Orientation-specific responses to sustained uniaxial stretching in focal adhesion growth and turnover

Abstract

Cells are mechanosensitive to extracellular matrix (ECM) deformation, which can be caused by muscle contraction or changes in hydrostatic pressure. Focal adhesions (FAs) mediate the linkage between the cell and the ECM and initiate mechanically stimulated signaling events. We developed a stretching apparatus in which cells grown on fibronectin-coated elastic substrates can be stretched and imaged live to study how FAs dynamically respond to ECM deformation. Human bone osteosarcoma epithelial cell line U2OS was transfected with GFP-paxillin as an FA marker and subjected to sustained uniaxial stretching. Two responses at different timescales were observed: rapid FA growth within seconds after stretching, and delayed FA disassembly and loss of cell polarity that occurred over tens of minutes. Rapid FA growth occurred in all cells; however, delayed responses to stretch occurred in an orientation-specific manner, specifically in cells with their long axes perpendicular to the stretching direction, but not in cells with their long axes parallel to stretch. Pharmacological treatments demonstrated that FA kinase (FAK) promotes but Src inhibits rapid FA growth, whereas FAK, Src, and calpain 2 all contribute to delayed FA disassembly and loss of polarity in cells perpendicular to stretching. Immunostaining for phospho-FAK after stretching revealed that FAK activation was maximal at 5 s after stretching, specifically in FAs oriented perpendicular to stretch. We hypothesize that orientation-specific activation of strain/stress-sensitive proteins in FAs upstream to FAK and Src promote orientation-specific responses in FA growth and disassembly that mediate polarity rearrangement in response to sustained stretch.mechanosensing | integrin | tissue mechanics

Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions

“…This is consistent with the observation that stretch induces disassembly of FAs oriented perpendicular to the strain vector and growth of FAs parallel to the strain (35). Such force-induced activation of oriented integrins could mediate direction-dependent mechanosensing that promotes cell polarization in response to stretch and/or blood flow in the vasculature.…”

A FAK-Cas-Rac-Lamellipodin Signaling Module Transduces Extracellular Matrix Stiffness into Mechanosensitive Cell Cycling

“…Sawada et al have demonstrated that stretch-mediated force extends Cas to open the substrate binding domain and permit Src-mediated phosphorylation (17). Chen et al have shown that activation of FAK in focal adhesions is induced by cellular stretching (54). We and others have shown that FAK activation is required for stiffness-sensitive cyclin D1 expression (6, 8, 13).…”

Uniaxial cell stretching device for live-cell imaging of mechanosensitive cellular functions

“…Our observation is in support of the theory of forcemediated FA dynamics 43 and is also consistent with results reported by others. 35 Given its unique capability for external stretch and subcellular live-cell imaging, our device provided a useful platform for studying mechanosensitive subcellular FA dynamics as a critical component of cellular mechanotransduction. …”

“…Numerous cell stretching devices have been developed in previous studies and applied to explore cellular behaviors in response to controlled external stretch. 9,14,17,21,[33][34][35][36][37][38][39][40] However, many of these implementations have significant limitations. With the exception of several state-of-the-art devices, such as the commercial Flexcell systems, which rely heavily on sophisticated pneumatic control systems for regulating air pressure, the regulation on the dynamic stretch is not demonstrated for most vacuum-driven cell stretchers.…”

“…9,33 So far, only a few studies have demonstrated cell stretching devices that are capable of real-time subcellular live-cell imaging, but all with limitations in applying dynamic, especially cyclic, directional stretch to cells. [34][35][36][37]39 Therefore, it is necessary to develop a novel device that can achieve both simple, versatile design of dynamic directional stretch while accessible to subcellular live-cell imaging in situ. In this study, by implementing a cam and follower mechanism for the first time in the development of the cell stretching device, we show that an easy and versatile design strategy can be achieved by using a standard mathematical model that enables the rational design of cam geometry and dimensions for dynamic stretch of desired waveforms.…”