FIGURE 6:

Mena regulates Golgi membrane fusion through F-actin elongation. (A, B) HeLa cells were transfected with control (A) or Mena (B) siRNA and incubated with nocodazole for 2 h. At 15 min after nocodazole removal, cells were stained for actin (red) and GRASP65 (white) and analyzed by confocal microscopy. Shown are single scans; parts of the images are enlarged to show the actin patches between Golgi membranes. Bars, 10 μm. (C) Mena was efficiently depleted from interphase cytosol, as shown by Western blot. (D–F) In vitro Golgi reassembly assay. Purified RLG membranes were disassembled by mitotic cytosol treatment, reisolated (D), and further incubated with interphase cytosol immunodepleted by control IgG (E) or by anti-Mena antibodies (F). Membranes were processed for EM and imaged. Bars, 500 nm. (G) Quantification of relative Golgi membrane fusion efficiency in E and F. ***p < 0.001. (H) Purified Golgi membranes that were first disassembled by mitotic cytosol treatment and then incubated with interphase cytosol in the presence of DMSO or 40 μM cytochalasin B (CytB). Membranes were processed for EM and quantified as in G.