Subcellular distribution of PDZ domains and effects of PtdInsP-modifying treatments compared to <i>in vitro</i> PtdIns(4,5)P2-binding.

<p><b>A.</b> Double reference subtracted sensorgrams of recombinant his-tagged PDZ domains injected, in a range of concentrations with the highest concentrations used indicated, over 5% PtdIns(4,5)P2 containing DOPC liposomes. <b>B–O.</b> Confocal or wide-field micrographs of MCF-7 cells transiently over-expressing selected eYFP-S1PDZ1-tagged PDZ domains (with the exception of SLC9A3R2_1* where S1PDZ1 was omitted). Sensorgrams are provided to the left of the micrographs for each PDZ domain under study. <b>B</b>. Co-expression of eYFP-S1PDZ1-MPP7 with PtdIns(4)P-5-kinase (PIPK; shown in the insets) to probe the effect of increased plasma membrane PtdIns(4,5)P2 levels on the distribution of the fluorescent protein. Note the absence of membrane enrichment of the fluorescence upon kinase over-expression. <b>C</b>. Serum stimulation of eYFP-S1PDZ1-MPP7 expressing cells to investigate the effect of increased plasma membrane PtdIns(3,4,5)P3 levels. Note the absence of membrane enrichment of the fluorescence upon serum stimulation. For control experiments of B and C, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054581#pone.0054581.s003" target="_blank">Fig. S3A</a>–B. <b>D–G</b> Ionomycin treatment (D, E), inhibition of PtdIns(4)-kinases by PAO (F) and rapamycin-induced membrane recruitment of PtdInsPs 5′ phosphatase to investigate how reducing PtdIns(4,5)P2 levels affect the membrane enrichment of eYFP-S1PDZ1-MPDZ_7 (D) and eYFP-S1PDZ1-CASK (E-G). <b>H–I.</b> Co-expression with mCherry-PTS1 identified the bright cytosolic spots enriched in eYFP-S1PDZ1-MAGI3_3 (H) and eYFP-S1PDZ1-IL16_1 (I) as peroxisomes. Co-expression with mCherry-B23 established that the subnuclear organelles enriched in eYPF-S1PDZ1-SCRIB4 (J) and eYFP-SLC9A3R2_1* (M) corresponded to nucleoli. Co-expression with a PtdIns(4,5)P2-4-phosphatase (PIP2 4 ptase, shown in the insets) or PCLΔNES, did not affect the nucleoli enrichment of eYFP-S1PDZ1-SCRIB4 (K, L) but induced a shift of eYFP-SLC9A3R2_1 (N, O) towards the nucleoplasm and the cytosol.