Fig 1.
Spindle length increases proportionally with cell volume.
(A) Left panel: Blastomeres of mouse 2-cell embryos were microinjected with cRNAs encoding Histone-H2B fused to mCherry and Tubulin fused to EGFP. The following cells were used for experiments: intact two cell embryos (Intact), cells with double volume obtained by fusion of two blastomeres (2 fused), and cells with triple volume obtained by fusion of three blastomeres (3 fused). Right panel shows representative movie frames from time lapse imaging of each cell type in interphase and in mitosis; chromosomes are in red, spindle is in green, scale bar represents 10 μm. (B) The length of the spindle in 2 fused cells (35.26 ± 5.52 μm, n = 14) was not significantly increased (p = 0.1236) in comparison to the length of the spindle in Intact cells (32.38 ± 5.63 μm, n = 28). (C) The length of the spindle in 3 fused cells (40.71 ± 3.57 μm, n = 14) was significantly increased (p < 0.0001) in comparison to the length of the spindle in Intact cells (34.68 ± 3.99 μm, n = 23).
Fig 2.
Changes in nuclear to cytoplasmic ratio have impact on spindle length.
(A) Left panel: Blastomeres of mouse 2-cell embryos were microinjected with cRNAs encoding Histone-H2B fused to mCherry and Tubulin fused to EGFP. The following cells were used for experiments: intact two cell embryos (Intact), cells with double volume obtained by fusion of two blastomeres (2 fused), and cells with double volume of the cytoplasm and impaired nuclear to cytoplasmic ratio resulting from enucleation (2 fused enucleated). Right panel shows representative movie frames from time lapse imaging of each cell type in interphase and in mitosis; chromosomes are in red, spindle is in green, scale bar represents 10 μm. (B) The length of the spindle in 2 fused enucleated cells (42.91 ± 3.35 μm, n = 12) was significantly increased (p < 0.0001) in comparison to the length of the spindle in intact cells (32.83 ± 2.95 μm, n = 22) or 2 fused cells (34.96 ± 3.37 μm, n = 14). (C) Left panel: haploid embryos were produced by parthenogenetic activation of metaphase II eggs. Following activation, cells were microinjected with cRNAs encoding Histone-H2B fused to mCherry and Tubulin fused to EGFP together with blastomeres of 2-cell embryos. Right panel shows representative movie frames from time lapse imaging of each cell type in interphase and in mitosis; chromosomes are in red, spindle is in green, scale bar is 10 μm. (D) The length of the spindle in Unfertilized haploid cells (38.04 ± 4.06 μm, n = 37) was significantly increased (p < 0.0001) in comparison to the length of the spindle in intact cells (Fertilized) (31.18 ± 3.32 μm, n = 26).
Fig 3.
Nuclear to cytoplasmic ratio in cells created by fusion, enucleation, or parthenogenetic activation.
Panel A: The chart shows nuclear to cytoplasmic ratio in all cells used in experiments described in Figs 1 and 2: Intact cells (0.046, n = 93), 2 fused cells (0.047, n = 28), 3 fused cells (0.048, n = 13), 2 fused enucleated cells (0.027, n = 12) and unfertilized cells (0.043, n = 40). N:C ratio of Intact cells is significantly different in comparison to 2 fused enucleated (p < 0.0001) and unfertilized cells (p = 0.0116). The diagram below the X-axis illustrates the proportional differences between cell and nuclear size of each cell type. Panel B: The plot shows a relationship between nuclear to cytoplasmic ratio and spindle length in all cells used in experiments described in Figs 1 and 2: Green dots represent intact cells (spindle length: 32.75 ± 4.30; NC ratio: 0.046 ± 0.006), red squares represent 2 fused cells (spindle length: 35.11 ± 4.49; NC ratio: 0.047 ± 0.008), orange triangles represent 3 fused cells (spindle length: 40.70 ± 3.56; NC ratio: 0.048 ± 0.003), blue rhombuses represent unfertilized cells (spindle length: 38.04 ± 4.06; NC ratio: 0.043 ± 0.006) and gray hexagons represent 2 fused enucleated cells (spindle length: 43.20 ± 3.69; NC ratio: 0.027 ± 0.003).
Fig 4.
Overexpression of TPX2 has no effect on spindle length.
(A) Left panel: Blastomeres of mouse 2-cell embryos were microinjected with cRNAs encoding Histone-H2B fused to mCherry and Tubulin fused to EGFP with (bottom) or without (top) cRNA encoding TPX2 protein. Following microinjection, one of the two blastomeres in each embryo was enucleated and then both fused together. Right panel shows representative movie frames from time lapse imaging of each cell type in interphase and in mitosis, chromosomes are in red, spindle is in green, scale bar represents 10 μm. (B) In some experiments, TXP2 was used with fluorescent tag. Representative images of cells before (left panels, 10 min) and during NEBD (right panel, 0 min) show the nuclear localization of exogenous TPX2 in interphase and its relocalization to the cytoplasm before spindle assembly. Chromosomes are in red, TPX2 in cyan, scale bar represents 10 μm. (C) The length of the spindle in 2 fused enucleated cells with overexpressed TPX2 (41.14 ± 4.07 μm, n = 18) was not significantly different (p = 0.3961) compared to the length of the spindle in 2 fused enucleated cells (40.14 ± 2.64 μm, n = 17).