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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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Vinculin mediates vinexin β recruitment to focal adhesions. Vinculin  cells were permeabilized with 0.05% digitonin and incubated with 10 μg/mL GST-vinexin β (residues 1–329, encoding full-length vinexin) and 25 μg/mL vinculin (A and B) or Vh (C and D) in 25 mM MES, pH 6.0, 3 mM MgCl2, and 1 mM EGTA. Vinculin localization was visualized by staining with 5 μg/mL of 3.24 monoclonal antivinculin, followed by Rhodamine red-X–conjugated donkey anti–mouse IgG (A and C). Vinexin was visualized by staining with 5 μg/mL of polyclonal anti-GST followed by Oregon green–conjugated donkey anti–rabbit IgG (B and D). In the presence of full-length vinculin, vinexin β becomes strongly enriched in focal adhesions. However, vinexin β fails to target to focal adhesions in the presence of Vh, which lacks the polyproline region required for a direct interaction with vinexin.
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fig10: Vinculin mediates vinexin β recruitment to focal adhesions. Vinculin cells were permeabilized with 0.05% digitonin and incubated with 10 μg/mL GST-vinexin β (residues 1–329, encoding full-length vinexin) and 25 μg/mL vinculin (A and B) or Vh (C and D) in 25 mM MES, pH 6.0, 3 mM MgCl2, and 1 mM EGTA. Vinculin localization was visualized by staining with 5 μg/mL of 3.24 monoclonal antivinculin, followed by Rhodamine red-X–conjugated donkey anti–mouse IgG (A and C). Vinexin was visualized by staining with 5 μg/mL of polyclonal anti-GST followed by Oregon green–conjugated donkey anti–rabbit IgG (B and D). In the presence of full-length vinculin, vinexin β becomes strongly enriched in focal adhesions. However, vinexin β fails to target to focal adhesions in the presence of Vh, which lacks the polyproline region required for a direct interaction with vinexin.

Mentions: Using a permeabilized cell model prepared from vin−/− MEC, we found that recruitment of exogenous vinexin β to focal adhesions was dependent on the presence of vinculin in the focal adhesions (Fig. 10, A and B). Consistent with a direct interaction between the two proteins, recruitment of vinexin β to focal adhesions depends on the presence of the vinexin binding site because Vh1-851, which lacks the site (Kioka et al., 1999), was unable to recruit vinexin (Fig. 10, C and D). Because vinculin must be activated to bind vinexin β (Fig. 9, A and B), we conclude that some of the endogenous vinculin at the focal adhesions must be in the open or activated conformation and that one functional consequence of this activated conformation is assembly of vinculin–vinexin β complexes at focal adhesions.


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)

Vinculin mediates vinexin β recruitment to focal adhesions. Vinculin  cells were permeabilized with 0.05% digitonin and incubated with 10 μg/mL GST-vinexin β (residues 1–329, encoding full-length vinexin) and 25 μg/mL vinculin (A and B) or Vh (C and D) in 25 mM MES, pH 6.0, 3 mM MgCl2, and 1 mM EGTA. Vinculin localization was visualized by staining with 5 μg/mL of 3.24 monoclonal antivinculin, followed by Rhodamine red-X–conjugated donkey anti–mouse IgG (A and C). Vinexin was visualized by staining with 5 μg/mL of polyclonal anti-GST followed by Oregon green–conjugated donkey anti–rabbit IgG (B and D). In the presence of full-length vinculin, vinexin β becomes strongly enriched in focal adhesions. However, vinexin β fails to target to focal adhesions in the presence of Vh, which lacks the polyproline region required for a direct interaction with vinexin.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171941&req=5

fig10: Vinculin mediates vinexin β recruitment to focal adhesions. Vinculin cells were permeabilized with 0.05% digitonin and incubated with 10 μg/mL GST-vinexin β (residues 1–329, encoding full-length vinexin) and 25 μg/mL vinculin (A and B) or Vh (C and D) in 25 mM MES, pH 6.0, 3 mM MgCl2, and 1 mM EGTA. Vinculin localization was visualized by staining with 5 μg/mL of 3.24 monoclonal antivinculin, followed by Rhodamine red-X–conjugated donkey anti–mouse IgG (A and C). Vinexin was visualized by staining with 5 μg/mL of polyclonal anti-GST followed by Oregon green–conjugated donkey anti–rabbit IgG (B and D). In the presence of full-length vinculin, vinexin β becomes strongly enriched in focal adhesions. However, vinexin β fails to target to focal adhesions in the presence of Vh, which lacks the polyproline region required for a direct interaction with vinexin.
Mentions: Using a permeabilized cell model prepared from vin−/− MEC, we found that recruitment of exogenous vinexin β to focal adhesions was dependent on the presence of vinculin in the focal adhesions (Fig. 10, A and B). Consistent with a direct interaction between the two proteins, recruitment of vinexin β to focal adhesions depends on the presence of the vinexin binding site because Vh1-851, which lacks the site (Kioka et al., 1999), was unable to recruit vinexin (Fig. 10, C and D). Because vinculin must be activated to bind vinexin β (Fig. 9, A and B), we conclude that some of the endogenous vinculin at the focal adhesions must be in the open or activated conformation and that one functional consequence of this activated conformation is assembly of vinculin–vinexin β complexes at focal adhesions.

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