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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 binding to SH3 domains of vinexin β is conformationally regulated. (A) Vinculin, at a 1-μM concentration, was incubated in 20 mM Pipes, pH 6.9, 100 mM KCl, and 0.1% Triton X-100 with GST-vinexin (residues 42–115, encoding the first two SH3 domains of vinexin) immobilized on glutathione-agarose beads in the presence of varying amounts of IpaA. After an overnight incubation at 4°C, supernatant (S) and pellet (P) were fractionated by centrifugation for 2 min at 10,000 g. The resin was washed twice with binding buffer before elution in Laemmli sample buffer. Equal loading of pellets and supernatants represent 10% of total reaction. Samples were analyzed by SDS-PAGE and Coomassie staining. (B) Densitometry-based quantification of vinculin–vinexin interaction based on digitized Coomassie blue–stained gel analyzed in NIH Image. (C) Coomassie-stained gel of negative controls for binding experiment shown in A. IpaA was incubated with GST-vinexin in the absence of vinculin, demonstrating that no direct interaction occurs. Furthermore, the vinculin–IpaA complex does not co-sediment with GST alone, demonstrating the specificity of the ternary complex with vinexin.
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fig9: Vinculin binding to SH3 domains of vinexin β is conformationally regulated. (A) Vinculin, at a 1-μM concentration, was incubated in 20 mM Pipes, pH 6.9, 100 mM KCl, and 0.1% Triton X-100 with GST-vinexin (residues 42–115, encoding the first two SH3 domains of vinexin) immobilized on glutathione-agarose beads in the presence of varying amounts of IpaA. After an overnight incubation at 4°C, supernatant (S) and pellet (P) were fractionated by centrifugation for 2 min at 10,000 g. The resin was washed twice with binding buffer before elution in Laemmli sample buffer. Equal loading of pellets and supernatants represent 10% of total reaction. Samples were analyzed by SDS-PAGE and Coomassie staining. (B) Densitometry-based quantification of vinculin–vinexin interaction based on digitized Coomassie blue–stained gel analyzed in NIH Image. (C) Coomassie-stained gel of negative controls for binding experiment shown in A. IpaA was incubated with GST-vinexin in the absence of vinculin, demonstrating that no direct interaction occurs. Furthermore, the vinculin–IpaA complex does not co-sediment with GST alone, demonstrating the specificity of the ternary complex with vinexin.

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 binding to SH3 domains of vinexin β is conformationally regulated. (A) Vinculin, at a 1-μM concentration, was incubated in 20 mM Pipes, pH 6.9, 100 mM KCl, and 0.1% Triton X-100 with GST-vinexin (residues 42–115, encoding the first two SH3 domains of vinexin) immobilized on glutathione-agarose beads in the presence of varying amounts of IpaA. After an overnight incubation at 4°C, supernatant (S) and pellet (P) were fractionated by centrifugation for 2 min at 10,000 g. The resin was washed twice with binding buffer before elution in Laemmli sample buffer. Equal loading of pellets and supernatants represent 10% of total reaction. Samples were analyzed by SDS-PAGE and Coomassie staining. (B) Densitometry-based quantification of vinculin–vinexin interaction based on digitized Coomassie blue–stained gel analyzed in NIH Image. (C) Coomassie-stained gel of negative controls for binding experiment shown in A. IpaA was incubated with GST-vinexin in the absence of vinculin, demonstrating that no direct interaction occurs. Furthermore, the vinculin–IpaA complex does not co-sediment with GST alone, demonstrating the specificity of the ternary complex with vinexin.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171941&req=5

fig9: Vinculin binding to SH3 domains of vinexin β is conformationally regulated. (A) Vinculin, at a 1-μM concentration, was incubated in 20 mM Pipes, pH 6.9, 100 mM KCl, and 0.1% Triton X-100 with GST-vinexin (residues 42–115, encoding the first two SH3 domains of vinexin) immobilized on glutathione-agarose beads in the presence of varying amounts of IpaA. After an overnight incubation at 4°C, supernatant (S) and pellet (P) were fractionated by centrifugation for 2 min at 10,000 g. The resin was washed twice with binding buffer before elution in Laemmli sample buffer. Equal loading of pellets and supernatants represent 10% of total reaction. Samples were analyzed by SDS-PAGE and Coomassie staining. (B) Densitometry-based quantification of vinculin–vinexin interaction based on digitized Coomassie blue–stained gel analyzed in NIH Image. (C) Coomassie-stained gel of negative controls for binding experiment shown in A. IpaA was incubated with GST-vinexin in the absence of vinculin, demonstrating that no direct interaction occurs. Furthermore, the vinculin–IpaA complex does not co-sediment with GST alone, demonstrating the specificity of the ternary complex with vinexin.
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