Measurements of strain on single stress fibers in living endothelial cells induced by fluid shear stress Yosuke Ueki a,b, * , Yuhei Uda c , Naoya Sakamoto a , Masaaki Sato a,c a Department of Bioengineering and Robotics, Graduate School of Engineering, Tohoku University, Japan b Research fellow of Japan Society for the Promotion of Science, Sumitomo-Ichibancho Bldg., 6 Ichibancho, Chiyoda-ku, Tokyo, Japan c Department of Biomedical Engineering, Graduate School of Biomedical Engineering, Tohoku University, 6-6-01, Aramaki-aoba, Aoba-ward, Sendai City, Japan article info Article history: Received 24 March 2010 Available online 10 April 2010 Keywords: Endothelial cell Fluid shear stress Force transmission Cytoskeleton Stress fiber abstract Fluid shear stress (FSS) acting on the apical surface of endothelial cells (ECs) can be sensed by mechano- sensors in adhesive protein complexes found in focal adhesions and intercellular junctions. This sensing occurs via force transmission through cytoskeletal networks. This study quantitatively evaluated the force transmitted through cytoskeletons to the mechano-sensors by measuring the FSS-induced strain on SFs using live-cell imaging for actin stress fibers (SFs). FSS-induced bending of SFs caused the SFs to align perpendicular to the direction of the flow. In addition, the displacement vectors of the SFs were detected using image correlation and the FSS-induced axial strain of the SFs was calculated. The results indicated that FSS-induced strain on SFs spanned the range 0.01–0.1% at FSSs ranging from 2 to 10 Pa. Together with the tensile property of SFs reported in a previous study, the force exerted on SFs was esti- mated to range from several to several tens of pN. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction Endothelial cells (ECs) lining the lumen of blood vessels are con- tinuously exposed to fluid shear stress (FSS) due to blood flow. It is well known that ECs exhibit various functional and morphological responses to FSS [1–4]. Many studies regarding the endothelial adaptation to FSS have recently focused on the mechanism by which ECs sense the FSS. Previous reports suggested that molecules in the protein complexes found in focal adhesions (FAs) [5] or intercellular junctions (IJs) [6,7] serve as the primary mechano- sensors, which convert the mechanical force into biochemical sig- nals in ECs. However, FSS would not directly act on these molecular complexes, which are embedded in the basal or lateral membrane of ECs. Rather, it is believed that the force due to FSS-exposure is transmitted from the apical surface in direct contact with the blood flow, via intracellular components such as actin cytoskeletons [4,8] intracellularly connected to FAs and IJs. Recent reports have dem- onstrated that locally applied forces to the apical area of a cell are transmitted via actin cytoskeletons to the basal side [9]. This in turn induces localized activations of Src and Rac1 at distal points [10,11], which then leads to various cellular responses. Therefore, to understand the mechanism of mechano-responses of ECs to FSS, it is necessary to understand how force transmission via actin cytoskeletons occurs. Previously, it was reported that FSS induces deformation of cytoskeletal networks in ECs, indicating the force is transmitted via cytoskeletons [12–14]. Deguchi et al. [15] reported the tensile property of a single SF, isolated from bovine aortic endothelial cells, which describes the relationship between the tensile force and the axial strain. In addition, they also indicated that there is a preexisting strain of about 20% on a SF in statically cultured living cells. Combining these findings with measurements for the change in strain on SFs should allow estimation of the force transmitted to the mechano-sensors in FAs and IJs in ECs exposed to FSS. Previous studies observed the deformation of cytoskeletal networks, but this is insufficient to elucidate the force transmitted because pas- sive deformation of cytoskeletons cannot be distinguished from their active reorganization. Thus, it is necessary to observe only passive structural deformation of cells in order to analyze the transmitted force quantitatively. We recently developed observational and analytical techniques for separating passive deformation from active cell movement in living ECs exposed to FSS using confocal microscopy and image processing [16]. In this report, we measure the passive change in the length of SFs in ECs exposed to FSS using image correlation, and calculate the incremental strain in order to quantitatively obtain the forces transmitted via SFs. 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.04.051 * Corresponding author at: Department of Bioengineering and Robotics, Graduate School of Engineering, Tohoku University, 6-6-01, Aramaki-aoba, Aoba-ward, Sendai City, Japan. Fax: +81 22 795 6945. E-mail address: ueki@bml.mech.tohoku.ac.jp (Y. Ueki). Biochemical and Biophysical Research Communications 395 (2010) 441–446 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc