Cellular force assay detects altered contractility caused by a nephritis‐associated mutation in nonmuscle myosin IIA

Abstract

Recent progress in understanding the essential roles of mechanical forces in regulating various cellular processes expands the field of biology to one where interdisciplinary approaches with engineering techniques become indispensable. Contractile forces or contractility-inherently present in proliferative cells due to the activity of ubiquitous nonmuscle myosin II (NMII)-are one of such mechano-regulators, but because NMII works downstream of diverse signaling pathways, it is often difficult to predict how the inherent cellular forces change upon perturbations to particular molecules. Here, we determine whether the contractility of individual cells is upregulated or downregulated based on an assay analyzing specific deformations of silicone gel substrates. We focus on the effect of mutations in the human MYH9 gene that encodes NMIIA, which have been implicated in the pathogenesis of various diseases including nephritis. Our assay equipped with a high-throughput data analysis capability reveals that a point mutation of E1841K but not I1816V significantly reduces the magnitude of the endogenous forces of human embryonic kidney (HEK293) cells. Given the increasingly recognized roles of the endogenous forces as a critical mechano-regulator as well as that no apparent morphological changes were induced to cells even by introducing the mutations, our findings suggest a possibility that the detected reduction in the force magnitude at the individual cellular level may underlie the pathogenesis of the kidney disease.

Wrinkle force microscopy: a new machine learning based approach to predict cell mechanics from images

“…The average traction is 50.3 ± 57.1 [Pa] (mean ± standard deviation). Figure 3(b) shows that the wrinkle length has a positive correlation with the mean traction of the images, which is in agreement with our previous experimental measurements (22), where the relationship between the wrinkle length and applied force was experimentally investigated using microneedles. The mean traction is simply obtained by averaging the norm of the traction of the image as…”

“…Based on our previous studies (16, 19, 22–24), we prepared the substrate that can reversibly generate wrinkles upon application of cellular forces. Firstly, a circular cover glass is treated with oxygen plasma (SEDE-GE, Meiwafosis) to hydrophilize the surface and is desiccated after fluorescent micro-beads (0.2 μ m in diameter, carboxylate yellow-green fluorescent beads; Invitrogen) in water solution are distributed on the surface.…”

“… where m is the index of the observation points. The wrinkle length is measured by counting the number of pixels after skeletonizing the wrinkle images (22). The wrinkle extincts when the mean traction in a image is less than 10 Pa, which is comparable to the noise level or the resolution of the current TFM.…”

“…As shown in Fig. 1(a), the cellular contractile forces are obtained using the traction force microscopy (TFM) (13), and cells generate wrinkles because the surface of PDMS layer is harden by the plasma irradiation (9,12,(14)(15)(16). Second, the wrinkle positions are extracted from the microscope images as shown in Fig.…”

Association betweenMYH9andAPOL1Gene Polymorphisms and the Risk of Diabetic Kidney Disease in Patients with Type 2 Diabetes in a Chinese Han Population

“…The MYH9 gene located on chromosome 22 q12.3-13.2 encodes nonmuscle myosin IIA. The approximately 224 kDa protein is widely expressed in most cells in the body [ 4 , 5 ]. Polymorphisms in MYH9 have been strongly associated with ESRD according to genome-wide association studies (GWAS), including human immunodeficiency virus- (HIV-) associated nephropathy in African Americans and idiopathic focal segmental glomerulosclerosis in European Americans and Hispanic Americans.…”