PAK1-dependent mechanotransduction enables myofibroblast nuclear adaptation and chromatin organization during fibrosis.

Myofibroblasts are responsible for scarring during fibrosis. The scar propagates mechanical signals inducing a radical transformation in myofibroblast cell state and increasing profibrotic phenotype. Here, we show mechanical stress from progressive scarring induces nuclear softening and de-repression of heterochromatin. The parallel loss of H3K9Me3 enables a permissive state for distinct chromatin accessibility and profibrotic gene regulation. Integrating chromatin accessibility profiles with RNA expression provides insight into the transcription network underlying the switch in profibrotic myofibroblast states, emphasizing mechanoadaptive regulation of PAK1 as key drivers. Through genetic manipulation in liver and lung fibrosis, loss of PAK1-dependent signaling impairs the mechanoadaptive response in vitro and dramatically improves fibrosis in vivo. Moreover, we provide human validation for mechanisms underpinning PAK1-mediated mechanotransduction in liver and lung fibrosis. Collectively, these observations provide insight into the nuclear mechanics driving the profibrotic chromatin landscape in fibrosis, highlighting actomyosin-dependent mechanisms as potential therapeutic targets in fibrosis.

Nanoindentation Response of 3D Printed PEGDA Hydrogels in a Hydrated Environment.

Hydrogels are commonly used materials in tissue engineering and organ-on-chip devices. This study investigated the nanomechanical properties of monolithic and multilayered poly(ethylene glycol) diacrylate (PEGDA) hydrogels manufactured using bulk polymerization and layer-by-layer projection lithography processes, respectively. An increase in the number of layers (or reduction in layer thickness) from 1 to 8 and further to 60 results in a reduction in the elastic modulus from 5.53 to 1.69 and further to 0.67 MPa, respectively. It was found that a decrease in the number of layers induces a lower creep index (CIT) in three-dimensional (3D) printed PEGDA hydrogels. This reduction is attributed to mesoscale imperfections that appear as pockets of voids at the interfaces of the multilayered hydrogels attributed to localized regions of unreacted prepolymers, resulting in variations in defect density in the samples examined. An increase in the degree of cross-linking introduced by a higher dosage of ultraviolet (UV) exposure leads to a higher elastic modulus. This implies that the elastic modulus and creep behavior of hydrogels are governed and influenced by the degree of cross-linking and defect density of the layers and interfaces. These findings can guide an optimal manufacturing pathway to obtain the desirable nanomechanical properties in 3D printed PEGDA hydrogels, critical for the performance of living cells and tissues, which can be engineered through control of the fabrication parameters.

Experimental long-term diabetes mellitus alters the transcriptome and biomechanical properties of the rat urinary bladder.

Diabetes mellitus (DM) is the leading cause of chronic kidney disease and diabetic nephropathy is widely studied. In contrast, the pathobiology of diabetic urinary bladder disease is less understood despite dysfunctional voiding being common in DM. We hypothesised that diabetic cystopathy has a characteristic molecular signature. We therefore studied bladders of hyperglycaemic and polyuric rats with streptozotocin (STZ)-induced DM. Sixteen weeks after induction of DM, as assessed by RNA arrays, wide-ranging changes of gene expression occurred in DM bladders over and above those induced in bladders of non-hyperglycaemic rats with sucrose-induced polyuria. The altered transcripts included those coding for extracellular matrix regulators and neural molecules. Changes in key genes deregulated in DM rat bladders were also detected in db/db mouse bladders. In DM rat bladders there was reduced birefringent collagen between detrusor muscle bundles, and atomic force microscopy showed a significant reduction in tissue stiffness; neither change was found in bladders of sucrose-treated rats. Thus, altered extracellular matrix with reduced tissue rigidity may contribute to voiding dysfunction in people with long-term DM. These results serve as an informative stepping stone towards understanding the complex pathobiology of diabetic cystopathy.

Nanoindentation of Molecular Crystals: Lessons Learned from Aspirin.

Nanoindentation enables the measurement of mechanical properties from single crystals with dimensions of a few micrometers. This experimental technique, however, has only recently been applied to molecular crystals. Key differences between the application of this technique to molecular crystals and metals and other inorganics are identified. From this, protocols for the measurement of hardness and elastic modulus of molecular crystals of pharmaceutical interest are proposed. Using form I aspirin as a model system, the impact of single crystal sample surface preparation (washing and cleaving) on the surface roughness is explored. We show the importance of using a calibration sample with hardness and stiffness close to that of molecular crystals for the acquisition of more accurate data. The issue of solvent occlusions formed during crystal growth is discussed as a source of material property variation as well as tip contamination. It is proposed that this in part explains the significantly larger variation of the measured mechanical properties among different single crystals compared to those performed on a unique sample. Because both the indentation modulus and the hardness can vary significantly for low depth indents, samples were tested over a wide range of depths, which revealed that a minimum depth of penetration is required for the acquisition of data. This experiment is crucial and needs to be carried out for every system under study since it allows for the determination of the minimum-working load. Post-indentation imaging combined with crystallographic analysis and molecular simulations allows for the characterization and rationalization of the material plastic deformation mechanisms.

WDR5 modulates cell motility and morphology and controls nuclear changes induced by a 3D environment.

Cell migration through extracellular matrices requires nuclear deformation, which depends on nuclear stiffness. In turn, chromatin structure contributes to nuclear stiffness, but the mechanosensing pathways regulating chromatin during cell migration remain unclear. Here, we demonstrate that WD repeat domain 5 (WDR5), an essential component of H3K4 methyltransferase complexes, regulates cell polarity, nuclear deformability, and migration of lymphocytes in vitro and in vivo, independent of transcriptional activity, suggesting nongenomic functions for WDR5. Similarly, depletion of RbBP5 (another H3K4 methyltransferase subunit) promotes similar defects. We reveal that a 3D environment increases the H3K4 methylation dependent on WDR5 and results in a globally less compacted chromatin conformation. Further, using atomic force microscopy, nuclear particle tracking, and nuclear swelling experiments, we detect changes in nuclear mechanics that accompany the epigenetic changes induced in 3D conditions. Indeed, nuclei from cells in 3D environments were softer, and thereby more deformable, compared with cells in suspension or cultured in 2D conditions, again dependent on WDR5. Dissecting the underlying mechanism, we determined that actomyosin contractility, through the phosphorylation of myosin by MLCK (myosin light chain kinase), controls the interaction of WDR5 with other components of the methyltransferase complex, which in turn up-regulates H3K4 methylation activation in 3D conditions. Taken together, our findings reveal a nongenomic function for WDR5 in regulating H3K4 methylation induced by 3D environments, physical properties of the nucleus, cell polarity, and cell migratory capacity.

Integrin α4ß1 controls G9a activity that regulates epigenetic changes and nuclear properties required for lymphocyte migration.

The mechanical properties of the cell nucleus change to allow cells to migrate, but how chromatin modifications contribute to nuclear deformability has not been defined. Here, we demonstrate that a major factor in this process involves epigenetic changes that underpin nuclear structure. We investigated the link between cell adhesion and epigenetic changes in T-cells, and demonstrate that T-cell adhesion to VCAM1 via α4ß1 integrin drives histone H3 methylation (H3K9me2/3) through the methyltransferase G9a. In this process, active G9a is recruited to the nuclear envelope and interacts with lamin B1 during T-cell adhesion through α4ß1 integrin. G9a activity not only reorganises the chromatin structure in T-cells, but also affects the stiffness and viscoelastic properties of the nucleus. Moreover, we further demonstrated that these epigenetic changes were linked to lymphocyte movement, as depletion or inhibition of G9a blocks T-cell migration in both 2D and 3D environments. Thus, our results identify a novel mechanism in T-cells by which α4ß1 integrin signaling drives specific chromatin modifications, which alter the physical properties of the nucleus and thereby enable T-cell migration.