PKC-Mediated ZYG1 Phosphorylation Induces Fusion of Myoblasts as well as of Dictyostelium Cells

Figure 4

Immunocytochemical detection of the PKC-mediated ZYG1 phosphorylation. Starved GFP^CONT and GFP/ZYG1^OE cells were developed for 2 h, fixed with methanol, and stained by the Phospho-(Ser) PKC Substrate antibody. This was followed by the rhodamine-conjugated anti-rabbit secondary antibody to detect the proteins phosphorylated by cPKC, as described in Section 2. Photographs were taken under DIC (a and b) and fluorescence microscope (GFP, (c) and (d); Rhodamine, (e) and (f); GFP and Rhodamine merged, (g) and (h)). Photographs represent the same fields of GFP^CONT cells (a, c, e, g) and of GFP/ZYG1^OE cells (b, d, f, h). GFP protein in GFP^CONT cells and GFP/ZYG1 fusion protein in GFP/ZYG1^OE cells are shown as the green color of GFP (c and d). In GFP/ZYG1^OE cells, GFP/ZYG1 fusion protein is localized at the region of cell-cell contacts (d; arrow). On the other hand, the localization of GFP protein at the region of cell-cell contacts is not observed in GFP^CONT cells (c; arrow). This indicates that its localization is due to the presence of ZYG1, but not to GFP itself. The protein phosphorylated by PKC is shown as the red color of Rhodamine in both cells ((e) and (f)). The protein phosphorylated by PKC is also localized at the region of cell-cell contacts in GFP/ZYG1^OE cells (f; arrow). This localization shown by Rhodamine is not observed in GFP^CONT cells (e; arrow). Since the merged color of GFP and Rhodamine shows yellow in GFP/ZYG1^OE cells (h; arrow), it is evident that ZYG1 protein is phosphorylated by cPKC at the regions of cell-cell contacts. Bar: 25 μm.

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