Figure 2.

Spatiotemporal regulation of CLIC4 by RhoA. (A) Visualization of GFP-CLIC4 translocation and cell morphology under various conditions affecting Ga13-RhoA signaling in N1E-115 cells. RNA interference-encoding vectors were transfected for 48 h together with GFP-CLIC4 (transfection marker mRFP-H2B was used in the cell rounding controls; not shown in the images). C3 transferase (30 μg/ml) was incubated for 16 h and the ROCK inhibitor Y-27632 (30 μM) for 30 min. Bars, 10 μm. (B) Introduction of the catalytic DH/PH domain of p190RhoGEF, which robustly stimulates GTP loading on RhoA (van Horck et al., 2001), leads to constitutive recruitment of GFP-CLIC4 at the plasma membrane. Bar, 10 μm. (C) Latrunculin A (1 μM; 4-min pretreatment) abolishes LPA-induced translocation of GFP-CLIC4. Bar, 20 μm.