By observing the dynamics of SFs in living cells, we identified novel acute, local SF damage events that occur spontaneously in response to increasing intrinsic stress within the fibers, or in response to mechanical perturbation, and defined a molecular mechanism for rapid SF repair and homeostasis of the actin cytoskeleton. SFs that exhibit acute, local strain events display a transient reduction of force transmitted though associated FAs. Zyxin rapidly accumulates at acute SF strain sites where free barbed ends of actin filaments are concentrated, and zyxin facilitates the recruitment of binding partners, VASP and α-actinin, which together comprise a repair complex that effects repair and mechanical stabilization of the SF. In the absence of zyxin, the incidence of catastrophic SF breaks is dramatically increased, and the capacity of the associated FAs to transmit forces to the substrate and retract a surrounding collagen gel is compromised (). The SF repair process restores the capacity of the SF to develop tension and convey traction forces to the ECM. Thus, SF strain events rapidly relieve cytoskeletal prestress, while simultaneously providing a clear target for a system that rebuilds the weakest region of the fiber, thereby enabling the cytoskeleton to tolerate dynamic increases in force load by sensing and repairing SF damage prior to failure. Without this repair system, cells exhibit a marked reduction in their ability to transmit force to remodel ECM, a process critical to tissue morphogenesis, maintenance and repair.
Both the direct application of mechanical stimulation or conditions of elevated internal SF contractility are sufficient to induce SF strain events. The zyxin-mediated repair complex is recruited within several seconds of the local strain event, suggesting a model in which new zyxin binding sites are generated at the SF strain site. Both FRAP and photoactivation of fluorescent zyxin (data not shown) indicate that zyxin on strain sites is rapidly exchanging with a cytoplasmic pool. However, how binding sites are created is not clear. While it has been proposed previously that a conformational change in α-actinin could be responsible for stress-induced recruitment of zyxin to SFs (Colombelli et al., 2009
), our data indicate that zyxin accumulation at strain sites is independent of α-actinin binding and precedes α-actinin accumulation by tens of seconds. Alternatively, it is possible that local actin filament breakage and the resultant generation of free filament barbed ends which are concentrated at SF strain sites triggers recruitment of zyxin. Indeed, zyxin is present at FAs where actin filament barbed ends are concentrated and has been shown to accumulate at sites of laser-induced SF severing (Colombelli et al., 2009
). Although a direct zyxin-actin interaction has not been reported, zyxin could be recruited to free barbed ends at strain sites as a co-complex with a barbed-end capping protein. Alternatively, strain-induced changes in the actin polymer or an associated protein other than α-actinin could reveal a new docking site for zyxin on the SF. Mechanical stimulation can reveal new epitopes on cytoskeleton-associated proteins, including talin (Lee et al., 2007
) and p130Cas (Sawada et al., 2006
). Zyxin accumulation could also be regulated by mechanically-induced local activation of kinases (Tamada et al., 2004
; Wang et al., 2005
), as is the movement of zyxin from FAs onto associated SF termini (Guo and Wang, 2007
Independent of the mechanism of zyxin recruitment, our data indicate that both α-actinin and VASP participate in stabilizing elongation of acute SF strain sites. The presence of actin barbed ends throughout the SF strain site suggests that SF repair is at least in part the result of actin polymerization. Indeed, zyxin and VASP have been shown to facilitate actin polymerization (Barzik et al., 2005
; Fradelizi et al., 2001
; Hirata et al., 2008
). However, within the sensitivity of our detection and analysis system, we did not observe a role for VASP in the restoration of actin at SF strain sites. Rather, VASP appeared to be coordinately recruited with zyxin to SF strain sites where it served to enhance the rate of zyxin accumulation, perhaps stabilizing a conformation that is compatible with its recruitment. The ability of α-actinin to crosslink actin filaments suggests that recruitment and bundling of preexisting actin polymer from the cytoplasm may restore actin density of the SF structure to restore mechanical integrity.
Cells that lack zyxin have enhanced migration relative to their wild-type counterparts (Hoffman et al., 2006
), however the reason for this altered behavior was not understood. The loss of zyxin’s contribution to the maintenance and structural stability of SFs in the knockout cells may contribute to this phenotype. In particular, it has long been appreciated that stationary cells are characterized by the presence of robust SFs whereas migratory cells display a much less robust complement of SFs. The increased migration velocity of zyxin (−/ −) cells may be attributable, at least in part, to the reduced stability and integrity of actin SFs in the null cells, a condition that would be expected to support enhanced migratory capacity.
Homeostasis is a central concept in animal physiology that describes the ability of living organisms to maintain constancy of their internal environment. Prior work extended this paradigm to the subcellular level, where it is evident in the maintenance of organelles such as the endoplasmic reticulum (Cox et al., 1993
; Kozutsumi et al., 1988
) and to macromolecular complexes such as the genetic material (Branzei and Foiani, 2008
). The novel mechanism of strain recognition and repair presented here demonstrates a cellular machinery for rapid adjustment of cytoskeletal tension in response to changes in cell contractility or extrinsic forces. The demonstration that SFs display an intrinsic capacity for self-monitoring and repair in response to mechanical stress further extends the concept of homeostasis to the cytoskeleton for the regulation of both cytoarchitecture and mechanical output.