Figure 1. A. thaliana HAP2 is sufficient... | Rockefeller University Press

Figure 1.

$Figure 1. A. thaliana HAP2 is sufficient to fuse mammalian BHK cells. (A) BHK cell–cell fusion assay: after discarding a possible failure in cell division (Table S1), cell–cell fusion is measured by the appearance of multinucleated cells labeled with either RFPcyto (magenta) or nuclear and cytoplasmic GFP (green; i). (ii) Fusion is also indicated by the appearance of multinucleated cells containing nuclear GFP and fluorescence from both RFPcyto and GFP in the cytoplasm. (iii) Nuclei are labeled with DAPI (blue) after fixation and permeabilization of the cells. (B, i) RFPcyto + GFP: negative control shows mononucleated cells expressing RFPcyto (magenta) or nuclear and cytoplasmic GFP (green). (ii) HAP2(RFPcyto) + HAP2(GFP): BHK cells were transfected with AtHAP2 and GFP (green) or RFPcyto (magenta); merged image of hybrid cell that contains mixed cytoplasm and three nuclei. (iii) EFF-1(RFPcyto) + EFF-1(GFP): hybrid binucleate cell emerged after EFF-1 expression and mixing of magenta and green cells (arrow). EFF-1(RFPcyto) binucleate cells (arrowheads). (iv) HAP2(RFPcyto) + HAP2(GFP): heterokaryons (hybrids) express magenta cytoplasm and green nuclei and cytoplasm (arrows). Multinucleate green cells (arrowheads). Bars: (B, i and ii) 10 µm; (B, iii and iv) 20 µm. (C) Quantification of multinucleation and content-mixing experiments. Magenta and green bars represent the fraction of multinucleated cells (two nuclei or higher) out of all the cells in contact (magenta or green, respectively). Black bars represent the RFPcyto and GFP content-mixing index. 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. Used unpaired t test comparing each color (RFPcyto, GFP, or mixed) for EFF-1 and HAP2 to the negative control (RFPcyto+GFP). *, P < 0.01; **, P < 0.005; ***, P < 0.001; ****, P < 0.0005. (D) Still images from time-lapse experiments reveal merging of two mononucleated (i) and three cells (ii) expressing RFPcyto and HAP2 (arrows and arrowheads, respectively). Time indicated in hours:minutes (see Videos 1 and 2 for panels i and ii in D, respectively). Note that the top nucleus (arrow in D, i) disappears because of defocus at 2:34. Two nuclei are out of focus at 4:57 (D, ii, bottom; see Fig. S1 A). Bars, 20 µm.$

A. thaliana HAP2 is sufficient to fuse mammalian BHK cells. (A) BHK cell–cell fusion assay: after discarding a possible failure in cell division (Table S1), cell–cell fusion is measured by the appearance of multinucleated cells labeled with either RFPcyto (magenta) or nuclear and cytoplasmic GFP (green; i). (ii) Fusion is also indicated by the appearance of multinucleated cells containing nuclear GFP and fluorescence from both RFPcyto and GFP in the cytoplasm. (iii) Nuclei are labeled with DAPI (blue) after fixation and permeabilization of the cells. (B, i) RFPcyto + GFP: negative control shows mononucleated cells expressing RFPcyto (magenta) or nuclear and cytoplasmic GFP (green). (ii) HAP2(RFPcyto) + HAP2(GFP): BHK cells were transfected with AtHAP2 and GFP (green) or RFPcyto (magenta); merged image of hybrid cell that contains mixed cytoplasm and three nuclei. (iii) EFF-1(RFPcyto) + EFF-1(GFP): hybrid binucleate cell emerged after EFF-1 expression and mixing of magenta and green cells (arrow). EFF-1(RFPcyto) binucleate cells (arrowheads). (iv) HAP2(RFPcyto) + HAP2(GFP): heterokaryons (hybrids) express magenta cytoplasm and green nuclei and cytoplasm (arrows). Multinucleate green cells (arrowheads). Bars: (B, i and ii) 10 µm; (B, iii and iv) 20 µm. (C) Quantification of multinucleation and content-mixing experiments. Magenta and green bars represent the fraction of multinucleated cells (two nuclei or higher) out of all the cells in contact (magenta or green, respectively). Black bars represent the RFPcyto and GFP content-mixing index. 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. Used unpaired t test comparing each color (RFPcyto, GFP, or mixed) for EFF-1 and HAP2 to the negative control (RFPcyto+GFP). *, P < 0.01; **, P < 0.005; ***, P < 0.001; ****, P < 0.0005. (D) Still images from time-lapse experiments reveal merging of two mononucleated (i) and three cells (ii) expressing RFPcyto and HAP2 (arrows and arrowheads, respectively). Time indicated in hours:minutes (see Videos 1 and 2 for panels i and ii in D, respectively). Note that the top nucleus (arrow in D, i) disappears because of defocus at 2:34. Two nuclei are out of focus at 4:57 (D, ii, bottom; see Fig. S1 A). Bars, 20 µm.

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