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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.

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Competition between BP-GTP and non-labeled GTP in GEF-induced GDP/GTP exchange on YFP-RhoA. (A) Normalized fluorescence emission spectra from the mixtures of YFP-RhoA, BP-GTP, and non-labeled GTP in the presence or absence of PH/DH domain of Vav1, a GEF for RhoA. Spectra at different time points after mixing are shown. The concentration of non-labeled GTP was varied in the presence of Vav1. (B) Changes in YFP fluorescence (530 nm) in the spectra shown in (A) were plotted to show the time course of the GDP/GTP exchange. A decrease in the YFP fluorescence indicates BP-GTP binding on YFP-Rho. Averages of four independent experiments are plotted with SE. Similar results were obtained using PH/DH-domains of LARG41, another GEF for Rho, instead of Vav1 (data not shown).
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f2-3_63: Competition between BP-GTP and non-labeled GTP in GEF-induced GDP/GTP exchange on YFP-RhoA. (A) Normalized fluorescence emission spectra from the mixtures of YFP-RhoA, BP-GTP, and non-labeled GTP in the presence or absence of PH/DH domain of Vav1, a GEF for RhoA. Spectra at different time points after mixing are shown. The concentration of non-labeled GTP was varied in the presence of Vav1. (B) Changes in YFP fluorescence (530 nm) in the spectra shown in (A) were plotted to show the time course of the GDP/GTP exchange. A decrease in the YFP fluorescence indicates BP-GTP binding on YFP-Rho. Averages of four independent experiments are plotted with SE. Similar results were obtained using PH/DH-domains of LARG41, another GEF for Rho, instead of Vav1 (data not shown).

Mentions: As shown in Figure 2, FRET was generated in a GEF-dependent manner. The FRET signal decreased in inverse proportion to the concentration of non-labeled GTP. This result indicates that GDP/GTP exchange was stimulated by GEF and that the binding of BP-GTP after the dissociation of GDP was competitively inhibited by the binding of non-labeled GTP. The inhibition rates inversely proportional to the concentration of non-labeled GTP at every time point of the binding timecourses (Fig. 2B) indicate that both the affinity and the on-rate of BP-GTP to YFP-RhoA were similar to those of non-labeled GTP. Other GEFs — Dbl and LARG — also increased FRET signal (data not shown). These results lead to the conclusion that YFP-RhoA with BP-GTP is useful as an indicator of GDP/GTP exchange on RhoA.


Rho small GTPase regulates the stability of individual focal adhesions: a FRET-based visualization of GDP/GTP exchange on small GTPases
Competition between BP-GTP and non-labeled GTP in GEF-induced GDP/GTP exchange on YFP-RhoA. (A) Normalized fluorescence emission spectra from the mixtures of YFP-RhoA, BP-GTP, and non-labeled GTP in the presence or absence of PH/DH domain of Vav1, a GEF for RhoA. Spectra at different time points after mixing are shown. The concentration of non-labeled GTP was varied in the presence of Vav1. (B) Changes in YFP fluorescence (530 nm) in the spectra shown in (A) were plotted to show the time course of the GDP/GTP exchange. A decrease in the YFP fluorescence indicates BP-GTP binding on YFP-Rho. Averages of four independent experiments are plotted with SE. Similar results were obtained using PH/DH-domains of LARG41, another GEF for Rho, instead of Vav1 (data not shown).
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Related In: Results  -  Collection

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f2-3_63: Competition between BP-GTP and non-labeled GTP in GEF-induced GDP/GTP exchange on YFP-RhoA. (A) Normalized fluorescence emission spectra from the mixtures of YFP-RhoA, BP-GTP, and non-labeled GTP in the presence or absence of PH/DH domain of Vav1, a GEF for RhoA. Spectra at different time points after mixing are shown. The concentration of non-labeled GTP was varied in the presence of Vav1. (B) Changes in YFP fluorescence (530 nm) in the spectra shown in (A) were plotted to show the time course of the GDP/GTP exchange. A decrease in the YFP fluorescence indicates BP-GTP binding on YFP-Rho. Averages of four independent experiments are plotted with SE. Similar results were obtained using PH/DH-domains of LARG41, another GEF for Rho, instead of Vav1 (data not shown).
Mentions: As shown in Figure 2, FRET was generated in a GEF-dependent manner. The FRET signal decreased in inverse proportion to the concentration of non-labeled GTP. This result indicates that GDP/GTP exchange was stimulated by GEF and that the binding of BP-GTP after the dissociation of GDP was competitively inhibited by the binding of non-labeled GTP. The inhibition rates inversely proportional to the concentration of non-labeled GTP at every time point of the binding timecourses (Fig. 2B) indicate that both the affinity and the on-rate of BP-GTP to YFP-RhoA were similar to those of non-labeled GTP. Other GEFs — Dbl and LARG — also increased FRET signal (data not shown). These results lead to the conclusion that YFP-RhoA with BP-GTP is useful as an indicator of GDP/GTP exchange on RhoA.

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