JCS032169 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. The total area of membranes in contact is conserved during fusion-pore growth. Total membrane fluorescence within initial contact zone (red squares) and total membrane fluorescence within the whole contact area (blue circles) for five cell pairs were analyzed as in Fig. 2. Fusion between S9^Op1D cells was triggered by a 1-minute low-pH pulse.

• Supplemental Figure S2 -

  Fig. S2. S9^Op1D syncytium formation is not accompanied by any detectable increase in cell membrane permeability to membrane-impermeable probe PI. The cells were treated (B) or not treated (A) with a low-pH pulse in the presence of 10 µM PI and imaged 10 minutes later. Red cells represent dead cells. Syncytia in B exclude PI indicating that cell-cell fusion is not accompanied by membrane permeabilization. Same scale bars, 20 µm.

• Supplemental Figure S3 -

  Fig. S3. Quantification procedure for final extents of syncytium formation. 30 minutes after a 1-minute pulse of low pH, S9^Op1D cells were fixed and stained with Hoechst. Analysis of the distribution of the nuclei between mono- and multi-nucleated cells (marked in the figure by blue and red arrows, respectively) was used to score the percentage of cells in syncytia as the ratio of nuclei within multinucleated cells to the total number of cell nuclei in the same field. In this example (a robust fusion between untreated S9^Op1D cells after a 1-minute pulse of low pH), 12 out of 22 nuclei are in syncytia and thus syncytium extent is quantified as 54.5%.

• Supplemental Table S1 -

  Table S1. Fusion pores in latrunculin-treated cells are less circular and more concave. Cells were incubated for 30 minutes with Grace medium with 2 µM LatA or without it prior to a 5-minute low-pH pulse. To quantify the changes of pore shape due to LatA treatment we manually outlined each pore. To control for effects due to pore area, this study was done only on pores of similar size with the area of 8-9 µm². For each pore we estimated eccentricity as an eccentricity of the ellipsoid defined as (1−b²/a²)^1/2, where a is semimajor axis and b is a semiminor axis having the same 2nd moments as the pore image. Pore eccentricity is a measure of how noncircular the pore is with eccentricity value of 0 corresponding to a circle and 1 to a line segment. We also estimated pore concavity that was defined as (S^cp-S^pore)/S^cp, where S^cp is area of smallest convex polygon which circumscribes the pore image and S^pore is the area of the pore.

• Movie 1 -

  Movie 1. Low-pH application to gp64-expressing cells triggers robust syncytia formation. The fusion reaction between Sf9^Op1D cells was triggered by a 1-minute application of low-pH medium. Images were acquired every 30 seconds.

• Movie 2 -

  Movie 2. Expansion of the fusion pores within a contact zone. Movie shows maximum intensity projection of the whole image stack along the z-axis on the left and maximum intensity projection along the x-axis of the contact zone region on the right. Image stacks were acquired every 88 seconds.