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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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Stretch-induced kinetics of GFP-zyxin, effects of inhibitors, and VASP localization. (A) Immunoblot analysis with antizyxin antibody in wild-type, zyxin −/−, and zyxin −/− cells expressing full-length GFP-tagged zyxin (GFP-zyx). GFP-zyxin was expressed at a level that was comparable to wild-type cells. FAK immunoblot illustrates equal protein loading. (B) Direct visualization of GFP-zyxin in unstretched and stretched cells. (C) Kinetics of zyxin mobilization to stress fibers in response to stretch. By using a live cell stretching system, GFP-zyxin signals were recorded during 150 s of cyclic stretching. Arrowheads indicate GFP-zyxin accumulation along actin filaments. (D) Cells were allowed to spread on poly-d-lysine–coated silicone for 3 h, stretched for 60 min, and immunostained to detect zyxin. (E) Cells were stretched in the presence of Gd3+, and zyxin was localized by immunostaining. (F) Wild-type (+/+), zyxin- (−/−), and zyxin- cells expressing GFP-zyxin were stimulated by cyclic stretch (+; 1 h at 15% and 0.5 Hz) and were immunostained to detect VASP.
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fig3: Stretch-induced kinetics of GFP-zyxin, effects of inhibitors, and VASP localization. (A) Immunoblot analysis with antizyxin antibody in wild-type, zyxin −/−, and zyxin −/− cells expressing full-length GFP-tagged zyxin (GFP-zyx). GFP-zyxin was expressed at a level that was comparable to wild-type cells. FAK immunoblot illustrates equal protein loading. (B) Direct visualization of GFP-zyxin in unstretched and stretched cells. (C) Kinetics of zyxin mobilization to stress fibers in response to stretch. By using a live cell stretching system, GFP-zyxin signals were recorded during 150 s of cyclic stretching. Arrowheads indicate GFP-zyxin accumulation along actin filaments. (D) Cells were allowed to spread on poly-d-lysine–coated silicone for 3 h, stretched for 60 min, and immunostained to detect zyxin. (E) Cells were stretched in the presence of Gd3+, and zyxin was localized by immunostaining. (F) Wild-type (+/+), zyxin- (−/−), and zyxin- cells expressing GFP-zyxin were stimulated by cyclic stretch (+; 1 h at 15% and 0.5 Hz) and were immunostained to detect VASP.

Mentions: To validate that the altered distribution of zyxin in response to stretch accurately reflects a physiological change and does not result from altered antibody accessibility in the stretched cells, we examined the impact of cyclic stretch on the distribution of GFP-zyxin that was expressed in fibroblasts derived from zyxin −/− mice (Fig. 3 A; Hoffman et al., 2003). The phenotype of zyxin −/− fibroblasts will be described in detail elsewhere (unpublished data). Direct visualization of GFP-zyxin revealed strong zyxin accumulation in focal adhesions before stretch and a mobilization of zyxin to stress fibers with a corresponding reduction in the focal adhesion resident zyxin after stretch (Fig. 3 B). Collectively, these data identify zyxin as a mechanosensitive protein at cell–substratum attachment sites. Given this evidence that the subcellular distribution of zyxin is dynamic and sensitive to physical cues, the validity of using GFP-zyxin as a focal adhesion marker should be reevaluated.


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)

Stretch-induced kinetics of GFP-zyxin, effects of inhibitors, and VASP localization. (A) Immunoblot analysis with antizyxin antibody in wild-type, zyxin −/−, and zyxin −/− cells expressing full-length GFP-tagged zyxin (GFP-zyx). GFP-zyxin was expressed at a level that was comparable to wild-type cells. FAK immunoblot illustrates equal protein loading. (B) Direct visualization of GFP-zyxin in unstretched and stretched cells. (C) Kinetics of zyxin mobilization to stress fibers in response to stretch. By using a live cell stretching system, GFP-zyxin signals were recorded during 150 s of cyclic stretching. Arrowheads indicate GFP-zyxin accumulation along actin filaments. (D) Cells were allowed to spread on poly-d-lysine–coated silicone for 3 h, stretched for 60 min, and immunostained to detect zyxin. (E) Cells were stretched in the presence of Gd3+, and zyxin was localized by immunostaining. (F) Wild-type (+/+), zyxin- (−/−), and zyxin- cells expressing GFP-zyxin were stimulated by cyclic stretch (+; 1 h at 15% and 0.5 Hz) and were immunostained to detect VASP.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171187&req=5

fig3: Stretch-induced kinetics of GFP-zyxin, effects of inhibitors, and VASP localization. (A) Immunoblot analysis with antizyxin antibody in wild-type, zyxin −/−, and zyxin −/− cells expressing full-length GFP-tagged zyxin (GFP-zyx). GFP-zyxin was expressed at a level that was comparable to wild-type cells. FAK immunoblot illustrates equal protein loading. (B) Direct visualization of GFP-zyxin in unstretched and stretched cells. (C) Kinetics of zyxin mobilization to stress fibers in response to stretch. By using a live cell stretching system, GFP-zyxin signals were recorded during 150 s of cyclic stretching. Arrowheads indicate GFP-zyxin accumulation along actin filaments. (D) Cells were allowed to spread on poly-d-lysine–coated silicone for 3 h, stretched for 60 min, and immunostained to detect zyxin. (E) Cells were stretched in the presence of Gd3+, and zyxin was localized by immunostaining. (F) Wild-type (+/+), zyxin- (−/−), and zyxin- cells expressing GFP-zyxin were stimulated by cyclic stretch (+; 1 h at 15% and 0.5 Hz) and were immunostained to detect VASP.
Mentions: To validate that the altered distribution of zyxin in response to stretch accurately reflects a physiological change and does not result from altered antibody accessibility in the stretched cells, we examined the impact of cyclic stretch on the distribution of GFP-zyxin that was expressed in fibroblasts derived from zyxin −/− mice (Fig. 3 A; Hoffman et al., 2003). The phenotype of zyxin −/− fibroblasts will be described in detail elsewhere (unpublished data). Direct visualization of GFP-zyxin revealed strong zyxin accumulation in focal adhesions before stretch and a mobilization of zyxin to stress fibers with a corresponding reduction in the focal adhesion resident zyxin after stretch (Fig. 3 B). Collectively, these data identify zyxin as a mechanosensitive protein at cell–substratum attachment sites. Given this evidence that the subcellular distribution of zyxin is dynamic and sensitive to physical cues, the validity of using GFP-zyxin as a focal adhesion marker should be reevaluated.

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