Actomyosin contractility is an essential element of many aspects of cellular biology and manifests as traction forces that cells exert on their surroundings. The central role of these forces makes them a novel principal therapeutic target in diverse diseases. This requires accurate and higher-capacity measurements of traction forces; however, existing methods are largely low throughput, limiting their utility in broader applications. To address this need, we employ Fourier-transform traction force microscopy in a parallelized 96-well format, which we refer to as contractile force screening. Critically, rather than the frequently employed hydrogel polyacrylamide, we fabricate these plates using polydimethylsiloxane rubber. Key to this approach is that the polydimethylsiloxane used is very compliant, with a lower-bound Young's modulus of ∼0.4 kPa. We subdivide these monolithic substrates spatially into biochemically independent wells, creating a uniform multiwell platform for traction force screening. We demonstrate the utility and versatility of this platform by quantifying the compound and dose-dependent contractility responses of human airway smooth muscle cells and retinal pigment epithelial cells. By directly quantifying the endpoint of therapeutic intent, airway-smooth-muscle contractile force, this approach fills an important methodological void in current screening approaches for bronchodilator drug discovery, and, more generally, in measuring contractile response for a broad range of cell types and pathologies.

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8Summary 9YAP is a key mechanotransduction protein with essential roles in diverse physiological processes. 10Dysregulation in YAP activity is associated with multiple diseases such as atherosclerosis, fibrosis, 11and cancer progression. Here we examine the physical stimuli that regulate dynamic YAP 12 translocation to the nucleus. Through a combination of biophysical studies, we demonstrate that 13 YAP localization is insensitive to cell substrate stiffness, but strongly determined by cellular 14contractile work, which in turn deforms the nucleus. We show that nuclear deformation from 15 LINC-mediated cytoskeletal contractility or extracellular osmotic forces triggers YAP nuclear 16 localization. By modulating the expression of lamin A and nuclear stiffness, we illustrate that 17 nuclear rigidity modulates deformation-mediated YAP nuclear localization. Finally, we show that 18 nuclear deformation causes relocalization of lamin A from the nuclear membrane to the 19 nucleoplasm, and this is essential in allowing YAP to enter the nucleus. These results reveal key 20 physical nuclear deformation mechanics that drive YAP nuclear import. 21

There is a need for reliable and quantitative real-time assessment of blood properties to study and treat a broad spectrum of disorders and cardiovascular diseases as well as to test the efficacy of hemostatic agents. In this study, the real-time changes in viscoelastic/rheological properties of bovine whole blood during coagulation induced by different concentrations of calcium chloride (CaCl 2 ; 15, 25, 35 and 45 mM) was investigated. For this purpose, a novel, contactless technique was used to accurately measure the clotting characteristics under controlled and sterile conditions. It was demonstrated that, increasing the calcium concentration from low values (i.e., 15 and 25 mM), led to shorter reaction time; however, a further increase in calcium concentration (i.e., 35 and 45 mM) favored longer reaction times. Additionally, increasing the CaCl 2 concentration resulted in higher shear storage modulus (i.e., stiffer clots). These results were also comparable to those generated by thromboelastrograph, a clinically established technique, as well as a conventional rheometer, which quantitatively verified the high correlation of the shear storage modulus data. In sum, the non-destructive testing technique used in this study is reproducible and sensitive in measuring clot formation kinetics, which could be applied to assess the efficacy of hemostatic agents, and may also contribute to better diagnosing relevant circulatory system diseases and conditions.

The sensing and generation of cellular forces are essential aspects of life. Traction force microscopy (TFM) has emerged as a standard broadly applicable methodology to measure cell contractility and its role in cell behavior. While TFM platforms have enabled diverse discoveries, their implementation remains limited in part due to various constraints, such as time-consuming substrate fabrication techniques, the need to detach cells to measure null force images, followed by complex imaging and analysis, and the unavailability of cells for postprocessing. Here we introduce a reference-free technique to measure cell contractile work in real time, with commonly available substrate fabrication methodologies, simple imaging, and analysis with the availability of the cells for postprocessing. In this technique, we confine the cells on fluorescent adhesive protein micropatterns of a known area on compliant silicone substrates and use the cell deformed pattern area to calculate cell contractile work. We validated this approach by comparing this pattern-based contractility screening (PaCS) with conventional bead-displacement TFM and show quantitative agreement between the methodologies. Using this platform, we measure the contractile work of highly metastatic MDA-MB-231 breast cancer cells that is significantly higher than the contractile work of noninvasive MCF-7 cells. PaCS enables the broader implementation of contractile work measurements in diverse quantitative biology and biomedical applications.