Using polyacrylamide hydrogels to model physiological aortic stiffness reveals that microtubules are critical regulators of isolated smooth muscle cell morphology and contractility.

Vascular smooth muscle cells (VSMCs) are the predominant cell type in the medial layer of the aortic wall and normally exist in a quiescent, contractile phenotype where actomyosin-derived contractile forces maintain vascular tone. However, VSMCs are not terminally differentiated and can dedifferentiate into a proliferative, synthetic phenotype. Actomyosin force generation is essential for the function of both phenotypes. Whilst much is already known about the mechanisms of VSMC actomyosin force generation, existing assays are either low throughput and time consuming, or qualitative and inconsistent. In this study, we use polyacrylamide hydrogels, tuned to mimic the physiological stiffness of the aortic wall, in a VSMC contractility assay. Isolated VSMC area decreases following stimulation with the contractile agonists angiotensin II or carbachol. Importantly, the angiotensin II induced reduction in cell area correlated with increased traction stress generation. Inhibition of actomyosin activity using blebbistatin or Y 27632 prevented angiotensin II mediated changes in VSMC morphology, suggesting that changes in VSMC morphology and actomyosin activity are core components of the contractile response. Furthermore, we show that microtubule stability is an essential regulator of isolated VSMC contractility. Treatment with either colchicine or paclitaxel uncoupled the morphological and/or traction stress responses of angiotensin II stimulated VSMCs. Our findings support the tensegrity model and we demonstrate that microtubules act to balance the actomyosin-derived traction stress generation and regulate the morphological responses of VSMCs.

The role of cell-matrix interactions in connective tissue mechanics

Living tissue is able to withstand large stresses in everyday life, yet it also actively adapts to dynamic loads. This remarkable mechanical behaviour emerges from the interplay between living cells and their non-living extracellular environment. Here we review recent insights into the biophysical mechanisms involved in the reciprocal interplay between cells and the extracellular matrix and how this interplay determines tissue mechanics, with a focus on connective tissues. We first describe the roles of the main macromolecular components of the extracellular matrix in regards to tissue mechanics. We then proceed to highlight the main routes via which cells sense and respond to their biochemical and mechanical extracellular environment. Next we introduce the three main routes via which cells can modify their extracellular environment: exertion of contractile forces, secretion and deposition of matrix components, and matrix degradation. Finally we discuss how recent insights in the mechanobiology of cell-matrix interactions are furthering our understanding of the pathophysiology of connective tissue diseases and cancer, and facilitating the design of novel strategies for tissue engineering.

Abnormal Aggregation of Invasive Cancer Cells Induced by Collective Polarization and ECM-Mediated Mechanical Coupling in Coculture Systems

Studies on pattern formation in coculture cell systems can provide insights into many physiological and pathological processes. Here, we investigate how the extracellular matrix (ECM) may influence the patterning in coculture systems. The model coculture system we use is composed of highly motile invasive breast cancer cells, initially mixed with inert nonmetastatic cells on a 2D substrate and covered with a Matrigel layer introduced to mimic ECM. We observe that the invasive cells exhibit persistent centripetal motion and yield abnormal aggregation, rather than random spreading, due to a “collective pulling” effect resulting from ECM-mediated transmission of active contractile forces generated by the polarized migration of the invasive cells along the vertical direction. The mechanism we report may open a new window for the understanding of biological processes that involve multiple types of cells.

Substrate stiffness controls the cell cycle of human mesenchymal stem cells via cellular traction

The microenvironment of human mesenchymal stem cells (hMSCs) regulates their self-renewal and differentiation properties. Previously it was shown that hMSCs remained quiescent on soft (0.25 kPa) polyacrylamide (PA) gels but re-entered into cell cycle on a stiff (7.5 kPa) gel. However, how cells behave on intermediate stiffness and what intracellular factors transmit mechanical changes to cell interior thereby regulating cell cycle remained unknown. In this work we demonstrated that PA gels between 1 and 5 kPa act as a mechanical switch in regulating cell cycle of hMSCs. By experiments on cell-cycle exit and re-entry, we found that hMSCs demonstrated a sharp transition from quiescence to proliferation between 1 and 5 kPa. Further studies with ROCK inhibitor Y-27632 revealed that contractile proteins, but not cell spread area, accounts for the sensitivity of hMSCs towards substrate stiffness and hence correlates with their changes in cell cycle. These observations therefore suggest that substrate stiffness regulates hMSC proliferation through contractile forces as generated by cellular contractile proteins in a unique pattern which is distinct from other cell types as studied.

Cardiac sarcomere mechanics in health and disease

The sarcomere is the fundamental structural and functional unit of striated muscle and is directly responsible for most of its mechanical properties. The sarcomere generates active or contractile forces and determines the passive or elastic properties of striated muscle. In the heart, mutations in sarcomeric proteins are responsible for the majority of genetically inherited cardiomyopathies. Here, we review the major determinants of cardiac sarcomere mechanics including the key structural components that contribute to active and passive tension. We dissect the molecular and structural basis of active force generation, including sarcomere composition, structure, activation, and relaxation. We then explore the giant sarcomere-resident protein titin, the major contributor to cardiac passive tension. We discuss sarcomere dynamics exemplified by the regulation of titin-based stiffness and the titin life cycle. Finally, we provide an overview of therapeutic strategies that target the sarcomere to improve cardiac contraction and filling.

Narrowing the Wide Tip via Endonasal Approach

Rhinoplasty is arguably the most complex and intricate surgery performed by facial plastic surgeons. Nasal tip refinement of a broad nasal tip has remained the most challenging part of rhinoplasty as sophisticated techniques are critical to achieve aesthetically pleasing and structurally sound nasal tips that can withstand the contractile forces of healing. Successful tip refinement relies on an in-depth preoperative and intraoperative understanding of the patient's nasal anatomy, well developed arsenal of techniques, the experience of the surgeon, and the aesthetic desires of the patient. Although the approach to gain access to the nasal tip so as to successfully reshape the tip has been a topic of debate over many years, the aim of this article is to outline and demonstrate how the broad nasal tip can be successfully recontoured through an endonasal approach using nondestructive techniques that have been effectively used in open rhinoplasty. We believe that there continues to be a place for endonasal tip rhinoplasty especially in this era in which patients desire less invasive procedures with shorter healing time.

Structural analysis of receptors and actin polarity in platelet protrusions

During activation the platelet cytoskeleton is reorganized, inducing adhesion to the extracellular matrix and cell spreading. These processes are critical for wound healing and clot formation. Initially, this task relies on the formation of strong cellular–extracellular matrix interactions, exposed in subendothelial lesions. Despite the medical relevance of these processes, there is a lack of high-resolution structural information on the platelet cytoskeleton controlling cell spreading and adhesion. Here, we present in situ structural analysis of membrane receptors and the underlying cytoskeleton in platelet protrusions by applying cryoelectron tomography to intact platelets. We utilized three-dimensional averaging procedures to study receptors at the plasma membrane. Analysis of substrate interaction-free receptors yielded one main structural class resolved to 26 Å, resembling the αIIbβ3 integrin folded conformation. Furthermore, structural analysis of the actin network in pseudopodia indicates a nonuniform polarity of filaments. This organization would allow generation of the contractile forces required for integrin-mediated cell adhesion.

Storage temperature determines platelet GPVI levels and function in mice and humans

Platelets are currently stored at room temperature before transfusion to maximize circulation time. This approach has numerous downsides, including limited storage duration, bacterial growth risk, and increased costs. Cold storage could alleviate these problems. However, the functional consequences of cold exposure for platelets are poorly understood. In the present study, we compared the function of cold-stored platelets (CSP) and room temperature-stored platelets (RSP) in vitro, in vivo, and post-transfusion. CSP formed larger aggregates under in vitro shear while generating similar contractile forces compared to RSP. We found significantly reduced GPVI levels after cold exposure of 5-7 days. After transfusion in humans, CSP were mostly equivalent to RSP yet aggregated significantly less to the GPVI agonist collagen. In a mouse model of platelet transfusion, we found a significantly lower response to the GPVI-dependent agonist convulxin and significantly lower GPVI levels on the surface of transfused platelets after cold storage. In summary, our data support an immediate but short-lived benefit of CSP and highlight the need for thorough investigations of this product. (NCT03787927)

Mechanical competition alters the cellular interpretation of an endogenous genetic program

The intrinsic genetic program of a cell is not sufficient to explain all of the cell’s activities. External mechanical stimuli are increasingly recognized as determinants of cell behavior. In the epithelial folding event that constitutes the beginning of gastrulation in Drosophila, the genetic program of the future mesoderm leads to the establishment of a contractile actomyosin network that triggers apical constriction of cells and thereby tissue folding. However, some cells do not constrict but instead stretch, even though they share the same genetic program as their constricting neighbors. We show here that tissue-wide interactions force these cells to expand even when an otherwise sufficient amount of apical, active actomyosin is present. Models based on contractile forces and linear stress–strain responses do not reproduce experimental observations, but simulations in which cells behave as ductile materials with nonlinear mechanical properties do. Our models show that this behavior is a general emergent property of actomyosin networks in a supracellular context, in accordance with our experimental observations of actin reorganization within stretching cells.