Chromatids segregate without centrosomes during Caenorhabditis elegans mitosis in a Ran- and CLASP-dependent manner

Abstract

During mitosis, chromosomes are connected to a microtubule-based spindle. Current models propose that displacement of the spindle poles and/or the activity of kinetochore microtubules generate mechanical forces that segregate sister chromatids. Using laser destruction of the centrosomes during Caenorhabditis elegans mitosis, we show that neither of these mechanisms is necessary to achieve proper chromatid segregation. Our results strongly suggest that an outward force generated by the spindle midzone, independently of centrosomes, is sufficient to segregate chromosomes in mitotic cells. Using mutant and RNAi analysis, we show that the microtubule-bundling protein SPD-1/MAP-65 and BMK-1/kinesin-5 act as a brake opposing the force generated by the spindle midzone. Conversely, we identify a novel role for two microtubule-growth and nucleation agents, Ran and CLASP, in the establishment of the centrosome-independent force during anaphase. Their involvement raises the interesting possibility that microtubule polymerization of midzone microtubules is continuously required to sustain chromosome segregation during mitosis.

FIGURE 1:

Chromatids separate in the absence of centrosomes. (A–C) Snapshots and kymographs of GFP::tubulin; GFP::histone embryos in wild-type controls (A), after laser destruction (OICD) of the anterior centrosome (B), and after OICD of both centrosomes (C). Red and white arrowheads point to chromatids and the plasma generated by the UV laser, respectively. In this and all subsequent figures, the anterior pole of the cell is to the left. Kymographs display centrosome (blue and red) and chromatid (cyan and magenta) trajectories as percentage of total cell length. (D) Average curves of the chromatid-to-chromatid distance in micrometers over time for wild-type embryos (blue) after OICD of one centrosome (black) or double OICD (red). Scale bar, 10 μm. t = 0 s: chromatid separation onset. Errors bars, SD.

FIGURE 2:

Midzone microtubules assemble after OICD. (A) Confocal image of a GFP::tubulin; GFP::histone embryo after IR laser destruction of the anterior centrosome. Exposure time is 6 s. (B) Snapshots of embryos coexpressing SPD-1::GFP and mCherry:HIS-58 in wild-type control (top) and after destruction of the anterior centrosome with a UV laser (bottom). Scale bar, 10 μm. (C) Chromatid-to-chromatid distance in micrometers measured 370 s after NEBD (this time was chosen because it corresponds to the end of mitosis) relative to the time elapsed between NEBD and centrosome ablation. The green area represents the time window used in the rest of our study. Bottom, snapshots of a one-cell stage GFP::tubulin; GFP::histone embryo for which the posterior centrosome was UV irradiated early during mitosis. t = 0 s corresponds to the time of OICD. White and red arrowheads point to the plasma and the chromosomes, respectively.

FIGURE 3:

Role of SPD-1 and ZEN-4 on the force generated independently of centrosomes. (A–D) Snapshots of GFP::tubulin; GFP::histone embryos carrying the spd-1(oj5) allele in intact cells (A) or after OICD of the anterior centrosome (B), or carrying the zen-4(or153) allele and treated with zen-4(RNAi) with intact centrosomes (C) or after OICD of the posterior centrosome (D). The red arrowheads point to DNA. (E, F) Average curves of the chromatid-to-chromatid distance in micrometers over time in intact cells or after OICD for wild-type (blue and black curves, respectively) and mutants cells (green and red curves, respectively). Right, only the curves corresponding to the OICD experiments are shown. spd-1(oj5) mutants are shown in E, and zen-4(or153) is shown in F. Scale bar, 10 μm. t = 0 s: chromatid separation onset. Errors bars, SD.

FIGURE 4:

BMK-1 acts as a brake to oppose the force generated independently of centrosomes. (A, B) Snapshots of GFP::tubulin; GFP::histone in a bmk-1(RNAi) embryo with intact centrosomes (A) or after OICD of the posterior centrosome (B). Red arrowheads point to the chromatids. (C) Average curves of the chromatid-to-chromatid distance in micrometers over time in intact cells or after OICD for wild-type (blue and black curves, respectively) and bmk-1(RNAi) embryos (green and red curves, respectively). Right, only the curves corresponding to the OICD experiments are shown. Scale bar, 10 μm. t = 0 s: chromatid separation onset. Errors bars, SD.

FIGURE 5:

CLASP is essential for chromatid separation in the absence of centrosomes. (A–C) Snapshots of GFP::tubulin; GFP::histone in a cls-2(RNAi) embryo (A), a gpr-1/2;cls-2(RNAi) embryo with intact centrosomes (B), or a gpr-1/2;cls-2(RNAi) embryo after OICD of the anterior centrosome (C). The red arrowhead points to the unsegregated chromatids. (D) Average curves of the chromatid-to-chromatid distance in micrometers over time in intact cells or after OICD for wild-type (blue and black curves, respectively) and cls-2;gpr-1/2(RNAi) embryos (green and red curves, respectively). Right, only the curves corresponding to the OICD experiments are shown. Scale bar, 10 μm. t = 0 s: chromatid separation onset. Errors bars, SD.

FIGURE 6:

RanGTP promotes chromatid separation in the absence of centrosomes. (A–D) Snapshots of GFP::tubulin; GFP::histone in an ran-3(RNAi) embryo with intact centrosomes (A) or after OICD of the posterior centrosome (B), and in a ran-2(t1598) homozygous mutant with intact centrosomes (C) or after OICD of the anterior centrosome (D). Red arrowheads point to the chromatids. (E, F) Average curves of the chromatid-to-chromatid distance in micrometers over time in intact cells or after OICD for wild-type (blue and black curves, respectively) and mutant or RNAi-treated embryos (green and red curves, respectively). Right, only the curves corresponding to the OICD experiments are shown. ran-3 (RNAi) embryos are shown in E, and ran-2(t1598) is shown in F. Scale bar, 10 μm. t = 0 s: chromatid separation onset. Errors bars, SD.