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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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Related in: MedlinePlus

Corrected emission ratios and FRET efficiencies of vinculin FRET probes. The mean emission ratios corrected for spectral cross talk (A), and the extracted FRET efficiencies (B) were obtained as described in Materials and methods. n = 3. Error bars are the SEM.
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fig4: Corrected emission ratios and FRET efficiencies of vinculin FRET probes. The mean emission ratios corrected for spectral cross talk (A), and the extracted FRET efficiencies (B) were obtained as described in Materials and methods. n = 3. Error bars are the SEM.

Mentions: Tail probe and various control constructs were expressed in HEK293 cells, and lysates were analyzed by spectrofluorimetry. Tail probe exhibited a strong FRET signal (Fig. 1 B), with a corrected emission ratio of 1.48 and an efficiency of 46% (see Fig. 4, A and B). To determine whether or not intermolecular FRET contributes to the FRET measurement, we compared a lysate containing tail probe at 5, 10, and 20 nM with mixtures of lysates containing vinculin/CFP and vinculin/YFP at 5, 10, or 20 nM each in the mixture. At all concentrations tested, the corrected FRET ratio of the mixed probes was close to that of CFP only, whereas the corrected FRET ratio of tail probe was insensitive to dilution. Thus, the FRET values obtained for tail probe report specifically on intramolecular FRET.


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)

Corrected emission ratios and FRET efficiencies of vinculin FRET probes. The mean emission ratios corrected for spectral cross talk (A), and the extracted FRET efficiencies (B) were obtained as described in Materials and methods. n = 3. Error bars are the SEM.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Corrected emission ratios and FRET efficiencies of vinculin FRET probes. The mean emission ratios corrected for spectral cross talk (A), and the extracted FRET efficiencies (B) were obtained as described in Materials and methods. n = 3. Error bars are the SEM.
Mentions: Tail probe and various control constructs were expressed in HEK293 cells, and lysates were analyzed by spectrofluorimetry. Tail probe exhibited a strong FRET signal (Fig. 1 B), with a corrected emission ratio of 1.48 and an efficiency of 46% (see Fig. 4, A and B). To determine whether or not intermolecular FRET contributes to the FRET measurement, we compared a lysate containing tail probe at 5, 10, and 20 nM with mixtures of lysates containing vinculin/CFP and vinculin/YFP at 5, 10, or 20 nM each in the mixture. At all concentrations tested, the corrected FRET ratio of the mixed probes was close to that of CFP only, whereas the corrected FRET ratio of tail probe was insensitive to dilution. Thus, the FRET values obtained for tail probe report specifically on intramolecular FRET.

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