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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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A FRET-based detection of GDP/GTP exchange on RhoA. (A) The principle for detecting GDP/GTP exchange on RhoA small GTPase based on FRET from YFP fused to RhoA (YFP-RhoA) to a fluorescent analogue of GTP, BODIPY(TR)-GTP (BP-GTP). (B) Fluorescence emission spectra of GST-YFP-RhoA (0.24 μM) in the presence of various concentrations of BP-GTP. The excitation wavelength was 488 nm. The dotted line is the emission spectrum from 0.24 μM BP-GTP excited at 488 nm in the absence of GST-YFP-RhoA. All spectra are the averages from three independent experiments.
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f1-3_63: A FRET-based detection of GDP/GTP exchange on RhoA. (A) The principle for detecting GDP/GTP exchange on RhoA small GTPase based on FRET from YFP fused to RhoA (YFP-RhoA) to a fluorescent analogue of GTP, BODIPY(TR)-GTP (BP-GTP). (B) Fluorescence emission spectra of GST-YFP-RhoA (0.24 μM) in the presence of various concentrations of BP-GTP. The excitation wavelength was 488 nm. The dotted line is the emission spectrum from 0.24 μM BP-GTP excited at 488 nm in the absence of GST-YFP-RhoA. All spectra are the averages from three independent experiments.

Mentions: Here, we have devised a technique that directly demonstrates the GDP/GTP exchange on small GTPases, in which FRET from YFP-fused small GTPases to a fluorescent analogue of GTP, BODIPY(TR)-GTP (BP-GTP) was used (Fig. 1A). Application of a similar technique has already been described by us28–30 and by another group31. In this report, we describe the detail of the technique and application of the technique to visualize the activation of RhoA during cytoskeletal remodeling of PC12D cells with high spatial resolution. FRET signals from both RhoA and Rac1 were confined in micrometer scale regions at the cell-substratum adhesion. FRET signals of RhoA accumulated only in long-lived focal adhesion but those of Rac1 were observed in all vinculin-positive adhesions. These results suggest that Rho is the major regulator in determining the stability of individual cell adhesion structures.


Rho small GTPase regulates the stability of individual focal adhesions: a FRET-based visualization of GDP/GTP exchange on small GTPases
A FRET-based detection of GDP/GTP exchange on RhoA. (A) The principle for detecting GDP/GTP exchange on RhoA small GTPase based on FRET from YFP fused to RhoA (YFP-RhoA) to a fluorescent analogue of GTP, BODIPY(TR)-GTP (BP-GTP). (B) Fluorescence emission spectra of GST-YFP-RhoA (0.24 μM) in the presence of various concentrations of BP-GTP. The excitation wavelength was 488 nm. The dotted line is the emission spectrum from 0.24 μM BP-GTP excited at 488 nm in the absence of GST-YFP-RhoA. All spectra are the averages from three independent experiments.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC5036652&req=5

f1-3_63: A FRET-based detection of GDP/GTP exchange on RhoA. (A) The principle for detecting GDP/GTP exchange on RhoA small GTPase based on FRET from YFP fused to RhoA (YFP-RhoA) to a fluorescent analogue of GTP, BODIPY(TR)-GTP (BP-GTP). (B) Fluorescence emission spectra of GST-YFP-RhoA (0.24 μM) in the presence of various concentrations of BP-GTP. The excitation wavelength was 488 nm. The dotted line is the emission spectrum from 0.24 μM BP-GTP excited at 488 nm in the absence of GST-YFP-RhoA. All spectra are the averages from three independent experiments.
Mentions: Here, we have devised a technique that directly demonstrates the GDP/GTP exchange on small GTPases, in which FRET from YFP-fused small GTPases to a fluorescent analogue of GTP, BODIPY(TR)-GTP (BP-GTP) was used (Fig. 1A). Application of a similar technique has already been described by us28–30 and by another group31. In this report, we describe the detail of the technique and application of the technique to visualize the activation of RhoA during cytoskeletal remodeling of PC12D cells with high spatial resolution. FRET signals from both RhoA and Rac1 were confined in micrometer scale regions at the cell-substratum adhesion. FRET signals of RhoA accumulated only in long-lived focal adhesion but those of Rac1 were observed in all vinculin-positive adhesions. These results suggest that Rho is the major regulator in determining the stability of individual cell adhesion structures.

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