Force-induced focal adhesion translocation: effects of force amplitude and frequency

Abstract

. Force-induced focal adhesion translocation: effects of force amplitude and frequency. Am J Physiol Cell Physiol 287: C954 -C962, 2004. First published June 9, 2004 10.1152/ajpcell. 00567.2003.-Vascular endothelial cells rapidly transduce local mechanical forces into biological signals through numerous processes including the activation of focal adhesion sites. To examine the mechanosensing capabilities of these adhesion sites, focal adhesion translocation was monitored over the course of 5 min with GFPpaxillin while applying nN-level magnetic trap shear forces to the cell apex via integrin-linked magnetic beads. A nongraded steady-load threshold for mechanotransduction was established between 0.90 and 1.45 nN. Activation was greatest near the point of forcing (Ͻ7.5 m), indicating that shear forces imposed on the apical cell membrane transmit nonuniformly to the basal cell surface and that focal adhesion sites may function as individual mechanosensors responding to local levels of force. Results from a continuum, viscoelastic finite element model of magnetocytometry that represented experimental focal adhesion attachments provided support for a nonuniform force transmission to basal surface focal adhesion sites. To further understand the role of force transmission on focal adhesion activation and dynamics, sinusoidally varying forces were applied at 0.1, 1.0, 10, and 50 Hz with a 1.45 nN offset and a 2.25 nN maximum. At 10 and 50 Hz, focal adhesion activation did not vary with spatial location, as observed for steady loading, whereas the response was minimized at 1.0 Hz. Furthermore, applying the tyrosine kinase inhibitors genistein and PP2, a specific Src family kinase inhibitor, showed tyrosine kinase signaling has a role in force-induced translocation. These results highlight the mutual importance of force transmission and biochemical signaling in focal adhesion mechanotransduction. mechanotransduction; endothelial cell; paxillin; viscoelastic model MECHANOTRANSDUCTION IS AN essential function of the cell, controlling its growth, proliferation, protein synthesis, and gene expression (8,18). Extensive data exist documenting the cellular responses to external force (15, 41, 44), but less is known about how force affects rapid biological signaling. Although integrins/focal adhesion sites (42), cytoskeleton constituents, G proteins (6), ion channels, intercellular junction proteins, and membrane biomolecules have all been identified as potential mechanosensors (6,16,42,43), we know little about the force level and frequency-dependent thresholds required to initiate mechanotransduction or the role of intracellular force transmission on mechanosensor activation. Biological readouts used to study mechanotransduction range from long-term gene expression and cell morphology changes (8, 26) to rapid variations in intracellular ion concentration and protein activity (8,26,42). Morphological and gene expression comparisons provide a robust marker of mechanotransduction, but the response is slow on the scale of hours, a...

Finite-Element Stress Analysis of a Multicomponent Model of Sheared and Focally-Adhered Endothelial Cells

“…However, stresses near the base of the cell from the bead twisting and pulling were comparable to those resulting from shear stress, a finding consistent with Ref. 34.…”

“…For example, Mack and colleagues recently used finite element analysis to assess force transmission from the translation of embedded beads from the cell surface to FAs. 34 They found that finite element analysis of a continuum model which included a cortical membrane and viscoelastic cytoplasm was able to predict FA stress directions that correlated well with FA translocation.…”

Cellular adaptation to mechanical stress: role of integrins, Rho, cytoskeletal tension and mechanosensitive ion channels

“…The reality is that cells commonly display both temporal and spatial variations in the level of stress they exert on their ECM adhesions (Beningo et al, 2001). The mechanical properties of cells also may differ depending on the frequency of force application (Fabry et al, 2001;Mack et al, 2004;Overby et al, 2005). Moreover, some cells exhibit distinct sensitivities to specific force frequencies (Goldschmidt et al, 2001;Hsieh and Turner, 2001;Coplen et al, 2003;Tanaka et al, 2003).…”

Visualizing the mechanical activation of Src

“…Green fluorescent protein (GFP)-tagged fluorescent markers have been used to study the displacement of cellular organelles and the formation of a focal adhesion complex induced by mechanical stimuli [22][23][24][25] , but these inert fluorescence markers cannot monitor the dynamic signal transduction process. Our FRET-based Src reporter enables the visualization and quantification of the mechano-activated Src with high temporal and spatial resolution in live cells.…”