Dataset Information

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

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.

SUBMITTER: Miyauchi T

PROVIDER: S-EPMC5036652 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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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.

Miyauchi Takayuki T Yanagida Toshio T Sako Yasushi Y

Biophysics (Nagoya-shi, Japan) 20071228

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 <i>et al.</i>, J. Cell Biol. 162, 223 (2003); Pertz <i>et al.</i>, Nature 440, 1069 (2006)]. These indicators use one of the interactions between RhoA (Rac1) and t ...[more]

PMID: 27857568

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Project description:The Plexin family of transmembrane receptors are unique in that their intracellular region interacts directly with small GTPases of the Rho family. The Rho GTPase binding domain of plexin (RBD)-which is responsible for these interactions-can bind with Rac1 as well as Rnd1 GTPases. GTPase complexes have been crystallized with the RBDs of plexinA1, -A2, and -B1. The protein association is thought to elicit different functional responses in a GTPase and plexin isoform specific manner, but the origin of this is unknown. In this project, we investigated complexes between several RBD and Rac1/Rnd1 GTPases using multimicrosecond length all atom molecular dynamics simulations, also with reference to the free forms of the RBDs and GTPases. In accord with the crystallographic data, the RBDs experience more structural changes than Rho-GTPases upon complex formation. Changes in protein dynamics and networks of correlated motions are revealed by analyzing dihedral angle fluctuations in the proteins. The extent of these changes differs between the different RBDs and also between the Rac1 and Rnd1 GTPases. While the RBDs in the free and bound states have similar-if not decreased-correlations, correlations within the GTPases are increased upon binding. Mapping highly correlated residues to the structures, it is found that the plexinA1, -B1, and -A2 RBDs all have similar communication pathways within the ubiquitin fold, but that different residues are involved. Dynamic network analyses indicate that plexinA1 and -B1 RBDs interact with small GTPases in a similar manner, whereas complexes with the plexinA2 RBD display different features. Importantly complexes with Rnd1 have a considerable number of dynamic correlations and network connections between the proteins, whereas such features are missing in the RBD-Rac1 complexes. Overall, the simulations suggest mechanisms that are consistent with the experimental data on plexinB1 and indicate RBD and GTPase isoform specific changes in protein dynamics upon complex formation.

Project description:Archaeal initiation factor 2 (aIF2) is a protein involved in the initiation of protein biosynthesis. In its GTP-bound, "ON" conformation, aIF2 binds an initiator tRNA and carries it to the ribosome. In its GDP-bound, "OFF" conformation, it dissociates from tRNA. To understand the specific binding of GTP and GDP and its dependence on the ON or OFF conformational state of aIF2, molecular dynamics free energy simulations (MDFE) are a tool of choice. However, the validity of the computed free energies depends on the simulation model, including the force field and the boundary conditions, and on the extent of conformational sampling in the simulations. aIF2 and other GTPases present specific difficulties; in particular, the nucleotide ligand coordinates a divalent Mg(2+) ion, which can polarize the electronic distribution of its environment. Thus, a force field with an explicit treatment of electronic polarizability could be necessary, rather than a simpler, fixed charge force field. Here, we begin by comparing a fixed charge force field to quantum chemical calculations and experiment for Mg(2+):phosphate binding in solution, with the force field giving large errors. Next, we consider GTP and GDP bound to aIF2 and we compare two fixed charge force fields to the recent, polarizable, AMOEBA force field, extended here in a simple, approximate manner to include GTP. We focus on a quantity that approximates the free energy to change GTP into GDP. Despite the errors seen for Mg(2+):phosphate binding in solution, we observe a substantial cancellation of errors when we compare the free energy change in the protein to that in solution, or when we compare the protein ON and OFF states. Finally, we have used the fixed charge force field to perform MDFE simulations and alchemically transform GTP into GDP in the protein and in solution. With a total of about 200 ns of molecular dynamics, we obtain good convergence and a reasonable statistical uncertainty, comparable to the force field uncertainty, and somewhat lower than the predicted GTP/GDP binding free energy differences. The sign and magnitudes of the differences can thus be interpreted at a semiquantitative level, and are found to be consistent with the experimental binding preferences of ON- and OFF-aIF2.

Dataset Information

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

Publications

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

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