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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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The conformation of vinculin during focal adhesion dynamics. 24 h after plating, a fully spread smooth muscle cell was imaged at time 0, 10, 40, and 45 min. Images were corrected for photobleaching before calculation of the FRET ratio image. (A) The positions of the cell at later time points (green) relative to 0 time point (red) were displayed as color joins of CFP images. (B) Enlargement of the retraction zone from region 1 in A. Notably, as mature focal adhesions disassemble, vinculin loses the actin-bound conformation in a gradient from the tip to the base of the focal adhesions. (C) Enlargement of the focal adhesion assembly zone from region 2 in A. As focal adhesions mature, recruited vinculin does not always adopt the actin-bound conformation.
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fig8: The conformation of vinculin during focal adhesion dynamics. 24 h after plating, a fully spread smooth muscle cell was imaged at time 0, 10, 40, and 45 min. Images were corrected for photobleaching before calculation of the FRET ratio image. (A) The positions of the cell at later time points (green) relative to 0 time point (red) were displayed as color joins of CFP images. (B) Enlargement of the retraction zone from region 1 in A. Notably, as mature focal adhesions disassemble, vinculin loses the actin-bound conformation in a gradient from the tip to the base of the focal adhesions. (C) Enlargement of the focal adhesion assembly zone from region 2 in A. As focal adhesions mature, recruited vinculin does not always adopt the actin-bound conformation.

Mentions: Fully spread smooth muscle cells were more photoresistant, enabling limited time-lapse analysis in cells that had spread for 24 h and were undergoing localized, asymmetric cell shape changes. We found that in retracting/contracting regions of the cell there is loss of the actin-binding conformation before loss of vinculin from focal adhesions (Fig. 8, A and B, region 1, compare 0- and 10-min time points). Interestingly, a gradient of vinculin conformation can be observed in which the actin binding conformation is found at the proximal edge of the gliding or disassembling focal adhesion even out to 45 min.


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)

The conformation of vinculin during focal adhesion dynamics. 24 h after plating, a fully spread smooth muscle cell was imaged at time 0, 10, 40, and 45 min. Images were corrected for photobleaching before calculation of the FRET ratio image. (A) The positions of the cell at later time points (green) relative to 0 time point (red) were displayed as color joins of CFP images. (B) Enlargement of the retraction zone from region 1 in A. Notably, as mature focal adhesions disassemble, vinculin loses the actin-bound conformation in a gradient from the tip to the base of the focal adhesions. (C) Enlargement of the focal adhesion assembly zone from region 2 in A. As focal adhesions mature, recruited vinculin does not always adopt the actin-bound conformation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171941&req=5

fig8: The conformation of vinculin during focal adhesion dynamics. 24 h after plating, a fully spread smooth muscle cell was imaged at time 0, 10, 40, and 45 min. Images were corrected for photobleaching before calculation of the FRET ratio image. (A) The positions of the cell at later time points (green) relative to 0 time point (red) were displayed as color joins of CFP images. (B) Enlargement of the retraction zone from region 1 in A. Notably, as mature focal adhesions disassemble, vinculin loses the actin-bound conformation in a gradient from the tip to the base of the focal adhesions. (C) Enlargement of the focal adhesion assembly zone from region 2 in A. As focal adhesions mature, recruited vinculin does not always adopt the actin-bound conformation.
Mentions: Fully spread smooth muscle cells were more photoresistant, enabling limited time-lapse analysis in cells that had spread for 24 h and were undergoing localized, asymmetric cell shape changes. We found that in retracting/contracting regions of the cell there is loss of the actin-binding conformation before loss of vinculin from focal adhesions (Fig. 8, A and B, region 1, compare 0- and 10-min time points). Interestingly, a gradient of vinculin conformation can be observed in which the actin binding conformation is found at the proximal edge of the gliding or disassembling focal adhesion even out to 45 min.

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