Fig. 2. Fusion-pore expansion does not decrease the total area of the cell-contact zone. Fusion was triggered by a 1-minute low-pH pulse. (A) Orthogonal planes through a 3D-stack image of two fusing cells before the onset of syncytia formation (no pores detectable) are shown. The xy and xz planes are placed to cut through the middle of fusing cells, whereas the yz plane is placed to coincide with the contact zone between two cells. The red and blue lines outline 3D regions of interest (ROI) used for the analysis that is presented in B. Red lines outline a cylinder that encloses the initial contact zone and extends 1.2 µm into each cell. Blue lines outline a box that also extends 1.2 µm into each cell and is large enough to enclose the growing contact zone throughout the whole process of fusion-pore expansion. (B) Total membrane fluorescence within the initial contact zone [outlined by the red cylinder in A (red squares on the graph)] and within the total contact area [outlined by the blue box in A (blue circles on the graph)] were corrected for bleaching using the total cell fluorescence (black triangles). The data were normalized to the corresponding fluorescence intensity value at the initial time point using the equation F_roi(t=T)F_tc(t=0)/[F_roi(t=0)F_tc(t=T)], where F_roi is an integrated pixel intensity of either initial-contact-zone or total-contact area, and F_tc is an integrated pixel intensity of the whole cell. Inset images show the contact zone at the time points that are indicated by the arrows. Cell membranes were labeled with FM4-64FX. The data are measurements from a single pair of fusing cells and represent one out of six experiments. (C) In this cartoon, fusion-pore growth is accompanied by an increase in the cell-contact diameter, whereas the total area of membranes in contact (shown in red) remains constant. As the contact zone expands, the total area of the initial contact zone (changes in the areas of the contact zone and fusion pore are drawn so as to be proportional to those of the fusing cells on A and B) is readily accommodated within a narrow rim (5% of total-contact-zone diameter in thickness) along the edges of the expanded contact zone.