Figure 1.

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 Table 1; 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.