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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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Vinculin tail probe rescues spreading and lamellipodial extension on fibronectin. (A) Vinculin  cells expressing a control CFP-YFP chimera, tail probe, or untagged vinculin were allowed to spread onto 20 μg/ml of FN for 2 h at 37°C. Cells expressing untagged vinculin were stained with Vin11-5 antibody and rhodamine-conjugated secondary antibody. Cells expressing CFP-YFP and tail probe were examined by GFP fluorescence. (B–D) The extent of spreading was quantified by measuring the ratio of major/minor axis of cells (B and D) and cell areas (C and E). (B and C) Mean of the axial ratio and cell area, respectively. Error bars are the SEM. (D and E) Box plots (Chase and Brown, 1997) of B and C. Each box encloses 50% of the data with median value displayed as a horizontal line. Top and bottom of box represent the limits of ±25% of the population. Lines extending from top and bottom of boxes mark the minimum and maximum values of the data set that fall within an acceptable range. Open circles denote outliers, points whose value is either >UQ + 1.5 × IQD or <LQ − 1.5 × IQD (UQ, upper quartile; IQD, inter-quartile distance; LQ, lower quartile). Asterisks mark histograms that are statistically different from the corresponding control. The tail probe (n = 56) or untagged vinculin (n = 49) reexpressing cells are significantly (P < 0.01) more spread than vin−/− cells (n = 44).
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fig5: Vinculin tail probe rescues spreading and lamellipodial extension on fibronectin. (A) Vinculin cells expressing a control CFP-YFP chimera, tail probe, or untagged vinculin were allowed to spread onto 20 μg/ml of FN for 2 h at 37°C. Cells expressing untagged vinculin were stained with Vin11-5 antibody and rhodamine-conjugated secondary antibody. Cells expressing CFP-YFP and tail probe were examined by GFP fluorescence. (B–D) The extent of spreading was quantified by measuring the ratio of major/minor axis of cells (B and D) and cell areas (C and E). (B and C) Mean of the axial ratio and cell area, respectively. Error bars are the SEM. (D and E) Box plots (Chase and Brown, 1997) of B and C. Each box encloses 50% of the data with median value displayed as a horizontal line. Top and bottom of box represent the limits of ±25% of the population. Lines extending from top and bottom of boxes mark the minimum and maximum values of the data set that fall within an acceptable range. Open circles denote outliers, points whose value is either >UQ + 1.5 × IQD or <LQ − 1.5 × IQD (UQ, upper quartile; IQD, inter-quartile distance; LQ, lower quartile). Asterisks mark histograms that are statistically different from the corresponding control. The tail probe (n = 56) or untagged vinculin (n = 49) reexpressing cells are significantly (P < 0.01) more spread than vin−/− cells (n = 44).

Mentions: Tail probe and endogenous vinculin differ in their sensitivity to IpaA (Fig. 2 B). This difference is abolished by inclusion of 1% Triton X-100 in the lysate (unpublished data). The requirement of Triton X-100 for IpaA activation of endogenous vinculin in cell lysates is unexpected because IpaA can activate at least 40% of purified smooth muscle vinculin and recombinant vinculin in vitro without the presence of Triton X-100 (Bourdet-Sicard et al., 1999; see Fig. 9). The differential response between the tail probe and endogenous vinculin reflects a more tightly closed conformation in endogenous vinculin, which may be mediated by a Triton X-100–sensitive component in the lysate. Despite this difference, the inability of the tail probe to cosediment with actin filaments shows that like endogenous vinculin, it adopts an autoinhibited conformation. Furthermore, tail probe and vinculin localize similarly in cells and are equally able to rescue spreading defects in vinculin cells (see Fig. 5).


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)

Vinculin tail probe rescues spreading and lamellipodial extension on fibronectin. (A) Vinculin  cells expressing a control CFP-YFP chimera, tail probe, or untagged vinculin were allowed to spread onto 20 μg/ml of FN for 2 h at 37°C. Cells expressing untagged vinculin were stained with Vin11-5 antibody and rhodamine-conjugated secondary antibody. Cells expressing CFP-YFP and tail probe were examined by GFP fluorescence. (B–D) The extent of spreading was quantified by measuring the ratio of major/minor axis of cells (B and D) and cell areas (C and E). (B and C) Mean of the axial ratio and cell area, respectively. Error bars are the SEM. (D and E) Box plots (Chase and Brown, 1997) of B and C. Each box encloses 50% of the data with median value displayed as a horizontal line. Top and bottom of box represent the limits of ±25% of the population. Lines extending from top and bottom of boxes mark the minimum and maximum values of the data set that fall within an acceptable range. Open circles denote outliers, points whose value is either >UQ + 1.5 × IQD or <LQ − 1.5 × IQD (UQ, upper quartile; IQD, inter-quartile distance; LQ, lower quartile). Asterisks mark histograms that are statistically different from the corresponding control. The tail probe (n = 56) or untagged vinculin (n = 49) reexpressing cells are significantly (P < 0.01) more spread than vin−/− cells (n = 44).
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fig5: Vinculin tail probe rescues spreading and lamellipodial extension on fibronectin. (A) Vinculin cells expressing a control CFP-YFP chimera, tail probe, or untagged vinculin were allowed to spread onto 20 μg/ml of FN for 2 h at 37°C. Cells expressing untagged vinculin were stained with Vin11-5 antibody and rhodamine-conjugated secondary antibody. Cells expressing CFP-YFP and tail probe were examined by GFP fluorescence. (B–D) The extent of spreading was quantified by measuring the ratio of major/minor axis of cells (B and D) and cell areas (C and E). (B and C) Mean of the axial ratio and cell area, respectively. Error bars are the SEM. (D and E) Box plots (Chase and Brown, 1997) of B and C. Each box encloses 50% of the data with median value displayed as a horizontal line. Top and bottom of box represent the limits of ±25% of the population. Lines extending from top and bottom of boxes mark the minimum and maximum values of the data set that fall within an acceptable range. Open circles denote outliers, points whose value is either >UQ + 1.5 × IQD or <LQ − 1.5 × IQD (UQ, upper quartile; IQD, inter-quartile distance; LQ, lower quartile). Asterisks mark histograms that are statistically different from the corresponding control. The tail probe (n = 56) or untagged vinculin (n = 49) reexpressing cells are significantly (P < 0.01) more spread than vin−/− cells (n = 44).
Mentions: Tail probe and endogenous vinculin differ in their sensitivity to IpaA (Fig. 2 B). This difference is abolished by inclusion of 1% Triton X-100 in the lysate (unpublished data). The requirement of Triton X-100 for IpaA activation of endogenous vinculin in cell lysates is unexpected because IpaA can activate at least 40% of purified smooth muscle vinculin and recombinant vinculin in vitro without the presence of Triton X-100 (Bourdet-Sicard et al., 1999; see Fig. 9). The differential response between the tail probe and endogenous vinculin reflects a more tightly closed conformation in endogenous vinculin, which may be mediated by a Triton X-100–sensitive component in the lysate. Despite this difference, the inability of the tail probe to cosediment with actin filaments shows that like endogenous vinculin, it adopts an autoinhibited conformation. Furthermore, tail probe and vinculin localize similarly in cells and are equally able to rescue spreading defects in vinculin cells (see Fig. 5).

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