Ist2 Mediates ER-Plasma Membrane Contacts functional coupling of Ist2 with proteins in specific PM domains. The yeast PM consists of two main compartments, namely the MCP and the MCC [16,22,25]. To test whether Ist2 colocalizes with proteins of the MCP we employed total internal reflection fluorescence (TIRF) and CLS microscopy. We replaced the PMA1 gene with PMA1-mCherry in ist2D cells with GFP-IST2 integrated into the his3D1 locus. TIRF microscopy and a surface view of CLS microscopy confirmed the network-like localization of GFP-Ist2 (Fig. 5A–D). The merged signals (white) showed a partial overlap of GFP-Ist2 (green) with Pma1-mCherry (magenta) (Fig. 5A, B, merge). A Pearson’s coefficient of 0.896 (n = 19) in TIRF microscopy is consistent with partial colocalization of Ist2 and Pma1. Equatorial views (Fig. 5A, B, fifth panel) also revealed a partial overlap of both signals in the periphery while internalized Pma1-mCherry localized to vacuoles. However, certain areas containing GFP-Ist2 were clearly separated from Pma1-mCherry. Partial colocalization of Ist2 and Pma1 was also illustrated by intensity profiles of GFP-Ist2 and Pma1-mCherry signals (Fig. 5A, B, fourth panel, indicated by white lines in third panel). We used Sur7-mCherry in order to test whether the GFP-Ist2 network overlaps with dot-like MCC structures. In TIRF micros- copy Sur7-mCherry localized in dots with a size of 1 or 262 pixels (Fig. 5C). With the given resolution of the camera, where pixel sizes correspond to 160 nm, this fits well to the reported dimension of a Sur7 domain when analyzed by light microscopy [21,22]. A similar distribution of Sur7-mCherry was observed in surface views of CLS microscopy (Fig. 5D and Fig. S5A–D). Consistent with previous reports the dot-like Sur7-mCherry domains were segregated from Figure 5. Partial colocalization of Ist2 and Pma1 in network-like structures. Cells were grown in complete HC media and imaged by TIRF (A, C and E) and CLS (B, D and F) microscopy. The third panel shows the merge image of the first and second panel with an intensity profile of the indicated cross section in the fourth panel. (A, C and E) Merged view of equatorial sections in epifluorescence mode (panel 5) and corresponding DIC images of the TIRF images (panel 6). (B, D and F) Merged view of equatorial sections and phase contrast images are shown in the fifth and sixth panels. (A and B) The PMA1 gene was replaced by PMA1-mCherry in ist2D cells with GFP-IST2 integrated into the his3D1 locus. The localization of GFP- Ist2 (green in the merged image) and Pma1-mCherry (magenta in the merged image) was analyzed. (C and D) The SUR7 gene was replaced by SUR7- mCherry in ist2D cells with GFP-IST2 integrated into the his3D1 locus. The localization of GFP-Ist2 (green in the merged image) and Sur7-mCherry (magenta in the merged image) was analyzed. (E and F) PMA1 and SUR7 were replaced by PMA1-GFP and SUR7-mCherry in WT cells and the localization of Pma1-GFP (green in the merged image) and Sur7-mCherry (magenta in the merged image) was analyzed. doi:10.1371/journal.pone.0039703.g005 PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e39703