PMC

Mutational analysis of CLIC4 translocation. (A and B) Mutation of residue Cys35 (YFP-CLIC4(C35A)) abolishes LPA-induced translocation (A) and colocalization with DsRed-NHERF2 (B). Bars, 10 μm. (C) Structural comparison of omega-GST (GSTO1) and CLIC4. The indicated residues were mutated, based on their equivalence to the residues that mediate the binding of glutathione and secondary substrates in GSTs. Results are summarized in D. See text for details.

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 (), 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.

Agonist-induced translocation of CLIC4 toward the plasma membrane. (A) LPA-induced translocation of GFP-CLIC4 in N1E-115 cells visualized by time-lapse microscopy. In resting cells, GFP-CLIC4 resides mainly in the cytosol with some patch-like accumulation at the cell periphery. LPA (1 μM) induces a rapid but transient recruitment of GFP-CLIC4 toward the plasma membrane. Similar responses were observed with fetal calf serum (5%), S1P, and TRP (see ; also see Supplemental Video 1). Bar, 10 μm. (B) GFP-CLIC4 translocation in response to 1 μM LPA measured semiquantitatively by monitoring the accumulation of GFP fluorescence at the PM (red trace) and the concomitant depletion of fluorescence from the cytosol (Cyt; blue trace). Net translocation is expressed as the ratio PM/Cyt (green trace). (C) Distribution of endogenous CLIC4 in resting versus LPA-stimulated N1E-115 cells. Plasma membrane accumulation was strongly increased after 1 min of 1 μM LPA addition. Bars, 10 μm.

LPA-induced membrane depolarization and inward currents. (A) LPA-stimulated N1E-115 cells undergo transient Cl⁻-mediated membrane depolarization as measured by a voltage-sensitive dye (see Materials and Methods). Indicated RNA interference or drug treatments correspond to those shown in A (n > 100) (transfection marker CFP-H2B). End-of-experiment calibration was done by using 150 mM KCl, which eliminates the transmembrane potential. (B) Latrunculin A (1 μM; up to 1 h pretreatment) does not affect LPA-induced membrane depolarization, whereas it abolishes CLIC4 translocation (see B). Recording is representative of eight experiments. (C) Representative patch-clamp recordings of inward Cl⁻ currents in LPA-stimulated N1E-115 cells. Membrane potential was clamped at −60 mV (for details, see ). Black trace, untreated cells. Red trace, CLIC4-depleted cells (three RNA interference targeting constructs). The bar diagram shows peak amplitudes (mean ± SEM). Transmembrane currents were corrected for membrane conductance, which is a measure for total cell surface area (pA/pF). Bottom, Western blots showing CLIC4 levels in control and knockdown N1E-115 cells. Actin was used as loading control.

CLIC4 translocation toward activated GPCRs and NHERF2. (A) Differential distribution of translocated GFP-CLIC4 in the same cell. N1E-115 cells were first stimulated with TRP (50 μM) and thereafter with LPA (1 μM). Also see Supplemental Video 2. Right, differential recruitment of endogenous CLIC4 by LPA and TRP. Bars, 10 μm. (B) Colocalization of GFP-CLIC4 and DsRed-NHERF2 at polarized regions of the plasma membrane in N1E-115 cells. Bar, 10 μm. (C) Triple colocalization of endogenous CLIC4 (Alexa488-conjugated secondary antibody; shown in blue), DsRed-NHERF2 (shown in green), and HA-LPA2 (Alexa633-conjugated secondary antibody; shown in red). Bar, 10 μm. (D) CLIC4 translocates to activated LPA receptor complexes. Pictures taken from time-lapse movies showing LPA1-GFP and CLIC4-mCherry before and after LPA stimulation. Arrows indicate plasma membrane regions where LPA1-GFP and CLIC4-mCherry transiently colocalize after LPA addition (also see Supplemental Video 3). Bar, 10 μm. (E) CLIC4 accumulates selectively in ligand-exposed membrane domains. TRP was locally applied by combining an application and a suction pipette. Besides TRP (3 μM), the application pipette contained calcium orange (0.2 mM) to continuously monitor the flux between both pipettes. After verifying the gradient steepness of the TRP/dye mix, a selected cell was positioned near the pipettes and GFP-CLIC4 was imaged during local TRP application. Top, fluorescence and transmission images before ligand application. Arrows indicate pipettes (cell debris was attracted by the suction pipette). Bottom, at t = 0, a brief pulse of TRP reaches a small area of the plasma membrane. At t = 40 s, GFP-CLIC4 accumulates selectively at the TRP-exposed area of the plasma membrane. At t = 50 s, a second TRP pulse is applied. At t = 90 s, further accumulation of GFP-CLIC4 is observed. Also see Supplemental Video 4. Pictures are representative of four independent experiments. Bar, 20 μm.