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Mechanical force mobilizes zyxin from focal adhesions to actin filaments and regulates cytoskeletal reinforcement.

Yoshigi M, Hoffman LM, Jensen CC, Yost HJ, Beckerle MC - J. Cell Biol. (2005)

Bottom Line: Organs and tissues adapt to acute or chronic mechanical stress by remodeling their actin cytoskeletons.Cells that are stimulated by cyclic stretch or shear stress in vitro undergo bimodal cytoskeletal responses that include rapid reinforcement and gradual reorientation of actin stress fibers; however, the mechanism by which cells respond to mechanical cues has been obscure.Our findings identify zyxin as a mechanosensitive protein and provide mechanistic insight into how cells respond to mechanical cues.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Utah, Salt Lake City, UT 84112, USA. masaaki.yoshigi@hsc.utah.edu

ABSTRACT
Organs and tissues adapt to acute or chronic mechanical stress by remodeling their actin cytoskeletons. Cells that are stimulated by cyclic stretch or shear stress in vitro undergo bimodal cytoskeletal responses that include rapid reinforcement and gradual reorientation of actin stress fibers; however, the mechanism by which cells respond to mechanical cues has been obscure. We report that the application of either unidirectional cyclic stretch or shear stress to cells results in robust mobilization of zyxin from focal adhesions to actin filaments, whereas many other focal adhesion proteins and zyxin family members remain at focal adhesions. Mechanical stress also induces the rapid zyxin-dependent mobilization of vasodilator-stimulated phosphoprotein from focal adhesions to actin filaments. Thickening of actin stress fibers reflects a cellular adaptation to mechanical stress; this cytoskeletal reinforcement coincides with zyxin mobilization and is abrogated in zyxin- cells. Our findings identify zyxin as a mechanosensitive protein and provide mechanistic insight into how cells respond to mechanical cues.

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Zyxin is required for actin stress fiber reinforcement in response to mechanical stimulation. (A) F-actin (phalloidin) in zyxin +/+ and −/− fibroblasts before (−) or after (+) cyclic stretch. Note the thinner stress fibers in stretched zyxin −/− cells. (B) SFTI was measured over a 1-h time course of stretch stimulation. Zyxin- (−/−) fibroblasts showed an impaired actin thickening response (*, P < 0.05). (C) Phalloidin staining and SFTI analysis indicated that the expression of GFP-zyxin in zyxin −/− cells rescued the stress fiber reinforcement response (*, P < 0.05 vs. +/+; †, P < 0.05 vs. −/−). (D) Comparison of the alignment index of zyxin +/+ and −/− cells over an 8-h time course of stretch stimulation. Error bars represent SEM.
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fig5: Zyxin is required for actin stress fiber reinforcement in response to mechanical stimulation. (A) F-actin (phalloidin) in zyxin +/+ and −/− fibroblasts before (−) or after (+) cyclic stretch. Note the thinner stress fibers in stretched zyxin −/− cells. (B) SFTI was measured over a 1-h time course of stretch stimulation. Zyxin- (−/−) fibroblasts showed an impaired actin thickening response (*, P < 0.05). (C) Phalloidin staining and SFTI analysis indicated that the expression of GFP-zyxin in zyxin −/− cells rescued the stress fiber reinforcement response (*, P < 0.05 vs. +/+; †, P < 0.05 vs. −/−). (D) Comparison of the alignment index of zyxin +/+ and −/− cells over an 8-h time course of stretch stimulation. Error bars represent SEM.

Mentions: To investigate mechanically stimulated molecular changes within individual cells, we subjected fibroblasts adhering to ECM on elastic silicone membranes to cyclic stretch. Phalloidin staining of actin filaments after unidirectional cyclic stretch revealed two significant responses: rapid thickening (reinforcement) of actin filaments and gradual reorientation of actin stress fibers that were perpendicular to the stretch axis, which resulted in cell alignment (Fig. 1, A and B). In this study, we have applied novel methods to quantitate the actin cytoskeletal remodeling that occurs in response to stretch. The actin thickening was quantitated based on the “erosion” rank filter to generate a stress fiber thickness index (SFTI) that reflects the thickness of actin filaments in regions, not on certain lines or points. The SFTI analysis demonstrated that 15 min of cyclic stretch induced significant thickening of actin filaments (Fig. 1 A). The gradual actin reorientation was also quantitated by using alignment index analysis (Yoshigi et al., 2003). We observed that cells dramatically reoriented their actin stress fibers from a random to a perpendicular alignment relative to the stretch axis (Fig. 1 B). As demonstrated below (see Fig. 5), our ability to separate and quantitate the two critical responses to stretch—cytoskeletal reinforcement and reorientation—allowed us to evaluate whether the two processes were parallel or sequential and whether they had distinct molecular requirements (Fig. 1 C).


Mechanical force mobilizes zyxin from focal adhesions to actin filaments and regulates cytoskeletal reinforcement.

Yoshigi M, Hoffman LM, Jensen CC, Yost HJ, Beckerle MC - J. Cell Biol. (2005)

Zyxin is required for actin stress fiber reinforcement in response to mechanical stimulation. (A) F-actin (phalloidin) in zyxin +/+ and −/− fibroblasts before (−) or after (+) cyclic stretch. Note the thinner stress fibers in stretched zyxin −/− cells. (B) SFTI was measured over a 1-h time course of stretch stimulation. Zyxin- (−/−) fibroblasts showed an impaired actin thickening response (*, P < 0.05). (C) Phalloidin staining and SFTI analysis indicated that the expression of GFP-zyxin in zyxin −/− cells rescued the stress fiber reinforcement response (*, P < 0.05 vs. +/+; †, P < 0.05 vs. −/−). (D) Comparison of the alignment index of zyxin +/+ and −/− cells over an 8-h time course of stretch stimulation. Error bars represent SEM.
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Related In: Results  -  Collection

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fig5: Zyxin is required for actin stress fiber reinforcement in response to mechanical stimulation. (A) F-actin (phalloidin) in zyxin +/+ and −/− fibroblasts before (−) or after (+) cyclic stretch. Note the thinner stress fibers in stretched zyxin −/− cells. (B) SFTI was measured over a 1-h time course of stretch stimulation. Zyxin- (−/−) fibroblasts showed an impaired actin thickening response (*, P < 0.05). (C) Phalloidin staining and SFTI analysis indicated that the expression of GFP-zyxin in zyxin −/− cells rescued the stress fiber reinforcement response (*, P < 0.05 vs. +/+; †, P < 0.05 vs. −/−). (D) Comparison of the alignment index of zyxin +/+ and −/− cells over an 8-h time course of stretch stimulation. Error bars represent SEM.
Mentions: To investigate mechanically stimulated molecular changes within individual cells, we subjected fibroblasts adhering to ECM on elastic silicone membranes to cyclic stretch. Phalloidin staining of actin filaments after unidirectional cyclic stretch revealed two significant responses: rapid thickening (reinforcement) of actin filaments and gradual reorientation of actin stress fibers that were perpendicular to the stretch axis, which resulted in cell alignment (Fig. 1, A and B). In this study, we have applied novel methods to quantitate the actin cytoskeletal remodeling that occurs in response to stretch. The actin thickening was quantitated based on the “erosion” rank filter to generate a stress fiber thickness index (SFTI) that reflects the thickness of actin filaments in regions, not on certain lines or points. The SFTI analysis demonstrated that 15 min of cyclic stretch induced significant thickening of actin filaments (Fig. 1 A). The gradual actin reorientation was also quantitated by using alignment index analysis (Yoshigi et al., 2003). We observed that cells dramatically reoriented their actin stress fibers from a random to a perpendicular alignment relative to the stretch axis (Fig. 1 B). As demonstrated below (see Fig. 5), our ability to separate and quantitate the two critical responses to stretch—cytoskeletal reinforcement and reorientation—allowed us to evaluate whether the two processes were parallel or sequential and whether they had distinct molecular requirements (Fig. 1 C).

Bottom Line: Organs and tissues adapt to acute or chronic mechanical stress by remodeling their actin cytoskeletons.Cells that are stimulated by cyclic stretch or shear stress in vitro undergo bimodal cytoskeletal responses that include rapid reinforcement and gradual reorientation of actin stress fibers; however, the mechanism by which cells respond to mechanical cues has been obscure.Our findings identify zyxin as a mechanosensitive protein and provide mechanistic insight into how cells respond to mechanical cues.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Utah, Salt Lake City, UT 84112, USA. masaaki.yoshigi@hsc.utah.edu

ABSTRACT
Organs and tissues adapt to acute or chronic mechanical stress by remodeling their actin cytoskeletons. Cells that are stimulated by cyclic stretch or shear stress in vitro undergo bimodal cytoskeletal responses that include rapid reinforcement and gradual reorientation of actin stress fibers; however, the mechanism by which cells respond to mechanical cues has been obscure. We report that the application of either unidirectional cyclic stretch or shear stress to cells results in robust mobilization of zyxin from focal adhesions to actin filaments, whereas many other focal adhesion proteins and zyxin family members remain at focal adhesions. Mechanical stress also induces the rapid zyxin-dependent mobilization of vasodilator-stimulated phosphoprotein from focal adhesions to actin filaments. Thickening of actin stress fibers reflects a cellular adaptation to mechanical stress; this cytoskeletal reinforcement coincides with zyxin mobilization and is abrogated in zyxin- cells. Our findings identify zyxin as a mechanosensitive protein and provide mechanistic insight into how cells respond to mechanical cues.

Show MeSH
Related in: MedlinePlus