The Intrinsic GDP/GTP Exchange Activities of Cdc42 and Rac1 Are Critical Determinants for Their Specific Effects on Mobilization of the Actin Filament System

Abstract

The Rho GTPases comprise a subfamily of the Ras superfamily of small GTPases. Their importance in regulation of cell morphology and cell migration is well characterized. According to the prevailing paradigm, Cdc42 regulates the formation of filopodia, Rac1 regulates the formation of lamellipodia, and RhoA triggers the assembly of focal adhesions. However, this scheme is clearly an oversimplification, as the Rho subfamily encompasses 20 members with diverse effects on a number of vital cellular processes, including cytoskeletal dynamics and cell proliferation, migration, and invasion. This article highlights the importance of the catalytic activities of the classical Rho GTPases Cdc42 and Rac1, in terms of their specific effects on the dynamic reorganization of the actin filament system. GTPase-deficient mutants of Cdc42 and Rac1 trigger the formation of broad lamellipodia and stress fibers, and fast-cycling mutations trigger filopodia formation and stress fiber dissolution. The filopodia response requires the involvement of the formin family of actin nucleation promotors. In contrast, the formation of broad lamellipodia induced by GTPase-deficient Cdc42 and Rac1 is mediated through Arp2/3-dependent actin nucleation.

Keywords: Cdc42; Rac; Rho GTPases; actin; fast-cycling; filopodia.

Figure 1

Cdc42 effects on actin dynamics. (A) Myc-tagged wt and mutant Cdc42 were exogenously expressed in BJ/hTERTSV40T cells. Myc-tagged proteins were detected with a rabbit anti-Myc antibody, followed by an Alexa Fluor 488-conjugated donkey anti-rabbit antibody. Filamentous actin was visualized using TRITC-conjugated phalloidin. Arrow-heads mark transfected cells. The boxed areas are enlarged at the right-hand-side of the corresponding image. Scale bar, 20 µm. (B,C) Quantification of formation of filopodia and broad lamellipodia (B), and of actin filament organization (C). At least 100 transfected cells were scored for each aspect (as indicated) from three independent experiments. Data are means ± standard deviation.

Figure 2

Cdc42 effects on actin dynamics (continued). (A) Myc-Cdc42/G12V, Myc-Cdc42/D118N, Myc-Cdc42/Q61LF37A, Myc-Cdc42/Q61L/Y40C, Myc-Cdc42/Q61LΔins, and Myc-Cdc42/Q61LT35A were exogenously expressed in BJ/hTERT SV40T cells. Myc-tagged Cdc42 was detected with a rabbit anti-Myc antibody, followed by an Alexa Fluor 488-conjugated donkey anti-rabbit antibody. Filamentous actin was visualized using TRITC-conjugated phalloidin. Arrow-heads mark transfected cells. The boxed areas are enlarged at the right-hand-side of the corresponding image. Scale bar, 20 µm. (B,C) Quantification of formation of filopodia and broad lamellipodia (B), and of actin filament organization (C). At least 100 transfected cells were scored for each aspect (as indicated) from three independent experiments. Data are means ± standard deviation.

Figure 3

The involvement of CAAX box and RhoGDI in the Cdc42-induced actin reorganization. (A) Myc-tagged Cdc42/Q61LR66A and Cdc42 variants with a mutation in the CAAX-box (Cdc42/Q61LSAAX, Cdc42/F28LSAAX) were exogenously expressed in BJ/hTERT SV40T cells. Myc-tagged proteins were detected with a rabbit anti-Myc antibody, followed by an Alexa Fluor 488-conjugated donkey anti-rabbit antibody. Filamentous actin was visualized using TRITC-conjugated phalloidin. Arrow-heads mark transfected cells. The boxed areas are enlarged at the right-hand-side of the corresponding image. Scale bar, 20 µm. (B,C) Quantification of formation of filopodia and broad lamellipodia (B), and of actin filament organization (C). At least 100 transfected cells were scored for each aspect (as indicated) from three independent experiments. Data are means ±standard deviation. Differences in cell shape between Cdc42/Q61L and Cdc42/Q61LSAAX, and Cdc42/F28L and Cdc42/F28LSAAX were determined using ImageJ as described in the Experimental section. (D), circularity, (E) perimeter and (F) cell area. Unpaired two-way Student’s t-tests with unequal variance were performed to calculate statistical significance. Histogram shows mean values and error bars standard deviation. *** = p < 0.001, ns = non-significant.

Figure 4

Rac1 effects on actin dynamics. (A) Myc-tagged wt and mutant Rac1 were exogenously expressed in BJ/hTERTSV40T cells. Myc-tagged proteins were detected with a rabbit anti-Myc antibody followed by an Alexa Fluor 488-conjugated donkey anti-rabbit antibody. Filamentous actin was visualized using TRITC-conjugated phalloidin. Arrow-heads mark transfected cells. The boxed areas are enlarged at the right-hand-side of the corresponding image. Scale bar, 20 µm. (B,C) Quantification of formation of filopodia and broad lamellipodia (B), and of actin filament organization (C). At least 100 transfected cells were scored for each phenotype (as indicated) from three independent experiments. Data are means ±standard deviation.

Figure 5

The involvement of formins and Arp2/3 in Cdc42- and Rac1-induced actin reorganization. (A,B) Myc-tagged Cdc42/Q61L or Myc-tagged Cdc42/F28L (A), and Myc-tagged Rac1/Q61L, Myc-tagged Rac1/F28L, or Myc-tagged Rac1/P29S (B), were exogenously expressed in BJ/hTERT SV40T cells. Six hours after transfection, the cells were treated with 30 μM SMIFH2 or 100 μM CK-666 for 18 h, and then fixed. Myc-tagged protein was detected with a rabbit anti-Myc antibody followed by an Alexa Fluor 488-conjugated donkey anti-rabbit antibody. Filamentous actin was visualized using TRITC-conjugated phalloidin. Arrow-heads mark transfected cells. The boxed areas are enlarged at the right-hand-side of the corresponding image. Scale bar, 20 µm. (C,D) Quantification of formation of filopodia and broad lamellipodia (C), and of actin filament organization (D). At least 200 transfected cells were scored for each phenotype (as indicated).