Figure 4

Golgi fragmentation. A. RGC5 cells transfected with pEGFP-N1 (mock control), GFP-tagged optineurin wild type, mutants or deletion fragment constructs were immunostained with anti-GM130 (Golgi marker, in red). Representative images of wild type, E50K, R96L, L157A, D474N, E478G, as well as deletion fragments 1–55, 1–148, 1–209, 1–398, 1–424, and 217–577 are shown. The nuclei were stained with DAPI in blue and transfected cells are in green. Severe Golgi fragmentation was observed in cells with prominent perinuclear foci formation such as wild type, E50K and R96L. The Golgi fragmentation however could still occur even without robust foci formation such as in Q398X (1–398). The percent of cells with fragmented Golgi was also increased with E478G, 1–424, and 217–577, but the values did not reach statistical significance. Higher magnification image of a transfected green cell indicated with a white star is shown in the inset. Scale bar, 10 μm. B. The percentage of cells with Golgi fragmentation was quantified. Compared to non-transfected normal or GFP mock controls, significantly more cells showed Golgi fragmentation in wild type, E50K, R96L and 1–398 transfected cells. **P < 0.033 compared to normal non-transfected or GFP controls. There was a moderate increase of cells with Golgi fragmentation in E478G, 1–424, and 217–577 transfected cells. All remaining mutations and deletion fragments showed no difference from controls.