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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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Spatial distribution of activated vinculin in living cells. Vin−/− MEC transfected with tail probe (A–F) or control probe (G–L) were imaged 1 h after plating. (A and G) Localization of tail probe and control probe in MECs imaged through CFP channel. (D and J) Pseudocolored ratio (FRET/CFP) image of the cells shown in A and G. (B, E, H, and K) Enlargement of boxed region in A, D, G, and J, respectively. (C and I) The average FRET ratio measured from segmented regions of cytoplasm or focal adhesions; all segmentable focal adhesions were included. (F and L) Histograms of FRET ratios measured from the segmented focal adhesions and cytoplasm. Notably, the tail probe gave a much lower average FRET ratio (corresponding to actin-binding conformation of vinculin) in focal adhesions (B and E, boxed region) than in cytoplasm even though not all focal adhesions are distinguishable from cytoplasm. The control probe did not distinguish between the two locations (H and K, boxed region). Similar results were obtained by analysis of three other cells from a separate experiment.
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fig6: Spatial distribution of activated vinculin in living cells. Vin−/− MEC transfected with tail probe (A–F) or control probe (G–L) were imaged 1 h after plating. (A and G) Localization of tail probe and control probe in MECs imaged through CFP channel. (D and J) Pseudocolored ratio (FRET/CFP) image of the cells shown in A and G. (B, E, H, and K) Enlargement of boxed region in A, D, G, and J, respectively. (C and I) The average FRET ratio measured from segmented regions of cytoplasm or focal adhesions; all segmentable focal adhesions were included. (F and L) Histograms of FRET ratios measured from the segmented focal adhesions and cytoplasm. Notably, the tail probe gave a much lower average FRET ratio (corresponding to actin-binding conformation of vinculin) in focal adhesions (B and E, boxed region) than in cytoplasm even though not all focal adhesions are distinguishable from cytoplasm. The control probe did not distinguish between the two locations (H and K, boxed region). Similar results were obtained by analysis of three other cells from a separate experiment.

Mentions: When focal adhesions were examined in vin−/− MEC transfected with tail probe, we found that the average FRET ratio is significantly lower in focal adhesions than in cytoplasm (Fig. 6, A–F), indicating enrichment of the actin-binding conformation in focal adhesions. In contrast, in vin−/− MEC transfected with the control probe (YC/V 1–400), the FRET ratio is similar in focal adhesions and in cytoplasm (Fig. 6, G–L). The FRET ratio of tail probe in cytoplasm is comparable to the global FRET ratio of control probe (Fig. 6, D, F, J, and L), indicating that vinculin is in the nonactin binding conformation in the cytoplasm. Although the actin-binding conformation of vinculin is enriched in focal adhesions, there were regions of the cell in which the conformation of vinculin in the focal adhesions was not readily distinguished from that in cytoplasm (Fig. 6, compare A with D). Similar results were obtained from analysis of three other cells in separate experiments.


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)

Spatial distribution of activated vinculin in living cells. Vin−/− MEC transfected with tail probe (A–F) or control probe (G–L) were imaged 1 h after plating. (A and G) Localization of tail probe and control probe in MECs imaged through CFP channel. (D and J) Pseudocolored ratio (FRET/CFP) image of the cells shown in A and G. (B, E, H, and K) Enlargement of boxed region in A, D, G, and J, respectively. (C and I) The average FRET ratio measured from segmented regions of cytoplasm or focal adhesions; all segmentable focal adhesions were included. (F and L) Histograms of FRET ratios measured from the segmented focal adhesions and cytoplasm. Notably, the tail probe gave a much lower average FRET ratio (corresponding to actin-binding conformation of vinculin) in focal adhesions (B and E, boxed region) than in cytoplasm even though not all focal adhesions are distinguishable from cytoplasm. The control probe did not distinguish between the two locations (H and K, boxed region). Similar results were obtained by analysis of three other cells from a separate experiment.
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Related In: Results  -  Collection

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fig6: Spatial distribution of activated vinculin in living cells. Vin−/− MEC transfected with tail probe (A–F) or control probe (G–L) were imaged 1 h after plating. (A and G) Localization of tail probe and control probe in MECs imaged through CFP channel. (D and J) Pseudocolored ratio (FRET/CFP) image of the cells shown in A and G. (B, E, H, and K) Enlargement of boxed region in A, D, G, and J, respectively. (C and I) The average FRET ratio measured from segmented regions of cytoplasm or focal adhesions; all segmentable focal adhesions were included. (F and L) Histograms of FRET ratios measured from the segmented focal adhesions and cytoplasm. Notably, the tail probe gave a much lower average FRET ratio (corresponding to actin-binding conformation of vinculin) in focal adhesions (B and E, boxed region) than in cytoplasm even though not all focal adhesions are distinguishable from cytoplasm. The control probe did not distinguish between the two locations (H and K, boxed region). Similar results were obtained by analysis of three other cells from a separate experiment.
Mentions: When focal adhesions were examined in vin−/− MEC transfected with tail probe, we found that the average FRET ratio is significantly lower in focal adhesions than in cytoplasm (Fig. 6, A–F), indicating enrichment of the actin-binding conformation in focal adhesions. In contrast, in vin−/− MEC transfected with the control probe (YC/V 1–400), the FRET ratio is similar in focal adhesions and in cytoplasm (Fig. 6, G–L). The FRET ratio of tail probe in cytoplasm is comparable to the global FRET ratio of control probe (Fig. 6, D, F, J, and L), indicating that vinculin is in the nonactin binding conformation in the cytoplasm. Although the actin-binding conformation of vinculin is enriched in focal adhesions, there were regions of the cell in which the conformation of vinculin in the focal adhesions was not readily distinguished from that in cytoplasm (Fig. 6, compare A with D). Similar results were obtained from analysis of three other cells in separate experiments.

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