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First published online October 22, 2008
doi: 10.1242/10.1242/jcs.032169

Journal of Cell Science 121, 3619-3628 (2008)
Published by The Company of Biologists 2008
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Fusion-pore expansion during syncytium formation is restricted by an actin network

Andrew Chen1, Eugenia Leikina1, Kamran Melikov1, Benjamin Podbilewicz2, Michael M. Kozlov3 and Leonid V. Chernomordik1,*

1 Section of Membrane Biology, Laboratory of Cellular and Molecular Biophysics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, 10 Center Drive, Bethesda, MD 20892-1855, USA
2 Department of Biology, Technion-Israel Institute of Technology, Haifa, 32000 Israel
3 Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, 69978 Tel Aviv, Israel


Figure 1
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  		Fig. 1. Fusion-pore opening and expansion in the cell-contact zone. (A-F) Time series of syncytia formation for cells that were labeled with FM4-64FX. For each time point, the top view (maximum intensity projection of the whole image stack along the z-axis) is shown on the right panel and the view of the contact zone as seen by looking from one fusing cell into another (maximum intensity projection along the x-axis of the region delimited by two vertical lines) is shown on the left panel. The arrow in B marks the location of the first pore that was observed in this series. (G,H) Pore-size (G) and pore-number per contact zone (H) distribution histograms based on analysis of 498 pores from 175 cell-cell contact zones. Because the average rates of fusion-pore expansion differed from day to day in more than ten experiments investigating the morphology of the fusion pores in fixed cells, these histograms include only the data collected in the same experiment. Cells were fixed 4 minutes after a 1-minute low-pH application. The height of each bar in G represents the number of pores with diameters in the interval between its lower and upper bounds on the x-axis.

 

Figure 2
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  		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 Froi(t=T)Ftc(t=0)/[Froi(t=0)Ftc(t=T)], where Froi is an integrated pixel intensity of either initial-contact-zone or total-contact area, and Ftc 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.

 

Figure 3
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  		Fig. 3. Syncytium formation is not driven by membrane tension and requires metabolic energy. (A) To explore the effects of a decrease in membrane tension on the extent of syncytium formation (black bars) and the mean radius of the cells (gray bars), immediately after a 1-minute application of acidic-pH buffer we placed Sf9Op1D cells into either PBS (control) or PBS supplemented with 250 mM stachyose (hypertonic medium). The syncytium-formation and cell-radii analyses were carried out 30 minutes after low-pH application for >1330 and >22 cells, respectively. The syncytium index and mean cell radius in hypertonic medium were normalized to those in the control experiments. (B,C) The metabolic inhibitor sodium azide (NaN3, 5 mM) did not prevent early fusion stages but blocked syncytium formation. (B) An overlay of a representative field of red and green fluorescence of the NaN3-treated cells fixed 1-hour after a 1-minute low-pH pulse. Membrane merger between cells that were pre-labeled with the red membrane dye Vybrant DiI and cells pre-labeled with content probe CellTracker Green yielded double-labeled cells. Unfused cells are seen as only red or only green ones. (C) Local fusion between cells that were pre-labeled with different probes in the presence of NaN3 and between controls was quantified as an average number of nuclei in double-labeled cells per field of view. Syncytium formation after a 1-minute low-pH pulse was quantified as the percentage of nuclei in syncytia. The total number of analyzed nuclei for each condition was >2500. A and C show means ± s.d. of ten replicates in a representative experiment, performed twice.

 

Figure 4
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  		Fig. 4. Fusion pores colocalize with openings in the actin cortex. Cells were labeled with FM4-64FX 9 minutes after a 2-minute low-pH pulse. 1 minute later, the cells were fixed and labeled with Alexa-Fluor-48–phalloidin. (A,B) xy-projections of representative contact zones (marked as 1-4) shown with membrane- and actin-labeling, respectively. (C,D) Maximum intensity projections of the image stacks of each of these zones along the z axis with membrane- (C) and, under it, actin- (D) labeling.

 

Figure 5
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  		Fig. 5. Actin-depolymerizing and -polymerizing reagents latrunculin A (LatA) and jasplakinolide (Jasp) promote and inhibit fusion-pore expansion, respectively. (A) Dissociation of the actin cortex by LatA. Sf9Op1D cells were incubated for 30 minutes with Grace medium with 2 µM LatA (`LatA') or without it (`Control') prior to a 5-minute low-pH pulse. In the former case, the cells were kept in the presence of LatA throughout the experiment. Fluorescence-microscopy and bright-field images of the cells, which were fixed and labeled with Alexa-Fluor-488–phalloidin 2 hours after low-pH application, are presented. Although LatA treatment resulted in a loss of actin-cortex labeling, LatA-treated cells, as did control cells, formed syncytia (marked by arrows). (B) LatA-treated cells have a larger average total area of fusion pores per contact zone than the control cells when fixed 1 minute after a 1-minute low-pH application. Each bar is based on analysis of 50 contact zones (152 and 127 pores for control and for LatA-treated cells, respectively). (C) Jasp slows down fusion-pore expansion. Cells treated with a 1-minute pulse of pH-4.9 medium followed by a 9-minute incubation in the presence of 0.5 µM Jasp and then fixed have a lower average total area of fusion pores per contact zone than the control Jasp-untreated cells. Results are based on the analysis of a total of 51 contact zones for control cells and 37 contact zones for Jasp-treated cells. B and C show the means ± s.e. based on the analysis of the data collected in the same experiment for the reagent-treated and untreated (control) FM4-64FX-labeled cells.

 

Figure 6
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  		Fig. 6. Effects of actin-modifying reagents on the final extents of syncytium formation. (A) Syncytium formation for Sf9Op1D cells treated with the cell-permeable actin-depolymerizing reagents LatA (2 µM, bar 2) and CD (0.1 µM, bar 3), and actin-polymerizing reagent Jasp (0.5 µM, bar 4), and for the untreated cells (bar 1). LatA was applied 30 minutes before a fusion-triggering low-pH pulse. CD and Jasp were applied immediately after the pulse (see Materials and Methods). (B) The membrane-impermeable actin-polymerizing reagent phalloidin that was delivered into Sf9Op1D cells by their fusion with phalloidin-loaded erythrocyte ghosts inhibited syncytium formation. In the control experiments we used unloaded erythrocyte ghosts. (A,B) Fusion was triggered by a 1-minute application of pH 4.9 medium. Syncytium-formation extents were scored 30 minutes later and presented as means+s.e. (n≥3).

 

Figure 7
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  		Fig. 7. Proposed mechanism of the fusion-pore expansion in syncytium formation. (A) Membrane curvature of an expanding fusion pore is similar to that in budding vesicles. (B) Proposed pathway of syncytium formation. Red networks under the contacting membranes in the pre-fusion state depict actin structures. Protein fusogens, such as baculovirus gp64, form nascent fusion pores. The subsequent expansion of these pores to yield syncytium is controlled by cell machinery. A dynamic resistance of the actin network disrupted by a pore slows down pore expansion, which is driven by membrane-bending proteins (banana-like shapes). These proteins, which are involved in the generation of highly curved intracellular membrane compartments, accumulate at the strongly bent rim of the fusion pores, lower the line tension of the pores and, thus, drive their expansion.

 

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© The Company of Biologists Ltd 2008