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Spatial distribution and functional significance of activated vinculin in living cells.

Chen H, Cohen DM, Choudhury DM, Kioka N, Craig SW - J. Cell Biol. (2005)

Bottom Line: However, nothing is known about vinculin's conformation in living cells.Time-lapse imaging reveals a gradient of conformational change that precedes loss of vinculin from focal adhesions in retracting regions.At stable or protruding regions, recruitment of vinculin is not necessarily coupled to the actin-binding conformation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

ABSTRACT
Conformational change is believed to be important to vinculin's function at sites of cell adhesion. However, nothing is known about vinculin's conformation in living cells. Using a Forster resonance energy transfer probe that reports on changes in vinculin's conformation, we find that vinculin is in the actin-binding conformation in a peripheral band of adhesive puncta in spreading cells. However, in fully spread cells with established polarity, vinculin's conformation is variable at focal adhesions. Time-lapse imaging reveals a gradient of conformational change that precedes loss of vinculin from focal adhesions in retracting regions. At stable or protruding regions, recruitment of vinculin is not necessarily coupled to the actin-binding conformation. However, a different measure of vinculin conformation, the recruitment of vinexin beta by activated vinculin, shows that autoinhibition of endogenous vinculin is relaxed at focal adhesions. Beyond providing direct evidence that vinculin is activated at focal adhesions, this study shows that the specific functional conformation correlates with regional cellular dynamics.

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Response of the control FRET probe YC-V1-400 to ligands. (A) Normalized fluorescence emission spectra of cell lysates from HEK 293 cells transfected with the control FRET probe in the absence or in the presence of 1 μM IpaA or 5 μM actin or both. Spectra were normalized to emission at 475 nm of control probe alone. The control probe preserves the IpaA binding site and a focal adhesion targeting signal of vinculin but lacks the actin binding site. It does not display FRET change in response to IpaA binding. (B) Actin cosedimentation assay, performed under the same conditions as in Fig. 2 B, showed that the control probe did not bind to actin filaments under conditions in which tail probe did bind.
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fig3: Response of the control FRET probe YC-V1-400 to ligands. (A) Normalized fluorescence emission spectra of cell lysates from HEK 293 cells transfected with the control FRET probe in the absence or in the presence of 1 μM IpaA or 5 μM actin or both. Spectra were normalized to emission at 475 nm of control probe alone. The control probe preserves the IpaA binding site and a focal adhesion targeting signal of vinculin but lacks the actin binding site. It does not display FRET change in response to IpaA binding. (B) Actin cosedimentation assay, performed under the same conditions as in Fig. 2 B, showed that the control probe did not bind to actin filaments under conditions in which tail probe did bind.

Mentions: For a control probe, we constructed an EYFP-ECFP chimera fused in frame to vinculin residues 1–400 (Fig. 1 A). This probe contains the binding site for IpaA (Bourdet-Sicard et al., 1999) and a focal adhesion targeting motif (Bendori et al., 1989) but lacks F-actin binding capacity (Menkel et al., 1994). Control probe had a corrected FRET ratio of 1.4 and a FRET efficiency of 44% (see Fig. 4, A and B). These values are similar, fortuitously, to unstimulated tail probe in cell lysates. This property was useful because it allowed us to use the FRET signal of control probe observed in cells to define the baseline FRET for the closed conformation of tail probe. There was no significant change in FRET for the control probe in cell lysates either before or after treatment with IpaA, actin, or both ligands together (Fig. 3 A; and Fig. 4, A and B); nor did the control probe cosediment with actin (Fig. 3 B). Therefore, we conclude that tail probe reports on conformational changes in vinculin that reflect its activation and binding to actin filaments, whereas control probe is insensitive to F-actin and to IpaA, an activator of the Vh.


Spatial distribution and functional significance of activated vinculin in living cells.

Chen H, Cohen DM, Choudhury DM, Kioka N, Craig SW - J. Cell Biol. (2005)

Response of the control FRET probe YC-V1-400 to ligands. (A) Normalized fluorescence emission spectra of cell lysates from HEK 293 cells transfected with the control FRET probe in the absence or in the presence of 1 μM IpaA or 5 μM actin or both. Spectra were normalized to emission at 475 nm of control probe alone. The control probe preserves the IpaA binding site and a focal adhesion targeting signal of vinculin but lacks the actin binding site. It does not display FRET change in response to IpaA binding. (B) Actin cosedimentation assay, performed under the same conditions as in Fig. 2 B, showed that the control probe did not bind to actin filaments under conditions in which tail probe did bind.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171941&req=5

fig3: Response of the control FRET probe YC-V1-400 to ligands. (A) Normalized fluorescence emission spectra of cell lysates from HEK 293 cells transfected with the control FRET probe in the absence or in the presence of 1 μM IpaA or 5 μM actin or both. Spectra were normalized to emission at 475 nm of control probe alone. The control probe preserves the IpaA binding site and a focal adhesion targeting signal of vinculin but lacks the actin binding site. It does not display FRET change in response to IpaA binding. (B) Actin cosedimentation assay, performed under the same conditions as in Fig. 2 B, showed that the control probe did not bind to actin filaments under conditions in which tail probe did bind.
Mentions: For a control probe, we constructed an EYFP-ECFP chimera fused in frame to vinculin residues 1–400 (Fig. 1 A). This probe contains the binding site for IpaA (Bourdet-Sicard et al., 1999) and a focal adhesion targeting motif (Bendori et al., 1989) but lacks F-actin binding capacity (Menkel et al., 1994). Control probe had a corrected FRET ratio of 1.4 and a FRET efficiency of 44% (see Fig. 4, A and B). These values are similar, fortuitously, to unstimulated tail probe in cell lysates. This property was useful because it allowed us to use the FRET signal of control probe observed in cells to define the baseline FRET for the closed conformation of tail probe. There was no significant change in FRET for the control probe in cell lysates either before or after treatment with IpaA, actin, or both ligands together (Fig. 3 A; and Fig. 4, A and B); nor did the control probe cosediment with actin (Fig. 3 B). Therefore, we conclude that tail probe reports on conformational changes in vinculin that reflect its activation and binding to actin filaments, whereas control probe is insensitive to F-actin and to IpaA, an activator of the Vh.

Bottom Line: However, nothing is known about vinculin's conformation in living cells.Time-lapse imaging reveals a gradient of conformational change that precedes loss of vinculin from focal adhesions in retracting regions.At stable or protruding regions, recruitment of vinculin is not necessarily coupled to the actin-binding conformation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

ABSTRACT
Conformational change is believed to be important to vinculin's function at sites of cell adhesion. However, nothing is known about vinculin's conformation in living cells. Using a Forster resonance energy transfer probe that reports on changes in vinculin's conformation, we find that vinculin is in the actin-binding conformation in a peripheral band of adhesive puncta in spreading cells. However, in fully spread cells with established polarity, vinculin's conformation is variable at focal adhesions. Time-lapse imaging reveals a gradient of conformational change that precedes loss of vinculin from focal adhesions in retracting regions. At stable or protruding regions, recruitment of vinculin is not necessarily coupled to the actin-binding conformation. However, a different measure of vinculin conformation, the recruitment of vinexin beta by activated vinculin, shows that autoinhibition of endogenous vinculin is relaxed at focal adhesions. Beyond providing direct evidence that vinculin is activated at focal adhesions, this study shows that the specific functional conformation correlates with regional cellular dynamics.

Show MeSH
Related in: MedlinePlus