Figure 3.

Modulation of cellular PIP₂ levels affects the distribution of syntenin-2 PDZ domains. (A) Time-lapse microscopy of MCF-7 cells expressing the tandem PDZ domains of syntenin-2 (tandem PDZ) fused to eYFP (a–c), and the PH domain of PLCδ fused to eCFP (d–f) before (a, d), 7 min after (b, e) or 8 min after (c, f) ionomycin-induced hydrolysis of plasma membrane PIP₂. (B) MCF-7 cells expressing the tandem PDZ fused to eYFP were treated with DNase I (a, b), RNase A (c, d) or α-amanitin (e, f). The subnuclear distributions of eYFP-tandem PDZ (a, c, e) and nuclear speckles/PIP₂ (b, d, f) were analyzed by confocal microscopy. In the merge panels, eYFP-tandem PDZ is in green and nuclear speckles/PIP₂ are in red. The structure below the asterisk in panels e and e-f most likely corresponds to a nucleolar fragment. (C) Distribution of the tandem PDZ after activation of nuclear PLCs. MCF-7 cells expressing the eGFP-tandem PDZ were scored for nuclear enrichment of the fluorescence. Diagram, left part: Swiss 3T3 cells were treated with IGF-I for 20 min and compared to nontreated cells (N). Diagram, right part: MCF-7 cells were treated with the PLC activator m-3M3 FBS for different periods, as indicated, and compared to nontreated cells (N), or cells that were allowed to recover in serum-supplemented medium for 1 h after removal of the activator (recov). The distribution of the fluorescence was scored on three separate experiments, with 100 cells counted for each condition. Bars indicate standard deviations. Pictures: Time-lapse micrographs of MCF-7 cells before and after 25 min incubation with m-3M3 FBS. Note that the treatment induces cytosolic distribution of the fluorescence. (D) Confocal micrograph of MCF-7 cells expressing the eYFP-yeast PLC1 with a mutated NES ‘eYFP-yPLC1-NES' (a) and a Myc fusion to the tandem PDZ (b). eYFP-yPLC1-NES was detected by eYFP fluorescence and the Myc-tandem PDZ using anti-Myc antibodies (9E10). In the merge (a-b), eYFP-yPLC1-NES is in green and the Myc-tandem PDZ is in red. Note the nuclear concentration of the Myc-tandem PDZ in the cell on the right side (below the asterisk) and the cytosolic/plasma membrane distribution in the cell expressing yPLC1-NES, on the left side. Size bars are 10 μm.