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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

Structure and spectral properties of the FRET probes. (A) Schematic structure of vinculin FRET probes. (B) The emission spectra of vinculin1-883-EYFP-vinculin884-1066-ECFP (Tail Probe), ECFP-vinculin-EYFP (CVY), EYFP-ECFP-vinculin1-400 (control probe), and vinculin-ECFP (VC). Numbers refer to amino acid residues in chicken vinculin (Coutu and Craig, 1988). Spectra were normalized to the emission of VC at 475 nm. (C) The structures of vinculin and GFP showing the size of each molecule. The arrow marks the site of YFP insertion between residues 883 and 884.
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fig1: Structure and spectral properties of the FRET probes. (A) Schematic structure of vinculin FRET probes. (B) The emission spectra of vinculin1-883-EYFP-vinculin884-1066-ECFP (Tail Probe), ECFP-vinculin-EYFP (CVY), EYFP-ECFP-vinculin1-400 (control probe), and vinculin-ECFP (VC). Numbers refer to amino acid residues in chicken vinculin (Coutu and Craig, 1988). Spectra were normalized to the emission of VC at 475 nm. (C) The structures of vinculin and GFP showing the size of each molecule. The arrow marks the site of YFP insertion between residues 883 and 884.

Mentions: To monitor activation of vinculin, we developed FRET probes using CFP and YFP as the donor-acceptor pair (Miyawaki and Tsien, 2000). Because the crystal structure of full-length vinculin was not known, we began by positioning ECFP and EYFP on the NH2- and COOH-terminal residues of vinculin (CVY; Fig. 1 A). In cell lysates prepared from HEK 293 cells transfected with CVY, the corrected FRET emission ratio (see Materials and methods) of CVY was 0.05, only slightly greater than the baseline for CFP alone which was set to zero for the calculation. The calculated FRET efficiency (see Materials and methods) for CVY was only 3%, indicating that the NH2 and COOH termini are not in proximity. In addition, there was little change in FRET of the CVY probe upon activation of vinculin by IpaA and binding to actin (unpublished data).


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)

Structure and spectral properties of the FRET probes. (A) Schematic structure of vinculin FRET probes. (B) The emission spectra of vinculin1-883-EYFP-vinculin884-1066-ECFP (Tail Probe), ECFP-vinculin-EYFP (CVY), EYFP-ECFP-vinculin1-400 (control probe), and vinculin-ECFP (VC). Numbers refer to amino acid residues in chicken vinculin (Coutu and Craig, 1988). Spectra were normalized to the emission of VC at 475 nm. (C) The structures of vinculin and GFP showing the size of each molecule. The arrow marks the site of YFP insertion between residues 883 and 884.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Structure and spectral properties of the FRET probes. (A) Schematic structure of vinculin FRET probes. (B) The emission spectra of vinculin1-883-EYFP-vinculin884-1066-ECFP (Tail Probe), ECFP-vinculin-EYFP (CVY), EYFP-ECFP-vinculin1-400 (control probe), and vinculin-ECFP (VC). Numbers refer to amino acid residues in chicken vinculin (Coutu and Craig, 1988). Spectra were normalized to the emission of VC at 475 nm. (C) The structures of vinculin and GFP showing the size of each molecule. The arrow marks the site of YFP insertion between residues 883 and 884.
Mentions: To monitor activation of vinculin, we developed FRET probes using CFP and YFP as the donor-acceptor pair (Miyawaki and Tsien, 2000). Because the crystal structure of full-length vinculin was not known, we began by positioning ECFP and EYFP on the NH2- and COOH-terminal residues of vinculin (CVY; Fig. 1 A). In cell lysates prepared from HEK 293 cells transfected with CVY, the corrected FRET emission ratio (see Materials and methods) of CVY was 0.05, only slightly greater than the baseline for CFP alone which was set to zero for the calculation. The calculated FRET efficiency (see Materials and methods) for CVY was only 3%, indicating that the NH2 and COOH termini are not in proximity. In addition, there was little change in FRET of the CVY probe upon activation of vinculin by IpaA and binding to actin (unpublished data).

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