Limits...
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.


Related in: MedlinePlus

Mobility of the accumulation of YFP-RhoA·BP-GTP. Time-lapse images of FRET signals of a PC12D cell expressing YFP-RhoA are shown. The cell was injected with BP-GTP and stimulated with NGF 10 min after the injection. Time 0 is the start point of the NGF stimulation. FRET signals were slight immediately after the injection (E). YFP-RhoA showed a defuse distribution in the cytoplasm throughout the observation as shown in (F). A bright spot of FRET was observed on the dorsal membrane when stimulation with NGF was started (arrows in A). The spot was immobile for 10 minutes but increased its FRET intensity (arrows in B–D). The microscope was focused on the dorsal cell surface in images A–F. FRET signals were weak at 2.5 μm above the substratum (G). Bar=10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC5036652&req=5

f6-3_63: Mobility of the accumulation of YFP-RhoA·BP-GTP. Time-lapse images of FRET signals of a PC12D cell expressing YFP-RhoA are shown. The cell was injected with BP-GTP and stimulated with NGF 10 min after the injection. Time 0 is the start point of the NGF stimulation. FRET signals were slight immediately after the injection (E). YFP-RhoA showed a defuse distribution in the cytoplasm throughout the observation as shown in (F). A bright spot of FRET was observed on the dorsal membrane when stimulation with NGF was started (arrows in A). The spot was immobile for 10 minutes but increased its FRET intensity (arrows in B–D). The microscope was focused on the dorsal cell surface in images A–F. FRET signals were weak at 2.5 μm above the substratum (G). Bar=10 μm.

Mentions: Within 10 minutes of the microinjection of BP-GTP, FRET signals appeared as spot-like distributions focused on the dorsal membrane, independent of any stimulation of cells (Figs. 4 and 6, and Supplement Fig. S1). FRET signals were slight in other regions of the cell. These FRET signals were caused by the spontaneous activation of RhoA in resting cells (Supplement Fig. S1). No FRET signal was observed in cells treated with genistein, an inhibitor of tyrosine kinases, or in cells microinjected with a dominant negative form of RhoA protein (GDP·RhoAAsn19; data not shown).


Rho small GTPase regulates the stability of individual focal adhesions: a FRET-based visualization of GDP/GTP exchange on small GTPases
Mobility of the accumulation of YFP-RhoA·BP-GTP. Time-lapse images of FRET signals of a PC12D cell expressing YFP-RhoA are shown. The cell was injected with BP-GTP and stimulated with NGF 10 min after the injection. Time 0 is the start point of the NGF stimulation. FRET signals were slight immediately after the injection (E). YFP-RhoA showed a defuse distribution in the cytoplasm throughout the observation as shown in (F). A bright spot of FRET was observed on the dorsal membrane when stimulation with NGF was started (arrows in A). The spot was immobile for 10 minutes but increased its FRET intensity (arrows in B–D). The microscope was focused on the dorsal cell surface in images A–F. FRET signals were weak at 2.5 μm above the substratum (G). Bar=10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

f6-3_63: Mobility of the accumulation of YFP-RhoA·BP-GTP. Time-lapse images of FRET signals of a PC12D cell expressing YFP-RhoA are shown. The cell was injected with BP-GTP and stimulated with NGF 10 min after the injection. Time 0 is the start point of the NGF stimulation. FRET signals were slight immediately after the injection (E). YFP-RhoA showed a defuse distribution in the cytoplasm throughout the observation as shown in (F). A bright spot of FRET was observed on the dorsal membrane when stimulation with NGF was started (arrows in A). The spot was immobile for 10 minutes but increased its FRET intensity (arrows in B–D). The microscope was focused on the dorsal cell surface in images A–F. FRET signals were weak at 2.5 μm above the substratum (G). Bar=10 μm.
Mentions: Within 10 minutes of the microinjection of BP-GTP, FRET signals appeared as spot-like distributions focused on the dorsal membrane, independent of any stimulation of cells (Figs. 4 and 6, and Supplement Fig. S1). FRET signals were slight in other regions of the cell. These FRET signals were caused by the spontaneous activation of RhoA in resting cells (Supplement Fig. S1). No FRET signal was observed in cells treated with genistein, an inhibitor of tyrosine kinases, or in cells microinjected with a dominant negative form of RhoA protein (GDP·RhoAAsn19; 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