RhoE regulates actin cytoskeleton organization and cell migration

Abstract

The actin cytoskeleton is regulated by Rho family proteins: in fibroblasts, Rho mediates the formation of actin stress fibers, whereas Rac regulates lamellipodium formation and Cdc42 controls filopodium formation. We have cloned the mouse RhoE gene, whose product is a member of the Rho family that shares (except in one amino acid) the conserved effector domain of RhoA, RhoB, and RhoC. RhoE is able to bind GTP but does not detectably bind GDP and has low intrinsic GTPase activity compared with Rac. The role of RhoE in regulating actin organization was investigated by microinjection in Bac1.2F5 macrophages and MDCK cells. In macrophages, RhoE induced actin reorganization, leading to the formation of extensions resembling filopodia and pseudopodia. In MDCK cells, RhoE induced the complete disappearance of stress fibers, together with cell spreading. However, RhoE did not detectably affect the actin bundles that run parallel to the outer membranes of cells at the periphery of colonies, which are known to be dependent on RhoA. In addition, RhoE induced an increase in the speed of migration of hepatocyte growth factor/scatter factor-stimulated MDCK cells, in contrast to the previously reported inhibition produced by activated RhoA. The subcellular localization of RhoE at the lateral membranes of MDCK cells suggests a role in cell-cell adhesion, as has been shown for RhoA. These results suggest that RhoE may act to inhibit signalling downstream of RhoA, altering some RhoA-regulated responses, such as stress fiber formation, but not affecting others, such as peripheral actin bundle formation.

FIG. 1

Phylogenetic analysis of Rho family proteins, including percentages of homology between mouse RhoE (asterisk) and the other proteins. Mouse RhoE cDNA was identified by screening of a 10-dpc mouse embryo library. It shares the five conserved GTP-binding domains that are found in all other Ras-related proteins and are known to be important for their function. It exhibits very strong sequence similarity to Rho6, Rho7, and Rho8. Sequences were aligned with the Geneworks program by use of the Clustal algorithm and a PAM250 table.

FIG. 2

RhoE binds and hydrolyzes GTP, but the intrinsic GTPase activity is low compared to that of other Rho proteins. (A) GTP-binding assay. Recombinant RhoE was expressed as a GST fusion protein in a protease-deficient strain (BL21), bound to glutathione-agarose beads, and cleaved with thrombin. RhoE at the indicated concentrations was incubated at 37°C with [³H]GTP for 30 min, and radioactivity bound to the protein was determined by a filter-binding assay. (B) Comparison of GTP and GDP binding. A nucleotide-binding assay with [³H]GDP showed background levels of radioactivity when GDP was incubated with RhoE at 25 μg/ml. (C) Intrinsic GTPase activity of thrombin-cleaved RhoE and Rac. Each protein was preloaded with [γ-³²P]GTP, incubated at 37°C in the presence of excess unlabeled GTP for the indicated times, and subjected to a filter-binding assay. The amount of radioactivity remaining bound to each protein was determined by scintillation counting. RhoEm, mouse RhoE.

FIG. 3

RhoE induces the disappearance of stress fibers in MDCK cells. Confocal micrographs of cells injected with recombinant RhoE at 25 μg/ml and rat IgG (0.5 mg/ml) (A, B, C, and D) or rat IgG alone at 0.5 mg/ml (E and F) (as a marker to identify injected cells) are shown. Actin filaments were localized (B, D, and F) by staining with TRITC-labelled phalloidin 30 min after RhoE injection at both the periphery (B) and within a cell colony (D) or after injection with IgG alone (F). Microinjected cells were detected with FITC-labelled goat anti-rat IgG (A, C, and E). Arrow in panel B, RhoE-injected cell with a lamellipodium. The scale bar represents 42 μm (A and B) or 25 μm (C, D, E, and F).

FIG. 4

Immunolocalization of RhoE in MDCK cells. A eukaryotic expression vector, pEXV3, containing myc epitope-tagged RhoE cDNA was microinjected into the nuclei of MDCK cells. Cells were analyzed by immunofluorescence to detect myc epitope-tagged RhoE with anti-myc epitope antibody 9E10 followed by incubation with FITC-conjugated goat anti-mouse IgG (A, C, and D) RhoE and TRITC-labelled phalloidin (B) to detect actin filaments. (A and B) Confocal images show that RhoE was localized mainly at the level of the plasma membrane as well as in some structures (arrows) inside the cytoplasm (A) that colocalized with aggregates of actin filaments (B). (C and D) Images show RhoE localization in basal (C) and near-apical (D) optical sections of the left-hand cell in panel A. The scale bar represents 25 μm (A and B) or 15 μm (C and D).

FIG. 5

Actin reorganization induced by RhoE in Bac1.2F5 macrophages. Confocal micrographs show growing (A) and CSF-1-starved (B) cells, cells injected with recombinant RhoE protein at 25 μg/ml (C and D), cells injected with rat IgG at 0.5 mg/ml as a control (E and F), and cells injected with expression vector pEXV3-myc-RhoE at 0.3 mg/ml (G and H). Bac1.2F5 cells were stained with TRITC-labelled phalloidin to reveal actin filaments (A, B, C, D, E, and G), with FITC-labeled goat anti-rabbit IgG (F) to detect microinjected cells, or with anti-myc epitope antibody 9E10 followed by FITC-labeled goat anti-mouse IgG (H). (H) A three-dimensional reconstruction shows the subcellular localization of RhoE 4 h after DNA microinjection. A maximum-intensity projection through a 768- by 512- by 25-voxel data set was generated with the intermediate axis (512 voxels) tilted 15°. Arrows indicate injected cells. The scale bar represents 20 μm (A, C, E, F, and G), 28 μm (B), 17 μm (D), or 14 μm (H).

FIG. 6

Enhancement of cell migration speed by RhoE. Cell migration was monitored by tracking injected cells from 5 to 15 h after the injection of RhoE. All cells monitored were microinjected with either RhoE (25 μg/ml) or rabbit IgG (1 mg/ml) as a control protein. HGF/SF (5 ng/ml) was added to MDCK cells 15 min after microinjection, and cells were monitored by time-lapse videomicroscopy. Ten cells were randomly selected, and the positions of their nuclei were tracked every hour. For MDCK cells, five cells at the outer edge and five cells within a colony were tracked. When a cell divided, both progeny were monitored and the mean of the distances that they moved was calculated. The final mean distance migrated was calculated from the average distance moved by each of the 10 cells per hour. The error bars represent the standard deviations of the means (four or five independent experiments for MDCK cells and two for R4.2 cells). The difference in migration rates between RhoE-injected cells and control cells was statistically significant (P ≤ 0.001 for MDCK and P ≤ 0.04 for R4.2).

FIG. 7

Comparison of the amino acid sequences of RhoA and RhoE in the effector domain and the insert helix. Sequence alignment between these two proteins showed very similar effector domains (except for one amino acid) but many differences in the insert helices. Letters in white represent identical residues.