Retrograde Fluxes of Focal Adhesion Proteins in Response to Cell Migration and Mechanical Signals

Abstract

Recent studies suggest that mechanical signals mediated by the extracellular matrix play an essential role in various physiological and pathological processes; yet, how cells respond to mechanical stimuli remains elusive. Using live cell fluorescence imaging, we found that actin filaments, in association with a number of focal adhesion proteins, including zyxin and vasodilator-stimulated phosphoprotein, undergo retrograde fluxes at focal adhesions in the lamella region. This flux is inversely related to cell migration, such that it is amplified in fibroblasts immobilized on micropatterned islands. In addition, the flux is regulated by mechanical signals, including stretching forces applied to flexible substrates and substrate stiffness. Conditions favoring the flux share the common feature of causing large retrograde displacements of the interior actin cytoskeleton relative to the substrate anchorage site, which may function as a switch translating mechanical input into chemical signals, such as tyrosine phosphorylation. In turn, the stimulation of actin flux at focal adhesions may function as part of a feedback mechanism, regulating structural assembly and force production in relation to cell migration and mechanical load. The retrograde transport of associated focal adhesion proteins may play additional roles in delivering signals from focal adhesions to the interior of the cell. INTRODUCTIONRecent studies suggest that mechanosensing is involved in several physiological processes, including embryogenesis (Newman and Comper, 1990;Beloussov et al., 1994) and wound healing (Hinz et al., 2001;Tomasek et al., 2002), and in pathological processes such as fibrosis and carcinogenesis (Paszek et al., 2005). Adherent cells seem capable of both responding to applied mechanical forces (Tzima et al., 2005) and applying contractile forces to probe mechanical properties of the environment (Discher et al., 2005). The downstream responses include changes in migration (Pelham and Wang, 1997;Sheetz et al., 1998;Lo et al., 2000), cell-cell interactions (Guo et al., 2006), proliferation (Wang et al., 2000Nelson et al., 2005), differentiation (Engler et al., 2004(Engler et al., , 2006, and apoptosis . For cultured adherent cells, focal adhesions are thought to mediate mechanosensing through integrin-mediated anchorage to the extracellular matrix (Larsen et al., 2006). The molecular repertoire of focal adhesions includes Ͼ100 adaptor and signaling proteins (Bershadsky et al., 2006), in addition to associated actomyosin bundles that provide mechanical forces for probing the environment (Choquet et al., 1997), inside-out signaling (Chrzanowska-Wodnicka and Burridge, 1996;Schwartz and Ginsberg, 2002), and cell migration (Ridley et al., 2003). However, few details are available concerning how these components work in concert to generate the responses to mechanical signals.Several aspects have emerged recently as the key features of integrin-mediated mechanosensing. First is the increase in cortical organization and contractility in resp...

Mechanosensing in actin stress fibers revealed by a close correlation between force and protein localization

“…However, no study has so far reported the formation of an adhesion contact triggered by pure mechanical friction of actin filaments along the plasma membrane. Instead, recent studies have shown the different levels of dynamic correlation between integrins, FA-related proteins such as vinculin and talin, and actin filaments (Guo and Wang, 2007;Brown et al, 2006;Hu et al, 2007). The hierarchical organization of FAs therefore constitutes a differential 'slippage-clutch' mechanism (Wang, 2007), which transmits forces from actin motion to the extracellular matrix to promote cell migration (Hu et al, 2007).…”

“…FAs incorporate a large number and diversity of compounds (Zaidel-bar et al, 2007), thus the precise description of force sensing and transmission in FAs is a great challenge. They assemble or reorganize directionally in response to external forces, as shown by single cell manipulation with micropipettes (Riveline et al, 2001;Guo and Wang, 2007), beads (Wang, 2007) or stretched substrates of adherent cells (Yoshigi et al, 2005;Kim-Kaneyama et al, 2005). Examples of mechanosensitive molecular mechanisms in FAs involve focal adhesion kinase (Mitra et al, 2005), paxillin in focal complexes (Zaidel-Bar et al, 2006) or the Src tyrosine kinase (Wang et al, 2005) and p130…”

“…Cytoplasmic-nuclear shuttling (Nix and Beckerle, 1997) of zyxin indicated a putative link between mechanotransduction and transcription. At the macromolecular level, it responds to external cellular stretch by reorganizing along the cytoskeleton (CSK) to further reinforce it (Yoshigi et al, 2005), correlates with the actin retrograde flux at FAs (Guo and Wang, 2007) …”

Integrins in Cell Migration

“…It is now clear that the molecules that comprise the clutch do not efficiently transmit all of the force to the substratum; but instead there is a molecular slippage (Brown et al 2006;Hu et al 2007). This is seen by the movement of adhesion molecules with actin during retrograde flow (Brown et al 2006;Guo et al 2007;Hu et al 2007). Some adhesion components like aactinin move with a velocity similar to actin, integrin generally does not move, and other components move at intermediate rates.…”

Intracellular Modelling of Cell-Matrix Adhesion during Cancer Cell Invasion

“…This phenomenon is known as retrograde flow. There is a balance between the actin cytoskeleton assembly and the retrograde flow that govern and control cell movement [1,27,54,20].…”