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

Nahaboo W, Zouak M, Askjaer P, Delattre M - Mol. Biol. Cell (2015)

Bottom Line: 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.Their involvement raises the interesting possibility that microtubule polymerization of midzone microtubules is continuously required to sustain chromosome segregation during mitosis.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology of the Cell, University of Lyon 1, CNRS UMR5239, Ecole Normale Supérieure de Lyon, 69007 Lyon, France.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: Finally, we tested the role of the small GTPase Ran/RAN-1 during C. elegans anaphase. Inhibition of Ran affects spindle assembly in many different species through the reduction of microtubule density around the DNA (Carazo-Salas et al., 1999; Kalab et al., 1999; Ohba et al., 1999; Wilde and Zheng, 1999; Zhang et al., 1999). Because of this early effect on spindle formation, it has been difficult to address the role of Ran in the subsequent steps of the cell cycle in vivo. Therefore whether Ran is also required for anaphase spindle elongation remains unclear (Yokoyama et al., 2009). To address the role of Ran during anaphase in the absence of centrosomes, we partially inactivated Ran guanine nucleotide exchange factor (GEF) RCC-1/RAN-3 or the RanGAP, RAN-2. On RanGEF inactivation, Ran remains in its inactive RanGDP-bound form. In ran-3(RNAi)–treated C. elegans embryos, RAN-3 is presumably only partially inactivated. As a consequence, although centrosomes first detach from the pronuclei in prometaphase, they eventually build a bipolar metaphase spindle, and anaphase proceeds (Askjaer et al., 2002; Figure 6, A and E). We found that chromatid segregation was reduced after OICD in ran-3(RNAi)–treated embryos compared with OICD performed in wild-type cells (Figure 6, B and E, and Supplemental Figure S5A). This result shows that a partial reduction of RanGTP is sufficient to affect the segregation of chromatids independently of centrosomes during anaphase.


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

Nahaboo W, Zouak M, Askjaer P, Delattre M - Mol. Biol. Cell (2015)

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.
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Related In: Results  -  Collection

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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.
Mentions: Finally, we tested the role of the small GTPase Ran/RAN-1 during C. elegans anaphase. Inhibition of Ran affects spindle assembly in many different species through the reduction of microtubule density around the DNA (Carazo-Salas et al., 1999; Kalab et al., 1999; Ohba et al., 1999; Wilde and Zheng, 1999; Zhang et al., 1999). Because of this early effect on spindle formation, it has been difficult to address the role of Ran in the subsequent steps of the cell cycle in vivo. Therefore whether Ran is also required for anaphase spindle elongation remains unclear (Yokoyama et al., 2009). To address the role of Ran during anaphase in the absence of centrosomes, we partially inactivated Ran guanine nucleotide exchange factor (GEF) RCC-1/RAN-3 or the RanGAP, RAN-2. On RanGEF inactivation, Ran remains in its inactive RanGDP-bound form. In ran-3(RNAi)–treated C. elegans embryos, RAN-3 is presumably only partially inactivated. As a consequence, although centrosomes first detach from the pronuclei in prometaphase, they eventually build a bipolar metaphase spindle, and anaphase proceeds (Askjaer et al., 2002; Figure 6, A and E). We found that chromatid segregation was reduced after OICD in ran-3(RNAi)–treated embryos compared with OICD performed in wild-type cells (Figure 6, B and E, and Supplemental Figure S5A). This result shows that a partial reduction of RanGTP is sufficient to affect the segregation of chromatids independently of centrosomes during anaphase.

Bottom Line: 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.Their involvement raises the interesting possibility that microtubule polymerization of midzone microtubules is continuously required to sustain chromosome segregation during mitosis.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology of the Cell, University of Lyon 1, CNRS UMR5239, Ecole Normale Supérieure de Lyon, 69007 Lyon, France.

No MeSH data available.


Related in: MedlinePlus