Mechanical forces play an important role in the regulation of physiological responses which affect cell structure and function, including local changes of adhesion sites and cytoskeletons, and alterations in cell motility, proliferation and survival. The traction forces exerted by an adherent cell on the substrate have been studied with traction force microscopy techniques. However, only two dimensional (2D) traction forces tangential to the substrate have been considered in the previous studies although forces are generally three-dimensional (3D) in nature. I have developed a novel technique to measure 3D traction forces, including the normal forces as well as tangential forces, exerted by cultured bovine aortic endothelial cells (BAECs) based on the image processing techniques and finite element method (FEM). Using this method, it has been demonstrated that not only tangential but also normal traction forces are related to the BAEC on the substrate. Upward normal traction force is shown at the edge of the BAEC while downward normal traction force is dominant under the nucleus. Combined with green fluorescent protein (GFP) technique to visualize the focal adhesion (FA) molecules including focal adhesion kinase (FAK) and paxillin, it has been demonstrated that 3D traction force is related to the FA dynamics of BAECs. It has been shown in migrating cells that upward normal traction force is related to the dynamic FAs (FAs that traverse a long distance or undergo turnover), and that downward normal traction force is related to the stable FAs (FAs do not change their position and size). This 3D traction force microscopy technique applied to BAECs and other types of cells provides a new way of assessing the full range of biomechanical dynamics of cells in conjunction with their biochemical activities and can contribute to the understanding of cellular functions in health and disease

The traction forces exerted by an adherent cell on a substrate have been studied only in the two-dimensions (2D) tangential to substrate surface (Txy). We developed a novel technique to measure the three-dimensional (3D) traction forces exerted by live bovine aortic endothelial cells (BAECs) on polyacrylamide deformable substrate. On 3D images acquired by confocal microscopy, displacements were determined with image-processing programs, and traction forces in tangential (XY) and normal (Z) directions were computed by finite element method (FEM). BAECs generated traction force in normal direction (Tz) with an order of magnitude comparable to Txy. Tz is upward at the cell edge and downward under the nucleus, changing continuously with a sign reversal between cell edge and nucleus edge. The method was evaluated regarding accuracy and precision of displacement measurements, effects of FE mesh size, displacement noises, and simple bootstrapping. These results provide new insights into cell-matrix interactions in terms of spatial and temporal variations in traction forces in 3D. This technique can be applied to study live cells to assess their biomechanical dynamics in conjunction with biochemical and functional activities, for investigating cellular functions in health and disease.

Mechanosensation is perhaps the last sensory modality not understood at the molecular level. Ion channels that sense mechanical force are postulated to play critical roles in a variety of biological processes including sensing touch/pain (somatosensation), sound (hearing), and shear stress (cardiovascular physiology); however, the identity of these ion channels has remained elusive. We previously identified Piezo1 and Piezo2 as mechanically activated cation channels that are expressed in many mechanosensitive cell types. Here, we show that Piezo1 is expressed in endothelial cells of developing blood vessels in mice. Piezo1-deficient embryos die at midgestation with defects in vascular remodeling, a process critically influenced by blood flow. We demonstrate that Piezo1 is activated by shear stress, the major type of mechanical force experienced by endothelial cells in response to blood flow. Furthermore, loss of Piezo1 in endothelial cells leads to deficits in stress fiber and cellular orientation in response to shear stress, linking Piezo1 mechanotransduction to regulation of cell morphology. These findings highlight an essential role of mammalian Piezo1 in vascular development during embryonic development.