Mechanosensitive FHL2 tunes endothelial function.

Endothelial tissues are essential mechanosensors in the vasculature and facilitate adaptation to various blood flow-induced mechanical cues. Defects in endothelial mechanoresponses can perturb tissue remodelling and functions leading to cardiovascular disease progression. In this context, the precise mechanisms of endothelial mechanoresponses contributing to normal and diseased tissue functioning remain elusive. Here, we sought to uncover how flow-mediated transcriptional regulation drives endothelial mechanoresponses in healthy and atherosclerotic-prone tissues. Using bulk RNA sequencing, we identify novel mechanosensitive genes in response to healthy unidirectional flow (UF) and athero-prone disturbed flow (DF). We find that the transcription as well as protein expression of Four-and-a-half LIM protein 2 (FHL2) are enriched in athero-prone DF both in vitro and in vivo. We then demonstrate that the exogenous expression of FHL2 is necessary and sufficient to drive discontinuous adherens junction morphology and increased tissue permeability. This athero-prone phenotype requires the force-sensitive binding of FHL2 to actin. In turn, the force-dependent localisation of FHL2 to stress fibres promotes microtubule dynamics to release the RhoGEF, GEF-H1, and activate the Rho-ROCK pathway. Thus, we unravelled a novel mechanochemical feedback wherein force-dependent FHL2 localisation promotes hypercontractility. This misregulated mechanoresponse creates highly permeable tissues, depicting classic hallmarks of atherosclerosis progression. Overall, we highlight crucial functions for the FHL2 force-sensitivity in tuning multi-scale endothelial mechanoresponses.

Limiting pool and actin architecture controls myosin cluster sizes in adherent cells.

The actomyosin cytoskeleton generates mechanical forces that power important cellular processes, such as cell migration, cell division, and mechanosensing. Actomyosin self-assembles into contractile networks and bundles that underlie force generation and transmission in cells. A central step is the assembly of the myosin II filament from myosin monomers, regulation of which has been extensively studied. However, myosin filaments are almost always found as clusters within the cell cortex. While recent studies characterized cluster nucleation dynamics at the cell periphery, how myosin clusters grow on stress fibers remains poorly characterized. Here, we utilize a U2OS osteosarcoma cell line with endogenously tagged myosin II to measure the myosin cluster size distribution in the lamella of adherent cells. We find that myosin clusters can grow with Rho-kinase (ROCK) activity alone in the absence of myosin motor activity. Time-lapse imaging reveals that myosin clusters grow via increased myosin association to existing clusters, which is potentiated by ROCK-dependent myosin filament assembly. Enabling myosin motor activity allows further myosin cluster growth through myosin association that is dependent on F-actin architecture. Using a toy model, we show that myosin self-affinity is sufficient to recapitulate the experimentally observed myosin cluster size distribution, and that myosin cluster sizes are determined by the pool of myosin available for cluster growth. Together, our findings provide new insights into the regulation of myosin cluster sizes within the lamellar actomyosin cytoskeleton.

Measuring response functions of active materials from data.

From flocks of birds to biomolecular assemblies, systems in which many individual components independently consume energy to perform mechanical work exhibit a wide array of striking behaviors. Methods to quantify the dynamics of these so-called active systems generally aim to extract important length or time scales from experimental fields. Because such methods focus on extracting scalar values, they do not wring maximal information from experimental data. We introduce a method to overcome these limitations. We extend the framework of correlation functions by taking into account the internal headings of displacement fields. The functions we construct represent the material response to specific types of active perturbation within the system. Utilizing these response functions we query the material response of disparate active systems composed of actin filaments and myosin motors, from model fluids to living cells. We show we can extract critical length scales from the turbulent flows of an active nematic, anticipate contractility in an active gel, distinguish viscous from viscoelastic dissipation, and even differentiate modes of contractility in living cells. These examples underscore the vast utility of this method which measures response functions from experimental observations of complex active systems.

Limiting Pool and Actin Architecture Controls Myosin Cluster Sizes in Adherent Cells.

The actomyosin cytoskeleton generates mechanical forces that power important cellular processes, such as cell migration, cell division, and mechanosensing. Actomyosin self-assembles into contractile networks and bundles that underlie force generation and transmission in cells. A central step is the assembly of the myosin II filament from myosin monomers, regulation of which has been extensively studied. However, myosin filaments are almost always found as clusters within the cell cortex. While recent studies characterized cluster nucleation dynamics at the cell periphery, how myosin clusters grow on stress fibers remains poorly characterized. Here, we utilize a U2OS osteosarcoma cell line with endogenously tagged myosin II to measure the myosin cluster size distribution in the lamella of adherent cells. We find that myosin clusters can grow with Rho-kinase (ROCK) activity alone in the absence of myosin motor activity. Time-lapse imaging reveals that myosin clusters grow via increased myosin association to existing clusters, which is potentiated by ROCK-dependent myosin filament assembly. Enabling myosin motor activity allows further myosin cluster growth through myosin association that is dependent on F-actin architecture. Using a toy model, we show that myosin self-affinity is sufficient to recapitulate the experimentally observed myosin cluster size distribution, and that myosin cluster sizes are determined by the pool of myosin available for cluster growth. Together, our findings provide new insights into the regulation of myosin cluster sizes within the lamellar actomyosin cytoskeleton.