Figure 2

CPN and FM4-64 treated BY-2 protoplasts examined using confocal microscopy. (A) Protoplast that was treated with medium (no CPNs) and then FM4-64 for 10 min or (B) more than 20 min. FM4-64 fluorescence is in the plasma membrane and vesicles budding from the plasma membrane at 10 min. Following a 20 min or longer incubation, red fluorescence is in the plasma membrane, perinuclear membranes, and intracellular vesicles. Arrow heads point to vesicles budding at the plasma membrane and in the cortical region. (C, D) Green and red fluorescent images of protoplasts treated with CPNs and then FM4-64 for 10 min. Arrow heads point to vesicles along the plasma membrane that contain both green and red fluorescence. There are a greater number of red than green fluorescent vesicles. However, most green granules also contain red fluorescence. (E, F, G) Protoplasts were treated with CPNs and then FM4-64 for 20 min showed green and red fluorescence in vesicles along the periphery of the cell. Repeated experiments showed similar outcomes. Arrows point to examples where green and red fluorescence overlap. Scale bars equal 20 μm.