RhoB regulates cell migration through altered focal adhesion dynamics

Abstract

The Rho GTPase RhoB has been shown to affect cell migration, but how it does this is not clear. Here we show that cells depleted of RhoB by RNAi are rounded and have defects in Rac-mediated spreading and lamellipodium extension, although they have active membrane ruffling around the periphery. Depletion of the exchange factor GEF-H1 induces a similar phenotype. RhoB-depleted cells migrate faster, but less persistently in a chemotactic gradient, and frequently round up during migration. RhoB-depleted cells have similar numbers of focal adhesions to control cells during spreading and migration, but show more diffuse and patchy contact with the substratum. They have lower levels of surface β1 integrin, and β1 integrin activity is reduced in actin-rich protrusions. We propose that RhoB contributes to directional cell migration by regulating β1 integrin surface levels and activity, thereby stabilizing lamellipodial protrusions.

Keywords: Rac1; Rho guanosine triphosphate; RhoB; cytoskeleton; focal adhesions; integrins.

Figure 1.

RhoB affects cell morphology. (a) Quantification of cell area and cell perimeter of siRNA-transfected PC3 cells (mean ± s.d. from four different experiments and >200 cells). Black bars denote siControl; light grey bars denote siRhoB-1; dark grey bars denote siRhoB-2. Values are shown relative to siControl (**p < 0.01). (b) Phase-contrast images of RhoB-depleted or control cells from the indicated cell lines. Scale bars, 40 µm. (c) Orthogonal view of a representative siControl or siRhoB-treated cell. Scale bars, z = 9.5 µm. (d) F-actin distribution in siRNA-transfected PC3 cells plated on uncoated plastic or glass, or plastic coated with Matrigel or fibronectin. Scale bars, 20 µm.

Figure 2.

RhoB knockdown impairs spreading. (a) PC3 cell adhesion after 20 min and (b) spread area after 30 min on uncoated plastic, or plastic coated with Matrigel or fibronectin. Black bars denote siControl, grey bars denote siRhoB. Graphs show mean ± s.e.m. (n = 5 with >100 cells per condition; **p < 0.01). (c) Analysis of β1 and β3 integrin cell surface expression in PC3 cells. Surface proteins were biotinylated, pulled down with streptavidin beads and then analysed by immunoblotting with β1 (black bars) and β3 (grey bars) integrin antibodies and GADPH antibodies as a loading control. (i) Representative Western blot; (ii) mean levels ± s.e.m. of surface integrin levels relative to total and expressed as percentage of siControl-1 levels. n = 6. *p < 0.05, **p < 0.01.

Figure 3.

RhoB is required for Rac1-driven lamellipodial protrusion. (a) (i) Kymographs of representative lamellipodial regions of migrating siRNA-transfected PC3 cells from the electronic supplementary material, movie S1. Black bars denote kymograph region. Scale bars, 20 µm. (ii) Lamellipodial protrusion rate measured from five to seven cells per condition, mean ± s.d. (b) (i) Representative images and (ii) quantification of GFP-positive PC3 cells after transfection with GFP-Rac1-L61 or control GFP and the indicated siRNAs. Scale bars, 20 µm. n > 300 cells per condition from three independent experiments; boxes of box and whisker plots show median, 25th and 75th percentile; whiskers show 95th percentile; **p < 0.01, ***p < 0.001.

Figure 4.

RhoB affects cell migration speed and directionality. (a) Migration speed of siRNA-transfected PC3 cells on tissue culture-treated plastic. n > 50 cells per condition from three different experiments. Results with two different siRNAs for RhoB and two siControl oligos are pooled; cells were tracked for 12–14 h, 58–60 h after transfection. (b) Montage from the electronic supplementary material, movie S3 showing representative GFP-actin-expressing PC3 cells. Tracks panels (right) show movement of the shown cells over time. Scale bars, 20 µm. (c) Forward migration index (FMI) and directionality of siRNA-transfected PC3 cells in a chemotaxis assay. n > 200 cells per condition from four different experiments using two different siRNAs for RhoB and two siControl oligos. Boxes of box and whisker plots show median, 25th and 75th percentile; whiskers show 95th percentile; ***p < 0.001. Rose plots show the proportion of cells with a migration direction lying within each 20° interval. Red arrow represents mean direction of cell migration; green segment, 95% CI.

Figure 5.

RhoB and GEF-H1 affect cell morphology, but not microtubule dynamics. (a) Confocal images showing F-actin and α-tubulin distribution in PC3 cells transfected with siRNAs targeting GEF-H1, RhoA or control siRNA. Scale bars, 20 µm. (b) Graph shows quantification of cell area (mean ± s.e.m.) relative to siControl from two different experiments with >40 cells per condition; **p < 0.01. (c) Quantification of microtubule tips per unit area from movies of EB3-GFP-expressing RhoB-depleted or control cells. Graph shows mean ± s.e.m. from 10 cells in two different experiments.

Figure 6.

Effects of mDia1 depletion on PC3 cells. Cells were transfected with two different siRNAs targeting mDia1, RhoB or control siRNA. Confocal images show F-actin and α-tubulin distribution. Scale bars, 20 µm.

Figure 7.

RhoB regulates focal adhesion dynamics. (a) Serum-starved PC3 cells stimulated with 10% FCS for 1 h to promote spreading were stained for F-actin and with antibodies to phospho-paxillin to show focal adhesions and focal complexes. Boxed regions (middle panels) are shown at higher magnification in the right panels. (b,c) Graphs show the number of phospho-paxillin-positive focal adhesions/focal complexes (FA) per cell or area occupied by FA relative to the total spread area. (d) PC3 cells under normal migratory conditions (1% FCS) were stained for F-actin and with antibodies to phospho-paxillin to show FA. Boxed regions (middle panels) are shown at higher magnification in the right panels. (e,f) Number of phospho-paxillin-positive FA per cell or area occupied by FA relative to the total spread area in normal migratory conditions. Results in graphs are from at least 10 cells from two different experiments. Boxes of box and whisker plots show median, 25th and 75th percentile; whiskers show 95th percentile; ***p < 0.001, **p < 0.01, *p < 0.05. (g) (i) Representative interference reflection microscopy images of PC3 cells after siRNA transfection. (ii) Dynamics images show a thresholded maximal projection of the same cells over 70 frames (15 s frame⁻¹). (iii) Detail: enlargement of boxed regions in (ii). (iv) Graph shows percentage of cells with stable focal contacts in interference reflection microscopy movies; n > 20 cells per condition. (a,d,g) Scale bars, 20 µm.

Figure 8.

RhoB affects β1 integrin activity in protrusions. (a) Confocal images showing active β1 integrin staining and F-actin. Boxed regions (merge panels) are shown at higher magnification in the right panels. Scale bars, 20 µm. (b) Quantification of active β1 integrin intensity in protrusions. Arb. units denotes arbitrary units. n = 18 cells from two different experiments; **p < 0.01.