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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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Recruitment of vinculin to the peripheral belt of adhesion puncta during cell spreading is associated with conformational activation of vinculin. Vin−/− MECs transfected with tail probe were replated into a dish (Bioptechs) coated with 20 μg/ml fibronectin heated at 37°C. (A and B) Images of a representative cell at the initial attachment stage ∼5 min after plating. (C–F) Images of two representative cells at ∼45–60 min after plating. (A, C, and E) Localization of tail probe in each Vin−/− MEC imaged through CFP channel. (B, D, and F) Pseudocolored ratio (FRET/CFP) image of the cells shown in A, C, and E. Notably, the adherent rounded cell (A and B) gave a high FRET ratio, similar to that of cells containing control probe (Fig. 6), indicating that vinculin was largely closed in conformation at the earliest stages of spreading. During spreading, tail probe showed lower FRET ratios correlating with actin-binding conformation in adhesion structures (C–F).
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fig7: Recruitment of vinculin to the peripheral belt of adhesion puncta during cell spreading is associated with conformational activation of vinculin. Vin−/− MECs transfected with tail probe were replated into a dish (Bioptechs) coated with 20 μg/ml fibronectin heated at 37°C. (A and B) Images of a representative cell at the initial attachment stage ∼5 min after plating. (C–F) Images of two representative cells at ∼45–60 min after plating. (A, C, and E) Localization of tail probe in each Vin−/− MEC imaged through CFP channel. (B, D, and F) Pseudocolored ratio (FRET/CFP) image of the cells shown in A, C, and E. Notably, the adherent rounded cell (A and B) gave a high FRET ratio, similar to that of cells containing control probe (Fig. 6), indicating that vinculin was largely closed in conformation at the earliest stages of spreading. During spreading, tail probe showed lower FRET ratios correlating with actin-binding conformation in adhesion structures (C–F).

Mentions: To explore the heterogeneity of vinculin conformation in focal adhesions, we asked whether or not the average conformation correlated with cellular activity. In vin−/− MECs that have attached to fibronectin but have not initiated spreading, vinculin is uniformly in the nonactin binding conformation (Fig. 7, A and B). At early phases of isotropic spreading, vinculin is recruited to puncta in the peripheral adhesion ring and to short central adhesions where it is largely in the actin-binding conformation (Fig. 7, C–F). Some cells (Fig. 7, C and D) showed the nonactin binding conformation of vinculin in a segment of the adhesion belt puncta (see bottom edge of cell in Fig. 7, C and D). Unfortunately, phototoxicity associated with acquiring the FRET image precluded correlating these asymmetric regions with subsequent events in cell spreading.


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)

Recruitment of vinculin to the peripheral belt of adhesion puncta during cell spreading is associated with conformational activation of vinculin. Vin−/− MECs transfected with tail probe were replated into a dish (Bioptechs) coated with 20 μg/ml fibronectin heated at 37°C. (A and B) Images of a representative cell at the initial attachment stage ∼5 min after plating. (C–F) Images of two representative cells at ∼45–60 min after plating. (A, C, and E) Localization of tail probe in each Vin−/− MEC imaged through CFP channel. (B, D, and F) Pseudocolored ratio (FRET/CFP) image of the cells shown in A, C, and E. Notably, the adherent rounded cell (A and B) gave a high FRET ratio, similar to that of cells containing control probe (Fig. 6), indicating that vinculin was largely closed in conformation at the earliest stages of spreading. During spreading, tail probe showed lower FRET ratios correlating with actin-binding conformation in adhesion structures (C–F).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171941&req=5

fig7: Recruitment of vinculin to the peripheral belt of adhesion puncta during cell spreading is associated with conformational activation of vinculin. Vin−/− MECs transfected with tail probe were replated into a dish (Bioptechs) coated with 20 μg/ml fibronectin heated at 37°C. (A and B) Images of a representative cell at the initial attachment stage ∼5 min after plating. (C–F) Images of two representative cells at ∼45–60 min after plating. (A, C, and E) Localization of tail probe in each Vin−/− MEC imaged through CFP channel. (B, D, and F) Pseudocolored ratio (FRET/CFP) image of the cells shown in A, C, and E. Notably, the adherent rounded cell (A and B) gave a high FRET ratio, similar to that of cells containing control probe (Fig. 6), indicating that vinculin was largely closed in conformation at the earliest stages of spreading. During spreading, tail probe showed lower FRET ratios correlating with actin-binding conformation in adhesion structures (C–F).
Mentions: To explore the heterogeneity of vinculin conformation in focal adhesions, we asked whether or not the average conformation correlated with cellular activity. In vin−/− MECs that have attached to fibronectin but have not initiated spreading, vinculin is uniformly in the nonactin binding conformation (Fig. 7, A and B). At early phases of isotropic spreading, vinculin is recruited to puncta in the peripheral adhesion ring and to short central adhesions where it is largely in the actin-binding conformation (Fig. 7, C–F). Some cells (Fig. 7, C and D) showed the nonactin binding conformation of vinculin in a segment of the adhesion belt puncta (see bottom edge of cell in Fig. 7, C and D). Unfortunately, phototoxicity associated with acquiring the FRET image precluded correlating these asymmetric regions with subsequent events in cell spreading.

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