially heterogeneous and shows a bimodal distribution, indicating regions along the lamina with strong chromatin binding and those exhibiting vanishing chromatin affinity interspersed with some regions exhibiting a relatively diminished chromatin interactions, in line with the presence of structures such as nuclear pores. Exploring the role of environmental cues on peripheral chromatin, we find that LAD thickness increases when hMSCs are cultured on a softer substrate, in correlation with contractility-dependent translocation of histone deacetylase 3 (HDAC3) from the cytosol to the nucleus. In soft microenvironments, chromatin becomes sequestered at the nuclear lamina, likely due to the interactions of HDAC3 with the chromatin anchoring protein LAP2 ß ,increasing chromatin-lamina affinity, as well as elevated levels of the intranuclear histone methylation. Our findings are further corroborated by pharmacological interventions that inhibit contractility, as well as by manipulating methylation levels using epigenetic drugs. Notably, in the context of tendinosis, a chronic condition characterized by collagen degeneration, we observed a similar increase in the thickness of peripheral chromatin akin to that of cells cultured on soft substrates consistent with theoretical predictions. Our findings underscore the pivotal role of the microenvironment in shaping genome organization and highlight its relevance in pathological conditions.

Microtubule forces drive nuclear damage in LMNA cardiomyopathy.

Nuclear homeostasis requires a balance of forces between the cytoskeleton and nucleus. Mutations in the LMNA gene, which encodes the nuclear envelope proteins lamin A/C, disrupt this balance by weakening the nuclear lamina. This results in nuclear damage in contractile tissues and ultimately muscle disease. Intriguingly, disrupting the LINC complex that connects the cytoskeleton to the nucleus has emerged as a promising strategy to ameliorate LMNA-associated cardiomyopathy. Yet how LINC complex disruption protects the cardiomyocyte nucleus remains unclear. To address this, we developed an assay to quantify the coupling of cardiomyocyte contraction to nuclear deformation and interrogated its dependence on the nuclear lamina and LINC complex. We found that, surprisingly, the LINC complex was mostly dispensable for transferring contractile strain to the nucleus, and that increased nuclear strain in lamin A/C-deficient cardiomyocytes was not rescued by LINC complex disruption. Instead, LINC complex disruption eliminated the cage of microtubules encircling the nucleus. Disrupting microtubules was sufficient to prevent nuclear damage and rescue cardiac function induced by lamin A/C deficiency. We computationally simulated the stress fields surrounding cardiomyocyte nuclei and show how microtubule forces generate local vulnerabilities that damage lamin A/C-deficient nuclei. Our work pinpoints localized, microtubule-dependent force transmission through the LINC complex as a pathological driver and therapeutic target for LMNA-cardiomyopathy.

Mechano-metabolism of metastatic breast cancer cells in 2D and 3D microenvironments.

Cells regulate their shape and metabolic activity in response to the mechano-chemical properties of their microenvironment. To elucidate the impact of matrix stiffness and ligand density on the bioenergetics of mesenchymal cells, we developed a nonequilibrium, active chemo-mechanical model that accounts for the mechanical energy of the cell and matrix, chemical energy from ATP hydrolysis, interfacial energy, and mechano-sensitive regulation of stress fiber assembly through signaling. By integrating the kinetics and energetics of these processes, we define the cell "metabolic potential" that, when minimized, provides testable predictions of cell contractility, shape, and ATP consumption. Specifically, we show that the morphology of MDA-MB-231 breast cancer cells in 3D collagen changes from spherical to elongated to spherical with increasing matrix stiffness, which is consistent with experimental observations. On 2D hydrogels, our model predicts a hemispherical-to-spindle-to-disc shape transition with increasing gel stiffness. In both cases, we show that these shape transitions emerge from competition between the energy of ATP hydrolysis associated with increased contractility that drives cell elongation and the interfacial energy that favors a rounded shape. Furthermore, our model can predict how increased energy demand in stiffer microenvironments is met by AMPK activation, which is confirmed experimentally in both 2D and 3D microenvironments and found to correlate with the upregulation of mitochondrial potential, glucose uptake, and ATP levels, as well as provide estimates of changes in intracellular adenosine nucleotide concentrations with changing environmental stiffness. Overall, we present a framework for relating adherent cell energy levels and contractility through biochemical regulation of underlying physical processes. Statement of Significance: Increasing evidence indicates that cellular metabolism is regulated by mechanical cues from the extracellular environment. Forces transmitted from the microenvironment activate mechanotransduction pathways in the cell, which trigger a cascade of biochemical events that impact cytoskeletal tension, cellular morphology and energy budget available to the cell. Using a nonequilibrium free energy-based theory, we can predict the ATP consumption, contractility, and shape of mesenchymal cancer cells, as well as how cells regulate energy levels dependent on the mechanosensitive metabolic regulator AMPK. The insights from our model can be used to understand the mechanosensitive regulation of metabolism during metastasis and tumor progression, during which cells experience dynamic changes in their microenvironment and metabolic state.

Lumen expansion is initially driven by apical actin polymerization followed by osmotic pressure in a human epiblast model.

Post-implantation, the pluripotent epiblast in a human embryo forms a central lumen, paving the way for gastrulation. Osmotic pressure gradients are considered the drivers of lumen expansion across development, but their role in human epiblasts is unknown. Here, we study lumenogenesis in a pluripotent-stem-cell-based epiblast model using engineered hydrogels. We find that leaky junctions prevent osmotic pressure gradients in early epiblasts and, instead, forces from apical actin polymerization drive lumen expansion. Once the lumen reaches a radius of ∼12 µm, tight junctions mature, and osmotic pressure gradients develop to drive further growth. Computational modeling indicates that apical actin polymerization into a stiff network mediates initial lumen expansion and predicts a transition to pressure-driven growth in larger epiblasts to avoid buckling. Human epiblasts show transcriptional signatures consistent with these mechanisms. Thus, actin polymerization drives lumen expansion in the human epiblast and may serve as a general mechanism of early lumenogenesis.

Active transcription and epigenetic reactions synergistically regulate meso-scale genomic organization.

In interphase nuclei, chromatin forms dense domains of characteristic sizes, but the influence of transcription and histone modifications on domain size is not understood. We present a theoretical model exploring this relationship, considering chromatin-chromatin interactions, histone modifications, and chromatin extrusion. We predict that the size of heterochromatic domains is governed by a balance among the diffusive flux of methylated histones sustaining them and the acetylation reactions in the domains and the process of loop extrusion via supercoiling by RNAPII at their periphery, which contributes to size reduction. Super-resolution and nano-imaging of five distinct cell lines confirm the predictions indicating that the absence of transcription leads to larger heterochromatin domains. Furthermore, the model accurately reproduces the findings regarding how transcription-mediated supercoiling loss can mitigate the impacts of excessive cohesin loading. Our findings shed light on the role of transcription in genome organization, offering insights into chromatin dynamics and potential therapeutic targets.

Vimentin is a key regulator of cell mechanosensing through opposite actions on actomyosin and microtubule networks.

The cytoskeleton is a complex network of interconnected biopolymers consisting of actin filaments, microtubules, and intermediate filaments. These biopolymers work in concert to transmit cell-generated forces to the extracellular matrix required for cell motility, wound healing, and tissue maintenance. While we know cell-generated forces are driven by actomyosin contractility and balanced by microtubule network resistance, the effect of intermediate filaments on cellular forces is unclear. Using a combination of theoretical modeling and experiments, we show that vimentin intermediate filaments tune cell stress by assisting in both actomyosin-based force transmission and reinforcement of microtubule networks under compression. We show that the competition between these two opposing effects of vimentin is regulated by the microenvironment stiffness. These results reconcile seemingly contradictory results in the literature and provide a unified description of vimentin's effects on the transmission of cell contractile forces to the extracellular matrix.

Microinterfaces in biopolymer-based bicontinuous hydrogels guide rapid 3D cell migration.

Cell migration is critical for tissue development and regeneration but requires extracellular environments that are conducive to motion. Cells may actively generate migratory routes in vivo by degrading or remodeling their environments or instead utilize existing extracellular matrix microstructures or microtracks as innate pathways for migration. While hydrogels in general are valuable tools for probing the extracellular regulators of 3-dimensional migration, few recapitulate these natural migration paths. Here, we develop a biopolymer-based bicontinuous hydrogel system that comprises a covalent hydrogel of enzymatically crosslinked gelatin and a physical hydrogel of guest and host moieties bonded to hyaluronic acid. Bicontinuous hydrogels form through controlled solution immiscibility, and their continuous subdomains and high micro-interfacial surface area enable rapid 3D migration, particularly when compared to homogeneous hydrogels. Migratory behavior is mesenchymal in nature and regulated by biochemical and biophysical signals from the hydrogel, which is shown across various cell types and physiologically relevant contexts (e.g., cell spheroids, ex vivo tissues, in vivo tissues). Our findings introduce a design that leverages important local interfaces to guide rapid cell migration.

Novel CHRNA3 variants identified in a patient with bladder dysfunction, dysautonomia, and gastrointestinal dysmotility.

Congenital anomalies of the kidney and urinary tract (CAKUT) are estimated to be responsible for 20%-50% of congenital anomalies and are also a leading etiology of early-onset renal disease. Primary CAKUT are caused by genetic factors that impair proper in-utero genitourinary tract development and secondary CAKUT result from the influence of environmental factors. The CHRNA3 gene, which encodes the Alpha-3 subunit of the nicotinic acetylcholine receptor, is hypothesized to be associated with Megacystis-microcolon-intestinal hyperperistalsis syndrome. More recently, pathogenic variants in CHRNA3 have been identified in individuals with CAKUT as well as individuals with panautonomic failure. Here we present a patient with neurogenic bladder, vesicoureteral reflux, mydriasis, and gastrointestinal dysmotility found to have novel compound heterozygous variants in CHRNA3. These findings support the consideration of CHRNA3 disruption in the differential for CAKUT with dysautonomia and gastrointestinal dysmotility.

CD44 and ß1-integrin are both engaged in cell traction force generation in hyaluronic acid-rich extracellular matrices.

Mechanical properties of the extracellular matrices (ECMs) critically regulate a number of important cell function including growth, differentiation and migration. Type I collagen and glycosaminoglycans (GAGs) are two primary components of ECMs that contribute to tissue mechanics with the collagen fiber network sustaining tension and GAGs withstanding compression. Collagen stiffness as well as its architecture are known to be important role players in cell-ECM mechanical interactions, however, much less is known about how GAGs within ECMs regulate cell force generation and invasion. Inspired by a recent theoretical work from the Shenoy lab that GAGs play important roles in cell - ECM interactions, we hereby present experimental studies on the role of hyaluronic acid (HA, an unsulfated GAG) in single tumor cell traction force generation within HA collagen cogels using a recently developed 3D cell traction force microscopy. Our work revealed that CD44, a cell surface adhesion receptor to HA, was engaged in cell traction force generation in conjunction with ß1-integrin. Furthermore, we found that HA significantly modified the architecture and mechanics of the collagen fiber network, decreased tumor cells' propensity to remodel the collagen network, decreased traction force generation and transmission distance, and attenuated tumor invasion in agreement with theoretical predictions. Our findings highlighted the significance of CD44 and HA engagement in cell-ECM mechanical interactions, providing new insights on the mechanical model of cellular force transmission.

Cell volume expansion and local contractility drive collective invasion of the basement membrane in breast cancer.

Breast cancer becomes invasive when carcinoma cells invade through the basement membrane (BM)-a nanoporous layer of matrix that physically separates the primary tumour from the stroma. Single cells can invade through nanoporous three-dimensional matrices due to protease-mediated degradation or force-mediated widening of pores via invadopodial protrusions. However, how multiple cells collectively invade through the physiological BM, as they do during breast cancer progression, remains unclear. Here we developed a three-dimensional in vitro model of collective invasion of the BM during breast cancer. We show that cells utilize both proteases and forces-but not invadopodia-to breach the BM. Forces are generated from a combination of global cell volume expansion, which stretches the BM, and local contractile forces that act in the plane of the BM to breach it, allowing invasion. These results uncover a mechanism by which cells collectively interact to overcome a critical barrier to metastasis.

Fibrillar adhesion dynamics govern the timescales of nuclear mechano-response via the vimentin cytoskeleton.

The cell nucleus is continuously exposed to external signals, of both chemical and mechanical nature. To ensure proper cellular response, cells need to regulate not only the transmission of these signals, but also their timing and duration. Such timescale regulation is well described for fluctuating chemical signals, but if and how it applies to mechanical signals reaching the nucleus is still unknown. Here we demonstrate that the formation of fibrillar adhesions locks the nucleus in a mechanically deformed conformation, setting the mechanical response timescale to that of fibrillar adhesion remodelling (~1 hour). This process encompasses both mechanical deformation and associated mechanotransduction (such as via YAP), in response to both increased and decreased mechanical stimulation. The underlying mechanism is the anchoring of the vimentin cytoskeleton to fibrillar adhesions and the extracellular matrix through plectin 1f, which maintains nuclear deformation. Our results reveal a mechanism to regulate the timescale of mechanical adaptation, effectively setting a low pass filter to mechanotransduction.

Microinterfaces in bicontinuous hydrogels guide rapid 3D cell migration.

Cell migration is critical for tissue development and regeneration but requires extracellular environments that are conducive to motion. Cells may actively generate migratory routes in vivo by degrading or remodeling their environments or may instead utilize existing ECM microstructures or microtracks as innate pathways for migration. While hydrogels in general are valuable tools for probing the extracellular regulators of 3D migration, few have recapitulated these natural migration paths. Here, we developed a biopolymer-based (i.e., gelatin and hyaluronic acid) bicontinuous hydrogel system formed through controlled solution immiscibility whose continuous subdomains and high micro-interfacial surface area enabled rapid 3D migration, particularly when compared to homogeneous hydrogels. Migratory behavior was mesenchymal in nature and regulated by biochemical and biophysical signals from the hydrogel, which was shown across various cell types and physiologically relevant contexts (e.g., cell spheroids, ex vivo tissues, in vivo tissues). Our findings introduce a new design that leverages important local interfaces to guide rapid cell migration.

Changes in fecal elastase-1 following initiation of CFTR modulator therapy in pediatric patients with cystic fibrosis.

BACKGROUND: Improvement in exocrine pancreatic function in persons with CF (pwCF) on cystic fibrosis transmembrane conductance regulator (CFTR) modulators has been documented in clinical trials using fecal pancreatic elastase-1 (FE-1). Our group endeavored to evaluate real-world data on FE-1 in children on CFTR modulator therapy at three pediatric cystic fibrosis (CF) centers. METHODS: Pediatric pwCF were offered FE-1 testing if they were on pancreatic enzyme replacement therapy (PERT) and on CFTR modulator therapy according to their center's guideline. FE-1 data were collected retrospectively. The primary outcome was absolute change in FE-1. RESULTS: 70 pwCF were included for analysis. 53 had baseline and post-modulator FE-1 values. There was a significant increase in FE-1 from median 25 mcg/g (IQR 25-60) at baseline to 57 mcg/g (IQR 20-228) post-modulator (p<0.001 by Wilcoxon matched pairs), with an absolute change in FE-1 of median 28 mcg/g (IQR -5-161) and mean 93.5 ± 146.8 mcg/g. Age was negatively correlated with change in FE-1 (Spearman r=-0.48, p<0.001). 15 pwCF (21%) had post-modulator FE-1 values ≥200 mcg/g, consistent with pancreatic sufficiency (PS). The PS group was significant for younger age at initiation of first CFTR modulator and a higher baseline FE-1. CONCLUSIONS: Most pwCF experienced an increase in FE-1 while receiving CFTR modulator treatment and a small percentage demonstrated values reflective of PS. These data suggest that PS may be attained in those that initiated modulator therapy at a younger age or had a higher baseline FE-1. FE-1 testing is suggested for children on any CFTR modulator therapy.

Chemo-mechanical diffusion waves explain collective dynamics of immune cell podosomes.

Immune cells, such as macrophages and dendritic cells, can utilize podosomes, mechanosensitive actin-rich protrusions, to generate forces, migrate, and patrol for foreign antigens. Individual podosomes probe their microenvironment through periodic protrusion and retraction cycles (height oscillations), while oscillations of multiple podosomes in a cluster are coordinated in a wave-like fashion. However, the mechanisms governing both the individual oscillations and the collective wave-like dynamics remain unclear. Here, by integrating actin polymerization, myosin contractility, actin diffusion, and mechanosensitive signaling, we develop a chemo-mechanical model for podosome dynamics in clusters. Our model reveals that podosomes show oscillatory growth when actin polymerization-driven protrusion and signaling-associated myosin contraction occur at similar rates, while the diffusion of actin monomers drives wave-like coordination of podosome oscillations. Our theoretical predictions are validated by different pharmacological treatments and the impact of microenvironment stiffness on chemo-mechanical waves. Our proposed framework can shed light on the role of podosomes in immune cell mechanosensing within the context of wound healing and cancer immunotherapy.

Lipid droplets are intracellular mechanical stressors that impair hepatocyte function.

Matrix stiffening and external mechanical stress have been linked to disease and cancer development in multiple tissues, including the liver, where cirrhosis (which increases stiffness markedly) is the major risk factor for hepatocellular carcinoma. Patients with nonalcoholic fatty liver disease and lipid droplet-filled hepatocytes, however, can develop cancer in noncirrhotic, relatively soft tissue. Here, by treating primary human hepatocytes with the monounsaturated fatty acid oleate, we show that lipid droplets are intracellular mechanical stressors with similar effects to tissue stiffening, including nuclear deformation, chromatin condensation, and impaired hepatocyte function. Mathematical modeling of lipid droplets as inclusions that have only mechanical interactions with other cellular components generated results consistent with our experiments. These data show that lipid droplets are intracellular sources of mechanical stress and suggest that nuclear membrane tension integrates cell responses to combined internal and external stresses.

Endothelium and Subendothelial Matrix Mechanics Modulate Cancer Cell Transendothelial Migration.

Cancer cell extravasation, a key step in the metastatic cascade, involves cancer cell arrest on the endothelium, transendothelial migration (TEM), followed by the invasion into the subendothelial extracellular matrix (ECM) of distant tissues. While cancer research has mostly focused on the biomechanical interactions between tumor cells (TCs) and ECM, particularly at the primary tumor site, very little is known about the mechanical properties of endothelial cells and the subendothelial ECM and how they contribute to the extravasation process. Here, an integrated experimental and theoretical framework is developed to investigate the mechanical crosstalk between TCs, endothelium and subendothelial ECM during in vitro cancer cell extravasation. It is found that cancer cell actin-rich protrusions generate complex push-pull forces to initiate and drive TEM, while transmigration success also relies on the forces generated by the endothelium. Consequently, mechanical properties of the subendothelial ECM and endothelial actomyosin contractility that mediate the endothelial forces also impact the endothelium's resistance to cancer cell transmigration. These results indicate that mechanical features of distant tissues, including force interactions between the endothelium and the subendothelial ECM, are key determinants of metastatic organotropism.

Aberrant chromatin reorganization in cells from diseased fibrous connective tissue in response to altered chemomechanical cues.

Changes in the micro-environment of fibrous connective tissue can lead to alterations in the phenotypes of tissue-resident cells, yet the underlying mechanisms are poorly understood. Here, by visualizing the dynamics of histone spatial reorganization in tenocytes and mesenchymal stromal cells from fibrous tissue of human donors via super-resolution microscopy, we show that physiological and pathological chemomechanical cues can directly regulate the spatial nanoscale organization and density of chromatin in these tissue-resident cell populations. Specifically, changes in substrate stiffness, altered oxygen tension and the presence of inflammatory signals drive chromatin relocalization and compaction into the nuclear boundary, mediated by the activity of the histone methyltransferase EZH2 and an intact cytoskeleton. In healthy cells, chemomechanically triggered changes in the spatial organization and density of chromatin are reversible and can be attenuated by dynamically stiffening the substrate. In diseased human cells, however, the link between mechanical or chemical inputs and chromatin remodelling is abrogated. Our findings suggest that aberrant chromatin organization in fibrous connective tissue may be a hallmark of disease progression that could be leveraged for therapeutic intervention.

Feedback between mechanosensitive signaling and active forces governs endothelial junction integrity.

The formation and recovery of gaps in the vascular endothelium governs a wide range of physiological and pathological phenomena, from angiogenesis to tumor cell extravasation. However, the interplay between the mechanical and signaling processes that drive dynamic behavior in vascular endothelial cells is not well understood. In this study, we propose a chemo-mechanical model to investigate the regulation of endothelial junctions as dependent on the feedback between actomyosin contractility, VE-cadherin bond turnover, and actin polymerization, which mediate the forces exerted on the cell-cell interface. Simulations reveal that active cell tension can stabilize cadherin bonds, but excessive RhoA signaling can drive bond dissociation and junction failure. While actin polymerization aids gap closure, high levels of Rac1 can induce junction weakening. Combining the modeling framework with experiments, our model predicts the influence of pharmacological treatments on the junction state and identifies that a critical balance between RhoA and Rac1 expression is required to maintain junction stability. Our proposed framework can help guide the development of therapeutics that target the Rho family of GTPases and downstream active mechanical processes.