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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 tail probe to ligands. The binding of actin filaments to IpaA-activated vinculin tail probe induced FRET loss, indicating a conformational change of vinculin in the tail domain. (A) Normalized fluorescence emission spectra of cell lysate from HEK 293 cells transfected with tail probe in the absence or presence of 1 μM IpaA or 5 μM actin or both. Spectra were normalized to the emission of tail probe at 475 nm. (B) Samples from A were spun in an Airfuge (Beckman Coulter) at 25 psi (130,000 g) for 35 min. Equivalent amounts of total sample before spin (T), supernatant (S), and pellet (P) fractions were subjected to SDS-PAGE and immunoblotted with hVIN1 and C4 mAbs (Sigma-Aldrich) to vinculin and actin, respectively.
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fig2: Response of tail probe to ligands. The binding of actin filaments to IpaA-activated vinculin tail probe induced FRET loss, indicating a conformational change of vinculin in the tail domain. (A) Normalized fluorescence emission spectra of cell lysate from HEK 293 cells transfected with tail probe in the absence or presence of 1 μM IpaA or 5 μM actin or both. Spectra were normalized to the emission of tail probe at 475 nm. (B) Samples from A were spun in an Airfuge (Beckman Coulter) at 25 psi (130,000 g) for 35 min. Equivalent amounts of total sample before spin (T), supernatant (S), and pellet (P) fractions were subjected to SDS-PAGE and immunoblotted with hVIN1 and C4 mAbs (Sigma-Aldrich) to vinculin and actin, respectively.

Mentions: To determine the conformational state of tail probe and to assess its ability to report on activation of vinculin, we measured FRET response as a function of ligand binding to vinculin. Neither tail probe nor endogenous vinculin cosedimented with F-actin, indicating that the probe and endogenous vinculin were in a conformationally closed and inactive state (Fig. 2 B). As expected, addition of F-actin caused no change in the FRET signal of the probe (Figs. 2 A). To stimulate vinculin to bind F-actin, we treated lysates with IpaA. IpaA is a Shigella flexneri virulence protein that binds to the D1 domain (residues 1–258; see Bakolitsa et al. [2004] for nomenclature reflecting new structure-based subdivisions of vinculin domains) of vinculin head and exposes the actin binding activity of vinculin tail (Bourdet-Sicard et al., 1999). Addition of IpaA alone did not cause a change in FRET, but subsequent addition of F-actin caused a 45% decrease in the corrected FRET ratio (1.48 to 0.81) of tail probe and 14% decrease in FRET efficiency (46% to 32%), reflecting a change in the conformation of vinculin (Fig. 2 A; and see Fig. 4, A and B). Tail probe cosedimented with actin filaments only in the presence of IpaA, demonstrating that the loss of FRET reports on binding of IpaA-activated tail probe to F-actin (Fig. 2 B).


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 tail probe to ligands. The binding of actin filaments to IpaA-activated vinculin tail probe induced FRET loss, indicating a conformational change of vinculin in the tail domain. (A) Normalized fluorescence emission spectra of cell lysate from HEK 293 cells transfected with tail probe in the absence or presence of 1 μM IpaA or 5 μM actin or both. Spectra were normalized to the emission of tail probe at 475 nm. (B) Samples from A were spun in an Airfuge (Beckman Coulter) at 25 psi (130,000 g) for 35 min. Equivalent amounts of total sample before spin (T), supernatant (S), and pellet (P) fractions were subjected to SDS-PAGE and immunoblotted with hVIN1 and C4 mAbs (Sigma-Aldrich) to vinculin and actin, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171941&req=5

fig2: Response of tail probe to ligands. The binding of actin filaments to IpaA-activated vinculin tail probe induced FRET loss, indicating a conformational change of vinculin in the tail domain. (A) Normalized fluorescence emission spectra of cell lysate from HEK 293 cells transfected with tail probe in the absence or presence of 1 μM IpaA or 5 μM actin or both. Spectra were normalized to the emission of tail probe at 475 nm. (B) Samples from A were spun in an Airfuge (Beckman Coulter) at 25 psi (130,000 g) for 35 min. Equivalent amounts of total sample before spin (T), supernatant (S), and pellet (P) fractions were subjected to SDS-PAGE and immunoblotted with hVIN1 and C4 mAbs (Sigma-Aldrich) to vinculin and actin, respectively.
Mentions: To determine the conformational state of tail probe and to assess its ability to report on activation of vinculin, we measured FRET response as a function of ligand binding to vinculin. Neither tail probe nor endogenous vinculin cosedimented with F-actin, indicating that the probe and endogenous vinculin were in a conformationally closed and inactive state (Fig. 2 B). As expected, addition of F-actin caused no change in the FRET signal of the probe (Figs. 2 A). To stimulate vinculin to bind F-actin, we treated lysates with IpaA. IpaA is a Shigella flexneri virulence protein that binds to the D1 domain (residues 1–258; see Bakolitsa et al. [2004] for nomenclature reflecting new structure-based subdivisions of vinculin domains) of vinculin head and exposes the actin binding activity of vinculin tail (Bourdet-Sicard et al., 1999). Addition of IpaA alone did not cause a change in FRET, but subsequent addition of F-actin caused a 45% decrease in the corrected FRET ratio (1.48 to 0.81) of tail probe and 14% decrease in FRET efficiency (46% to 32%), reflecting a change in the conformation of vinculin (Fig. 2 A; and see Fig. 4, A and B). Tail probe cosedimented with actin filaments only in the presence of IpaA, demonstrating that the loss of FRET reports on binding of IpaA-activated tail probe to F-actin (Fig. 2 B).

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