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Rho small GTPase regulates the stability of individual focal adhesions: a FRET-based visualization of GDP/GTP exchange on small GTPases

View Article: PubMed Central - PubMed

ABSTRACT

RhoA and Rac1 are small GTPases primarily involved in cytoskeletal remodeling. Many biochemical studies have suggested that they are also key organizers of cell-substrate adhesion. Recently, fluorescence resonance energy transfer (FRET)-based indicators have been developed to visualize RhoA and Rac1 activity in living cells [Yoshizaki et al., J. Cell Biol. 162, 223 (2003); Pertz et al., Nature 440, 1069 (2006)]. These indicators use one of the interactions between RhoA (Rac1) and the RhoA (Rac1)-binding domain of their effector proteins. However, distribution of RhoA activity in single cells has not yet been observed with micrometer-scale resolution. Here, we employed an approach that detects GDP/GTP exchange on small GTPases by using FRET from YFP-fused small GTPases to a fluorescent analogue of GTP, BODIPY(TR)-GTP. This approach allowed us to visualize confined localization of active (GTP-bound forms of) RhoA and Rac1 in individual focal adhesions. Activated RhoA accumulated in immobile and long-lived focal adhesions but was not evident in unstable and temporary adhesions, while activated Rac1 was observed at every adhesion. Our results suggest that RhoA is the major regulator determining the stability of individual cell adhesion structures.

No MeSH data available.


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Interaction of BP-GTP·RhoA with an effector protein Rhotekin. (A) A GST fusion of the Rho-binding domain (RBD) of Rhotekin was attached on GSH-beads. MBP-RhoA was incubated with the beads in the presence of various concentrations of BP-GTP. Proteins bound to the beads were dissociated from the beads with a buffer containing 20 mM glutathione and analyzed by SDS-PAGE. Arrow-heads represent MBP-RhoA (silver-stained) bound to GST-Rhotekin. Arrows represent GST-Rhotekin (CBB-stained). (B) The amount of MBP-RhoA bound to GST-Rhotekin was plotted as a function of the BP-GTP concentration. The amount of MBP-RhoA was normalized to that in the presence of 1 μM BP-GTP after subtraction of the non-specific binding observed in the absence of BP-GTP. The average and SE of four independent experiments are shown. Similar results were obtained using RBD of Rho-kinase instead of RBD of Rhotekin (data not shown). The difference between the amount of the bound MBP-RhoA with BP-GTP and that with non-labeled GTP is probably due to structural difference between RhoA·GTP and RhoA·BP-GTP. A hydrogen bond network is formed between 2′-OH of GTP and Cys20 and Pro31 of RhoA47. BP-GTP is a mixture of 2′- and 3′-isoforms and 2′-OH is blocked in the 2′-isoform.
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f3-3_63: Interaction of BP-GTP·RhoA with an effector protein Rhotekin. (A) A GST fusion of the Rho-binding domain (RBD) of Rhotekin was attached on GSH-beads. MBP-RhoA was incubated with the beads in the presence of various concentrations of BP-GTP. Proteins bound to the beads were dissociated from the beads with a buffer containing 20 mM glutathione and analyzed by SDS-PAGE. Arrow-heads represent MBP-RhoA (silver-stained) bound to GST-Rhotekin. Arrows represent GST-Rhotekin (CBB-stained). (B) The amount of MBP-RhoA bound to GST-Rhotekin was plotted as a function of the BP-GTP concentration. The amount of MBP-RhoA was normalized to that in the presence of 1 μM BP-GTP after subtraction of the non-specific binding observed in the absence of BP-GTP. The average and SE of four independent experiments are shown. Similar results were obtained using RBD of Rho-kinase instead of RBD of Rhotekin (data not shown). The difference between the amount of the bound MBP-RhoA with BP-GTP and that with non-labeled GTP is probably due to structural difference between RhoA·GTP and RhoA·BP-GTP. A hydrogen bond network is formed between 2′-OH of GTP and Cys20 and Pro31 of RhoA47. BP-GTP is a mixture of 2′- and 3′-isoforms and 2′-OH is blocked in the 2′-isoform.

Mentions: To examine the activity of RhoA after association with BP-GTP, a pull-down assay of MBP-RhoA·BP-GTP was carried out using an effector protein bound to GSH-beads (Fig. 3). Depending on the dose of BP-GTP, MBP-RhoA bound with the Rho binding domain of Rhotekin fused with GST. This suggests that the FRET indicates the production of active Rho. A BP-GTP dependent interaction of MBP-YFP-Rac1 with the GST fused Rac binding domain of p21-activated kinase (PAK) was also observed (data not shown).


Rho small GTPase regulates the stability of individual focal adhesions: a FRET-based visualization of GDP/GTP exchange on small GTPases
Interaction of BP-GTP·RhoA with an effector protein Rhotekin. (A) A GST fusion of the Rho-binding domain (RBD) of Rhotekin was attached on GSH-beads. MBP-RhoA was incubated with the beads in the presence of various concentrations of BP-GTP. Proteins bound to the beads were dissociated from the beads with a buffer containing 20 mM glutathione and analyzed by SDS-PAGE. Arrow-heads represent MBP-RhoA (silver-stained) bound to GST-Rhotekin. Arrows represent GST-Rhotekin (CBB-stained). (B) The amount of MBP-RhoA bound to GST-Rhotekin was plotted as a function of the BP-GTP concentration. The amount of MBP-RhoA was normalized to that in the presence of 1 μM BP-GTP after subtraction of the non-specific binding observed in the absence of BP-GTP. The average and SE of four independent experiments are shown. Similar results were obtained using RBD of Rho-kinase instead of RBD of Rhotekin (data not shown). The difference between the amount of the bound MBP-RhoA with BP-GTP and that with non-labeled GTP is probably due to structural difference between RhoA·GTP and RhoA·BP-GTP. A hydrogen bond network is formed between 2′-OH of GTP and Cys20 and Pro31 of RhoA47. BP-GTP is a mixture of 2′- and 3′-isoforms and 2′-OH is blocked in the 2′-isoform.
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Related In: Results  -  Collection

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f3-3_63: Interaction of BP-GTP·RhoA with an effector protein Rhotekin. (A) A GST fusion of the Rho-binding domain (RBD) of Rhotekin was attached on GSH-beads. MBP-RhoA was incubated with the beads in the presence of various concentrations of BP-GTP. Proteins bound to the beads were dissociated from the beads with a buffer containing 20 mM glutathione and analyzed by SDS-PAGE. Arrow-heads represent MBP-RhoA (silver-stained) bound to GST-Rhotekin. Arrows represent GST-Rhotekin (CBB-stained). (B) The amount of MBP-RhoA bound to GST-Rhotekin was plotted as a function of the BP-GTP concentration. The amount of MBP-RhoA was normalized to that in the presence of 1 μM BP-GTP after subtraction of the non-specific binding observed in the absence of BP-GTP. The average and SE of four independent experiments are shown. Similar results were obtained using RBD of Rho-kinase instead of RBD of Rhotekin (data not shown). The difference between the amount of the bound MBP-RhoA with BP-GTP and that with non-labeled GTP is probably due to structural difference between RhoA·GTP and RhoA·BP-GTP. A hydrogen bond network is formed between 2′-OH of GTP and Cys20 and Pro31 of RhoA47. BP-GTP is a mixture of 2′- and 3′-isoforms and 2′-OH is blocked in the 2′-isoform.
Mentions: To examine the activity of RhoA after association with BP-GTP, a pull-down assay of MBP-RhoA·BP-GTP was carried out using an effector protein bound to GSH-beads (Fig. 3). Depending on the dose of BP-GTP, MBP-RhoA bound with the Rho binding domain of Rhotekin fused with GST. This suggests that the FRET indicates the production of active Rho. A BP-GTP dependent interaction of MBP-YFP-Rac1 with the GST fused Rac binding domain of p21-activated kinase (PAK) was also observed (data not shown).

View Article: PubMed Central - PubMed

ABSTRACT

RhoA and Rac1 are small GTPases primarily involved in cytoskeletal remodeling. Many biochemical studies have suggested that they are also key organizers of cell-substrate adhesion. Recently, fluorescence resonance energy transfer (FRET)-based indicators have been developed to visualize RhoA and Rac1 activity in living cells [Yoshizaki et al., J. Cell Biol. 162, 223 (2003); Pertz et al., Nature 440, 1069 (2006)]. These indicators use one of the interactions between RhoA (Rac1) and the RhoA (Rac1)-binding domain of their effector proteins. However, distribution of RhoA activity in single cells has not yet been observed with micrometer-scale resolution. Here, we employed an approach that detects GDP/GTP exchange on small GTPases by using FRET from YFP-fused small GTPases to a fluorescent analogue of GTP, BODIPY(TR)-GTP. This approach allowed us to visualize confined localization of active (GTP-bound forms of) RhoA and Rac1 in individual focal adhesions. Activated RhoA accumulated in immobile and long-lived focal adhesions but was not evident in unstable and temporary adhesions, while activated Rac1 was observed at every adhesion. Our results suggest that RhoA is the major regulator determining the stability of individual cell adhesion structures.

No MeSH data available.


Related in: MedlinePlus