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. 2004 Aug 2;166(3):317-23.
doi: 10.1083/jcb.200312168.

Active Rho is localized to podosomes induced by oncogenic Src and is required for their assembly and function

Rebecca L Berdeaux  1 , Begoña Díaz, Lomi Kim, G Steven Martin
Affiliations

Active Rho is localized to podosomes induced by oncogenic Src and is required for their assembly and function

Rebecca L Berdeaux et al. J Cell Biol. .

Abstract

Transformation of fibroblasts by oncogenic Src causes disruption of actin stress fibers and formation of invasive adhesions called podosomes. Because the small GTPase Rho stimulates stress fiber formation, Rho inactivation by Src has been thought to be necessary for stress fiber disruption. However, we show here that Rho[GTP] levels do not decrease after transformation by activated Src. Inactivation of Rho in Src-transformed fibroblasts by dominant negative RhoA or the Rho-specific inhibitor C3 exoenzyme disrupted podosome structure as judged by localization of podosome components F-actin, cortactin, and Fish. Inhibition of Rho strongly inhibited Src-induced proteolytic degradation of the extracellular matrix. Furthermore, development of an in situ Rho[GTP] affinity assay allowed us to detect endogenous Rho[GTP] at podosomes, where it colocalized with F-actin, cortactin, and Fish. Therefore, Rho is not globally inactivated in Src-transformed fibroblasts, but is necessary for the assembly and function of structures implicated in tumor cell invasion.

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Figures

Figure 1.
Figure 1.
Endogenous Rho is active in cells expressing oncogenic Src. (A–C) Rho affinity pull-down assays were used to determine the level of active Rho. In each panel, an anti-RhoA/B/C antibody was used to visualize Rho[GTP] in pellet fractions (top blot) or total Rho in 5% of the cell lysate (bottom blot). (A) Rho activity in duplicate samples of control (vector, lanes 1, 2, 5, and 6) and v-Src–transformed (lanes 3, 4, 7, and 8) NIH3T3 fibroblasts growing in 10% FCS under subconfluent (lanes 1–4) or confluent conditions (lanes 5–8). (B) Rho[GTP] levels in duplicate lysates from NIH3T3 fibroblasts expressing empty vector (lanes 1–2) or c-Src(Y527F) (lanes 3–4). (C) Rho pull-down assays of lysates from subconfluent cultures of NIH3T3 cells expressing ts-v-Src(tsLA90). Cells were serum-starved for 20 h at 39.5°C (lanes 1–8) or at 37°C (lane 9) and then shifted to 37°C for the indicated times.
Figure 2.
Figure 2.
Inhibition of Rho disrupts F-actin in Src-transformed fibroblasts. Src(Y527F) fibroblasts exponentially growing on glass were left untreated (A, E, and I) or treated with 0.5 μM Tat-C3 for 18 h (B, F, and J) and processed for immunofluorescence. Another set of Src(Y527F) fibroblasts were transfected with myc-RhoA(S19N) for 24 h, and plated on glass for an additional 12 h (C, D, G, H, and K) before processing for immunofluorescence. (A–C) F-actin staining at low magnification. (D) myc costaining corresponding to field in C. Arrows in C and D indicate transfected cell. (E–H) High magnification images of representative cells from the same experiments. (E–G) F-actin; (H) myc. G and H correspond to the same cell. (I–K) DAPI staining of corresponding nuclei in E–G. Bars: (D) 30 μm; (H) 9.5 μm.
Figure 3.
Figure 3.
Inhibition of Rho in Src-transformed fibroblasts causes podosome disassembly. Mouse fibroblasts expressing Src(Y527F) exponentially growing on glass were left untreated (A and B) or treated with 0.5 μM Tat-C3 for 18 h (C and D). Another set of Src(Y527F) fibroblasts were transfected with myc-RhoA(S19N) for 24 h, and plated on glass for an additional 12 h (E). Samples were processed for immunostaining for cortactin (A, C, and E) or Fish (B and D). Arrowheads in A and B indicate podosome rosettes. Inset in E shows myc costaining of the cell in E (arrows). Bar, 17.5 μm.
Figure 4.
Figure 4.
Inhibition of Rho but not ROCK impairs Src-induced protease secretion. Src(Y527F) cells exponentially growing on plastic in complete medium were treated with vehicle (A and D), 0.5 μM Tat-C3 (B and E), or 10 μM Y-27632 (C and F) for 20 h. Cells were then trypsinized and plated on glass coverslips coated with cross-linked Oregon green®–labeled gelatin in medium containing the indicated inhibitor. After 3 h, cells were fixed and stained for F-actin and DAPI. (A–C) Superimposed images of gelatin (green) and DAPI (blue). (D–F) Superimposed images of the same fields in A–C corresponding to F-actin (red) and DAPI (blue). (G–I) High magnification images of untreated control samples in the same experiment. Degradation patches in Oregon green®–labeled gelatin (G, arrows) and F-actin condensation at podosomes (H, arrows) colocalize when red and green channels are merged (I, arrows). (J) Gelatin degradation was quantified and is represented as the percent of cells able to form degradation patches (see Materials and methods). Results are the mean ± SD of three different experiments. Bars: (F) 32 μm; (I) 6 μm.
Figure 5.
Figure 5.
Active Rho[GTP] at podosome rosettes colocalizes with podosome components. Src(Y527F) cells exponentially growing on glass were left untreated (A–I) or were treated with 0.5 μM Tat-C3 for 18 h (J–L). After fixation, cells were incubated with 50 μg/ml of soluble GST-RBD. Rho[GTP]-bound RBD was detected by GST immunostaining (A, D, G, and J, green). The same samples were costained for the podosome components cortactin (B), Fish (E), or F-actin (H, control; K, Tat-C3), red. Merged green (Rho[GTP]) and red (podosome component) channels are shown in C, F, I, and L (yellow). Arrows in A–I indicate rosettes; arrowheads in J–L indicate disrupted rosettes. Bar, 14 μm.
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