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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.


FRET signals of RhoA in a PC12D cell stimulated with NGF. (A) Intracellular distribution of FRET signals were observed in a cell stimulated with 50 ng ml−1 NGF. BP-GTP was microinjected into the cell 10 min prior to the stimulation with NGF starting at time 0. Upper row: fluorescence from YFP-Rho. Middle row: FRET signals. Lower row: merged images of YFP Rho (green) and FRET signals (red). White arrows indicate positions with prominent FRET signals. Red arrowheads indicate locations of evident morphological changes of the cell observed after stimulation with NGF. (B) Co-localization of FRET signals with the vinculin. The same cell shown in (A) were fixed with paraformaldehyde 20 min after stimulation, incubated with anti-vinculin antibody, and stained with a secondary antibody conjugated with Alexa555. Upper: the image of vinculin staining. Lower: the merged image of FRET at 20 min (red) and the vinculin staining (green). Arrows indicate co-localization of FRET signal and vinculin staining. Arrowheads indicate vinculin staining not co-localized with a FRET signal.
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f5-3_63: FRET signals of RhoA in a PC12D cell stimulated with NGF. (A) Intracellular distribution of FRET signals were observed in a cell stimulated with 50 ng ml−1 NGF. BP-GTP was microinjected into the cell 10 min prior to the stimulation with NGF starting at time 0. Upper row: fluorescence from YFP-Rho. Middle row: FRET signals. Lower row: merged images of YFP Rho (green) and FRET signals (red). White arrows indicate positions with prominent FRET signals. Red arrowheads indicate locations of evident morphological changes of the cell observed after stimulation with NGF. (B) Co-localization of FRET signals with the vinculin. The same cell shown in (A) were fixed with paraformaldehyde 20 min after stimulation, incubated with anti-vinculin antibody, and stained with a secondary antibody conjugated with Alexa555. Upper: the image of vinculin staining. Lower: the merged image of FRET at 20 min (red) and the vinculin staining (green). Arrows indicate co-localization of FRET signal and vinculin staining. Arrowheads indicate vinculin staining not co-localized with a FRET signal.

Mentions: Our technique was used to visualize the correlation between cell-substratum adhesion and activation of RhoA and Rac1 in PC12D cells. PC12D is a flat-shaped variant of the PC12 cell sub-cloned by Sano35. PC12D continually changes its shape even in conditions without any stimulation, remodeling the cell-substratum adhesion structures. PC12D cells do not show thick stress fibers (data not shown) and show only sparse vinculin-positive anchoring apparatus, i.e., focal complexes and focal adhesions (Figs. 5 and 7). These characteristics make it easier for us to observe the activities of the Rho family at individual focal complexes or focal adhesions.


Rho small GTPase regulates the stability of individual focal adhesions: a FRET-based visualization of GDP/GTP exchange on small GTPases
FRET signals of RhoA in a PC12D cell stimulated with NGF. (A) Intracellular distribution of FRET signals were observed in a cell stimulated with 50 ng ml−1 NGF. BP-GTP was microinjected into the cell 10 min prior to the stimulation with NGF starting at time 0. Upper row: fluorescence from YFP-Rho. Middle row: FRET signals. Lower row: merged images of YFP Rho (green) and FRET signals (red). White arrows indicate positions with prominent FRET signals. Red arrowheads indicate locations of evident morphological changes of the cell observed after stimulation with NGF. (B) Co-localization of FRET signals with the vinculin. The same cell shown in (A) were fixed with paraformaldehyde 20 min after stimulation, incubated with anti-vinculin antibody, and stained with a secondary antibody conjugated with Alexa555. Upper: the image of vinculin staining. Lower: the merged image of FRET at 20 min (red) and the vinculin staining (green). Arrows indicate co-localization of FRET signal and vinculin staining. Arrowheads indicate vinculin staining not co-localized with a FRET signal.
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Related In: Results  -  Collection

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f5-3_63: FRET signals of RhoA in a PC12D cell stimulated with NGF. (A) Intracellular distribution of FRET signals were observed in a cell stimulated with 50 ng ml−1 NGF. BP-GTP was microinjected into the cell 10 min prior to the stimulation with NGF starting at time 0. Upper row: fluorescence from YFP-Rho. Middle row: FRET signals. Lower row: merged images of YFP Rho (green) and FRET signals (red). White arrows indicate positions with prominent FRET signals. Red arrowheads indicate locations of evident morphological changes of the cell observed after stimulation with NGF. (B) Co-localization of FRET signals with the vinculin. The same cell shown in (A) were fixed with paraformaldehyde 20 min after stimulation, incubated with anti-vinculin antibody, and stained with a secondary antibody conjugated with Alexa555. Upper: the image of vinculin staining. Lower: the merged image of FRET at 20 min (red) and the vinculin staining (green). Arrows indicate co-localization of FRET signal and vinculin staining. Arrowheads indicate vinculin staining not co-localized with a FRET signal.
Mentions: Our technique was used to visualize the correlation between cell-substratum adhesion and activation of RhoA and Rac1 in PC12D cells. PC12D is a flat-shaped variant of the PC12 cell sub-cloned by Sano35. PC12D continually changes its shape even in conditions without any stimulation, remodeling the cell-substratum adhesion structures. PC12D cells do not show thick stress fibers (data not shown) and show only sparse vinculin-positive anchoring apparatus, i.e., focal complexes and focal adhesions (Figs. 5 and 7). These characteristics make it easier for us to observe the activities of the Rho family at individual focal complexes or focal adhesions.

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.