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The conformational state of Tes regulates its zyxin-dependent recruitment to focal adhesions.

Garvalov BK, Higgins TE, Sutherland JD, Zettl M, Scaplehorn N, Köcher T, Piddini E, Griffiths G, Way M - J. Cell Biol. (2003)

Bottom Line: The COOH-terminal half recruits zyxin as well as Mena and VASP from cell extracts.These differences suggest that the ability of Tes to associate with alpha-actinin, paxillin, and zyxin is dependent on the conformational state of the molecule.Consistent with this hypothesis, we demonstrate that the two halves of Tes interact with each other in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, D-69117 Heidelberg, Germany.

ABSTRACT
The function of the human Tes protein, which has extensive similarity to zyxin in both sequence and domain organization, is currently unknown. We now show that Tes is a component of focal adhesions that, when expressed, negatively regulates proliferation of T47D breast carcinoma cells. Coimmunoprecipitations demonstrate that in vivo Tes is complexed with actin, Mena, and vasodilator-stimulated phosphoprotein (VASP). Interestingly, the isolated NH2-terminal half of Tes pulls out alpha-actinin and paxillin from cell extracts in addition to actin. The COOH-terminal half recruits zyxin as well as Mena and VASP from cell extracts. These differences suggest that the ability of Tes to associate with alpha-actinin, paxillin, and zyxin is dependent on the conformational state of the molecule. Consistent with this hypothesis, we demonstrate that the two halves of Tes interact with each other in vitro and in vivo. Using fibroblasts lacking Mena and VASP, we show that these proteins are not required to recruit Tes to focal adhesions. However, using RNAi ablation, we demonstrate that zyxin is required to recruit Tes, as well as Mena and VASP, but not vinculin or paxillin, to focal adhesions.

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Analysis of the cellular localization of GFP-tagged Tes domains. Immunofluorescence analysis of HeLa cells expressing the indicated GFP-Tes colabeled for paxillin and phalloidin. The NH2-terminal half of Tes (Tes-N-term) is associated with actin stress fibers, lamellipodia, and focal adhesions. The COOH-terminal half of Tes (Tes-C-term) is largely observed at focal adhesions and weakly observed along stress fibers. The LIM1, but not the LIM2 (not shown) or LIM3 domain, is strongly recruited to focal adhesions and weakly recruited along stress fibers. Disruption of LIM1 (Tes-C265A) or LIM2 (Tes-C328A) (not depicted) in full-length Tes does not affect recruitment of the protein to focal adhesions but results in increased localization along stress fibers. Disruption of the LIM3 domain (Tes-C391A) results in a diffuse cytoplasmic localization. Bar, 20 μm.
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fig2: Analysis of the cellular localization of GFP-tagged Tes domains. Immunofluorescence analysis of HeLa cells expressing the indicated GFP-Tes colabeled for paxillin and phalloidin. The NH2-terminal half of Tes (Tes-N-term) is associated with actin stress fibers, lamellipodia, and focal adhesions. The COOH-terminal half of Tes (Tes-C-term) is largely observed at focal adhesions and weakly observed along stress fibers. The LIM1, but not the LIM2 (not shown) or LIM3 domain, is strongly recruited to focal adhesions and weakly recruited along stress fibers. Disruption of LIM1 (Tes-C265A) or LIM2 (Tes-C328A) (not depicted) in full-length Tes does not affect recruitment of the protein to focal adhesions but results in increased localization along stress fibers. Disruption of the LIM3 domain (Tes-C391A) results in a diffuse cytoplasmic localization. Bar, 20 μm.

Mentions: To determine which region of Tes is responsible for recruitment of the protein to focal adhesions, we examined the localization of a series of GFP-tagged Tes constructs (Fig. 1 A). We found that the NH2 terminus of the protein was recruited to stress fibers and focal adhesions (Fig. 2 ; and Fig. S2, available at www.jcb.org/cgi/content/full/jcb.200211015/DC1). This half of Tes, in contrast to the full-length Tes or COOH-terminal half of the protein was also recruited to actin-rich ruffles and lamellipodia as well as Shigella and vaccinia induced actin tails (Fig. 2; unpublished data). The COOH-terminal half of the molecule, corresponding to the three LIM domains, was strongly concentrated at focal adhesions and weakly observed along stress fibers (Fig. 2; and Fig. S2). To further delineate if a single LIM domain is required for recruitment to focal adhesions, we expressed individual GFP-tagged LIM domains in cells (Fig. 1 A). Immunofluorescence analysis of transfected cells revealed that only GFP-LIM1 is recruited to focal adhesions and also observed weakly along stress fibers (Figs. 1 A and 2; and Fig. S2). Disruption of the LIM1 domain by the introduction of a single point mutation (C265A) completely abolished this recruitment (Fig. 1 A). Likewise, only the absence of a functional LIM1 domain in the isolated COOH-terminal half of the molecule resulted in a loss of focal adhesion recruitment (Fig. 1 A). Unexpectedly, when the same point mutations were introduced into full-length Tes, we found that disruption of LIM1 or LIM2 had no effect on the targeting of Tes to focal adhesions (Figs. 1 A and 2). These mutations did, however, result in a noticeable recruitment of Tes to stress fibers (Fig. 2; and Fig. S2). In contrast, disruption of LIM3 led to a diffuse cytoplasmic localization and an absence of recruitment to focal adhesions (Fig. 2). Our observations indicate that, although the LIM3 domain is required for targeting, interaction sites in the NH2-terminal half and LIM1 contribute to recruitment and stabilization of Tes at focal adhesions.


The conformational state of Tes regulates its zyxin-dependent recruitment to focal adhesions.

Garvalov BK, Higgins TE, Sutherland JD, Zettl M, Scaplehorn N, Köcher T, Piddini E, Griffiths G, Way M - J. Cell Biol. (2003)

Analysis of the cellular localization of GFP-tagged Tes domains. Immunofluorescence analysis of HeLa cells expressing the indicated GFP-Tes colabeled for paxillin and phalloidin. The NH2-terminal half of Tes (Tes-N-term) is associated with actin stress fibers, lamellipodia, and focal adhesions. The COOH-terminal half of Tes (Tes-C-term) is largely observed at focal adhesions and weakly observed along stress fibers. The LIM1, but not the LIM2 (not shown) or LIM3 domain, is strongly recruited to focal adhesions and weakly recruited along stress fibers. Disruption of LIM1 (Tes-C265A) or LIM2 (Tes-C328A) (not depicted) in full-length Tes does not affect recruitment of the protein to focal adhesions but results in increased localization along stress fibers. Disruption of the LIM3 domain (Tes-C391A) results in a diffuse cytoplasmic localization. Bar, 20 μm.
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Related In: Results  -  Collection

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fig2: Analysis of the cellular localization of GFP-tagged Tes domains. Immunofluorescence analysis of HeLa cells expressing the indicated GFP-Tes colabeled for paxillin and phalloidin. The NH2-terminal half of Tes (Tes-N-term) is associated with actin stress fibers, lamellipodia, and focal adhesions. The COOH-terminal half of Tes (Tes-C-term) is largely observed at focal adhesions and weakly observed along stress fibers. The LIM1, but not the LIM2 (not shown) or LIM3 domain, is strongly recruited to focal adhesions and weakly recruited along stress fibers. Disruption of LIM1 (Tes-C265A) or LIM2 (Tes-C328A) (not depicted) in full-length Tes does not affect recruitment of the protein to focal adhesions but results in increased localization along stress fibers. Disruption of the LIM3 domain (Tes-C391A) results in a diffuse cytoplasmic localization. Bar, 20 μm.
Mentions: To determine which region of Tes is responsible for recruitment of the protein to focal adhesions, we examined the localization of a series of GFP-tagged Tes constructs (Fig. 1 A). We found that the NH2 terminus of the protein was recruited to stress fibers and focal adhesions (Fig. 2 ; and Fig. S2, available at www.jcb.org/cgi/content/full/jcb.200211015/DC1). This half of Tes, in contrast to the full-length Tes or COOH-terminal half of the protein was also recruited to actin-rich ruffles and lamellipodia as well as Shigella and vaccinia induced actin tails (Fig. 2; unpublished data). The COOH-terminal half of the molecule, corresponding to the three LIM domains, was strongly concentrated at focal adhesions and weakly observed along stress fibers (Fig. 2; and Fig. S2). To further delineate if a single LIM domain is required for recruitment to focal adhesions, we expressed individual GFP-tagged LIM domains in cells (Fig. 1 A). Immunofluorescence analysis of transfected cells revealed that only GFP-LIM1 is recruited to focal adhesions and also observed weakly along stress fibers (Figs. 1 A and 2; and Fig. S2). Disruption of the LIM1 domain by the introduction of a single point mutation (C265A) completely abolished this recruitment (Fig. 1 A). Likewise, only the absence of a functional LIM1 domain in the isolated COOH-terminal half of the molecule resulted in a loss of focal adhesion recruitment (Fig. 1 A). Unexpectedly, when the same point mutations were introduced into full-length Tes, we found that disruption of LIM1 or LIM2 had no effect on the targeting of Tes to focal adhesions (Figs. 1 A and 2). These mutations did, however, result in a noticeable recruitment of Tes to stress fibers (Fig. 2; and Fig. S2). In contrast, disruption of LIM3 led to a diffuse cytoplasmic localization and an absence of recruitment to focal adhesions (Fig. 2). Our observations indicate that, although the LIM3 domain is required for targeting, interaction sites in the NH2-terminal half and LIM1 contribute to recruitment and stabilization of Tes at focal adhesions.

Bottom Line: The COOH-terminal half recruits zyxin as well as Mena and VASP from cell extracts.These differences suggest that the ability of Tes to associate with alpha-actinin, paxillin, and zyxin is dependent on the conformational state of the molecule.Consistent with this hypothesis, we demonstrate that the two halves of Tes interact with each other in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, D-69117 Heidelberg, Germany.

ABSTRACT
The function of the human Tes protein, which has extensive similarity to zyxin in both sequence and domain organization, is currently unknown. We now show that Tes is a component of focal adhesions that, when expressed, negatively regulates proliferation of T47D breast carcinoma cells. Coimmunoprecipitations demonstrate that in vivo Tes is complexed with actin, Mena, and vasodilator-stimulated phosphoprotein (VASP). Interestingly, the isolated NH2-terminal half of Tes pulls out alpha-actinin and paxillin from cell extracts in addition to actin. The COOH-terminal half recruits zyxin as well as Mena and VASP from cell extracts. These differences suggest that the ability of Tes to associate with alpha-actinin, paxillin, and zyxin is dependent on the conformational state of the molecule. Consistent with this hypothesis, we demonstrate that the two halves of Tes interact with each other in vitro and in vivo. Using fibroblasts lacking Mena and VASP, we show that these proteins are not required to recruit Tes to focal adhesions. However, using RNAi ablation, we demonstrate that zyxin is required to recruit Tes, as well as Mena and VASP, but not vinculin or paxillin, to focal adhesions.

Show MeSH
Related in: MedlinePlus