Figure 3.
Figure 3. HAP2 is a bilateral fusogen. (A) Quantification of content mixing experiments showing that HAP2 and EFF-1 are required in both interacting cells to form hybrids. Control RFPcyto + GFP is the same as in Fig. 1 B. The fusion and mixing indexes are presented as means ± SEM of three independent experiments. Total number of nuclei counted in multinucleated cells and in cells in contact, n ≥ 1,000 for each experimental condition. ***, P < 0.001; ****, P < 0.0005. P > 0.05 is not significant. (B) Representative fields used to determine percentages of multinucleation and content mixing. RFPcyto + GFP: mixed control cells; nuclear staining DAPI (blue). Dividing green cell with two nuclei is marked by arrowhead. EFF-1(RFPcyto) + GFP: BHK–EFF-1 (magenta) do not mix with GFP-expressing cells, revealing that EFF-1–mediated fusion is bilateral (homotypic). Multinucleate cells expressing RFPcyto (arrowheads). HAP2(RFPcyto) + GFP: BHK-HAP2 multinucleation (arrowheads) and failure to mix with GFP-expressing cells revealing HAP2-mediated fusion is bilateral. HAP2(RFPcyto) + EFF-1(GFP): hybrids (arrows) between BHK–EFF-1 (green) and BHK-HAP2 (magenta) reveal heterotypic merger of cells. Multinucleated green or red cells (arrowheads). Some hybrids are mononucleate probably because of cell division or nuclear fusion after merger. Bars, 20 µm. (C) Examples of multinucleate cells containing two to four nuclei are marked with arrows. The cells are able to divide after fusion; therefore, the number of nuclei per cells is usually smaller than six. EFF-1(RFPcyto) + GFP and HAP2(RFPcyto) + GFP images show no mixing indicating that HAP2-mediated fusion is bilateral (homotypic) in BHK cells. Bars, 10 µm. (D) Immunoblot of vector (negative control) and EFF-1 and HAP2 proteins carrying a V5 epitope fused to the cytoplasmic tail. Surface biotinylation of BHK cells expressing the different proteins. S indicates surface expression after affinity purification using neutravidin agarose beads; L is lysate. The amount of sample of HAP2 “S” is 600 times higher than EFF-1. The amount of lysate for HAP2 “L” is 12 times higher than for EFF-1. (E) Immunofluorescence of HAP2-YFP shows surface expression in cells revealed by anti-HAP2Extracellular (HAP2EC) polyclonal antibody. Without permeabilization, 20% of the cells expressing HAP2-YFP showed punctate expression on the surface; merged image (top, magenta). Untransfected BHK cells showed no immunoreactivity. After permeabilization with detergent, there is colocalization between immunostaining with anti-HAP2EC and the YFP signal revealing the specificity of the antibody that recognizes HAP2 in the reticular ER and perinuclear localization (merged image; bottom, yellow). Immunofluorescence in permeabilized BHK-HAP2-V5 cells using anti-V5 antibody shows similar localization (Fig. S1, B and C). Bar, 20 µm.

HAP2 is a bilateral fusogen. (A) Quantification of content mixing experiments showing that HAP2 and EFF-1 are required in both interacting cells to form hybrids. Control RFPcyto + GFP is the same as in Fig. 1 B. The fusion and mixing indexes are presented as means ± SEM of three independent experiments. Total number of nuclei counted in multinucleated cells and in cells in contact, n ≥ 1,000 for each experimental condition. ***, P < 0.001; ****, P < 0.0005. P > 0.05 is not significant. (B) Representative fields used to determine percentages of multinucleation and content mixing. RFPcyto + GFP: mixed control cells; nuclear staining DAPI (blue). Dividing green cell with two nuclei is marked by arrowhead. EFF-1(RFPcyto) + GFP: BHK–EFF-1 (magenta) do not mix with GFP-expressing cells, revealing that EFF-1–mediated fusion is bilateral (homotypic). Multinucleate cells expressing RFPcyto (arrowheads). HAP2(RFPcyto) + GFP: BHK-HAP2 multinucleation (arrowheads) and failure to mix with GFP-expressing cells revealing HAP2-mediated fusion is bilateral. HAP2(RFPcyto) + EFF-1(GFP): hybrids (arrows) between BHK–EFF-1 (green) and BHK-HAP2 (magenta) reveal heterotypic merger of cells. Multinucleated green or red cells (arrowheads). Some hybrids are mononucleate probably because of cell division or nuclear fusion after merger. Bars, 20 µm. (C) Examples of multinucleate cells containing two to four nuclei are marked with arrows. The cells are able to divide after fusion; therefore, the number of nuclei per cells is usually smaller than six. EFF-1(RFPcyto) + GFP and HAP2(RFPcyto) + GFP images show no mixing indicating that HAP2-mediated fusion is bilateral (homotypic) in BHK cells. Bars, 10 µm. (D) Immunoblot of vector (negative control) and EFF-1 and HAP2 proteins carrying a V5 epitope fused to the cytoplasmic tail. Surface biotinylation of BHK cells expressing the different proteins. S indicates surface expression after affinity purification using neutravidin agarose beads; L is lysate. The amount of sample of HAP2 “S” is 600 times higher than EFF-1. The amount of lysate for HAP2 “L” is 12 times higher than for EFF-1. (E) Immunofluorescence of HAP2-YFP shows surface expression in cells revealed by anti-HAP2Extracellular (HAP2EC) polyclonal antibody. Without permeabilization, 20% of the cells expressing HAP2-YFP showed punctate expression on the surface; merged image (top, magenta). Untransfected BHK cells showed no immunoreactivity. After permeabilization with detergent, there is colocalization between immunostaining with anti-HAP2EC and the YFP signal revealing the specificity of the antibody that recognizes HAP2 in the reticular ER and perinuclear localization (merged image; bottom, yellow). Immunofluorescence in permeabilized BHK-HAP2-V5 cells using anti-V5 antibody shows similar localization (Fig. S1, B and C). Bar, 20 µm.

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