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Direct interaction between centralspindlin and PRC1 reinforces mechanical resilience of the central spindle.

Lee KY, Esmaeili B, Zealley B, Mishima M - Nat Commun (2015)

Bottom Line: The central spindle should be flexible enough for efficient chromosome segregation while maintaining its structural integrity for reliable cytokinesis.How the cell balances these potentially conflicting requirements is poorly understood.This mechanism involves the direct interaction of two different types of conserved microtubule bundlers that are crucial for central spindle formation, PRC1 and centralspindlin.

View Article: PubMed Central - PubMed

Affiliation: 1] Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK [2] Division of Biomedical Cell Biology, Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK.

ABSTRACT
During animal cell division, the central spindle, an anti-parallel microtubule bundle structure formed between segregating chromosomes during anaphase, cooperates with astral microtubules to position the cleavage furrow. Because the central spindle is the only structure linking the two halves of the mitotic spindle, it is under mechanical tension from dynein-generated cortical pulling forces, which determine spindle positioning and drive chromosome segregation through spindle elongation. The central spindle should be flexible enough for efficient chromosome segregation while maintaining its structural integrity for reliable cytokinesis. How the cell balances these potentially conflicting requirements is poorly understood. Here, we demonstrate that the central spindle in C. elegans embryos has a resilient mechanism for recovery from perturbations by excess tension derived from cortical pulling forces. This mechanism involves the direct interaction of two different types of conserved microtubule bundlers that are crucial for central spindle formation, PRC1 and centralspindlin.

No MeSH data available.


Related in: MedlinePlus

Mutations in the C-terminal tail of CYK-4 affect the SPD-1–CYK-4 interaction and cause mid-anaphase spindle rupture.(a) An alignment of the CYK-4 C-terminal tail region, which corresponds to amino acids 644–675 of the C. elegans protein. The conserved motifs with the mutations assessed are highlighted. Ce, C. elegans; Cb, C. briggsae (nematode); Nv, N. vectensis (sea anemone); Hv, H. vulgaris (hydra); Ct, C. teleta (annelid); Cg, C. gigas (oyster); Sp, S. purpuratus (sea urchin); Dr, D. rerio (zebrafish); Xl, X. laevis (frog); Cl, C. livia (bird) and Hs, H. sapiens. (b) Yeast two-hybrid assay assessing the effects of mutations within the above conserved motifs. (c) An SPD-1 1-228 fragment was evaluated for in vitro binding with wild-type or mutant (AA and EAE) CYK-4 constructs immobilized on beads via a maltose-binding protein (MBP) tag (bottom panel, Coomassie staining) and detected with an anti-SPD-1 antibody (top panel). (d) Rescue of the embryonic lethality of cyk-4  embryos by the indicated cyk-4::gfp transgenes. For each strain (two independent strains each for wild-type, AA, EAE and ΔTail), at least 690 embryos produced by seven hermaphrodites homozygous for the cyk-4  allele and the cyk-4::gfp transgene were scored. (e,f) Spinning disk confocal time-lapse imaging of embryos expressing mCherry::tubulin and the indicated cyk-4::gfp transgene in the  background for the cyk-4 endogenous locus (n=25 and 23 for EAE and ΔTail, respectively). Mid-anaphase spindle rupture was observed in the mutants defective for the SPD-1–CYK-4 interaction (t=210 s). No recovery of the midzone accumulation of CYK-4 mutants was observed, although in some embryos, the transient association of mutant CYK-4 near the microtubule plus ends was detected (arrows). Scale bar, 10 μm. In f, the data for the mutants are displayed in colour, whereas those for the wild-type control (same as in Fig. 1; n=33) are plotted in grey.
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f3: Mutations in the C-terminal tail of CYK-4 affect the SPD-1–CYK-4 interaction and cause mid-anaphase spindle rupture.(a) An alignment of the CYK-4 C-terminal tail region, which corresponds to amino acids 644–675 of the C. elegans protein. The conserved motifs with the mutations assessed are highlighted. Ce, C. elegans; Cb, C. briggsae (nematode); Nv, N. vectensis (sea anemone); Hv, H. vulgaris (hydra); Ct, C. teleta (annelid); Cg, C. gigas (oyster); Sp, S. purpuratus (sea urchin); Dr, D. rerio (zebrafish); Xl, X. laevis (frog); Cl, C. livia (bird) and Hs, H. sapiens. (b) Yeast two-hybrid assay assessing the effects of mutations within the above conserved motifs. (c) An SPD-1 1-228 fragment was evaluated for in vitro binding with wild-type or mutant (AA and EAE) CYK-4 constructs immobilized on beads via a maltose-binding protein (MBP) tag (bottom panel, Coomassie staining) and detected with an anti-SPD-1 antibody (top panel). (d) Rescue of the embryonic lethality of cyk-4 embryos by the indicated cyk-4::gfp transgenes. For each strain (two independent strains each for wild-type, AA, EAE and ΔTail), at least 690 embryos produced by seven hermaphrodites homozygous for the cyk-4 allele and the cyk-4::gfp transgene were scored. (e,f) Spinning disk confocal time-lapse imaging of embryos expressing mCherry::tubulin and the indicated cyk-4::gfp transgene in the background for the cyk-4 endogenous locus (n=25 and 23 for EAE and ΔTail, respectively). Mid-anaphase spindle rupture was observed in the mutants defective for the SPD-1–CYK-4 interaction (t=210 s). No recovery of the midzone accumulation of CYK-4 mutants was observed, although in some embryos, the transient association of mutant CYK-4 near the microtubule plus ends was detected (arrows). Scale bar, 10 μm. In f, the data for the mutants are displayed in colour, whereas those for the wild-type control (same as in Fig. 1; n=33) are plotted in grey.

Mentions: To further assess the physiological importance of the interaction between SPD-1 and CYK-4, we introduced point mutations in CYK-4 that affected its interaction with SPD-1. Sequence comparison of the C-terminal tails of CYK-4 orthologues revealed the presence of two motifs, SILGPVTT and K/R-X-K/R, which have been widely conserved throughout metazoan evolution (Fig. 3a and Supplementary Fig. 5). Mutations in these motifs weakened the SPD-1–CYK-4 interaction, either moderately (IL to NN, IL to AA and SIL to AAA) or more severely (RAR to EAE; Fig. 3b). Similar effects were also observed in an in vitro pull-down assay (Fig. 3c). We generated transgenic animals carrying these mutations (AA and EAE) as well as a deletion of the tail domain (ΔTail) in a cyk-4::gfp transgene and expressed the transgenes in a strain in which endogenous cyk-4 had been deleted. Although the wild-type transgene efficiently suppressed the embryonic lethality caused by the deletion of endogenous cyk-4, embryos expressing the mutant transgenes showed different levels of lethality, according to the severity of the SPD-1-binding defect (Fig. 3d). Although the mild AA mutation caused mild lethality, the more severe EAE mutation caused much higher lethality. The ΔTail mutation resulted in the most severe phenotype (>90% lethality). This close correlation between the in vitro and in vivo phenotypes clearly shows that the SPD-1–CYK-4 interaction plays a crucial role in early C. elegans development.


Direct interaction between centralspindlin and PRC1 reinforces mechanical resilience of the central spindle.

Lee KY, Esmaeili B, Zealley B, Mishima M - Nat Commun (2015)

Mutations in the C-terminal tail of CYK-4 affect the SPD-1–CYK-4 interaction and cause mid-anaphase spindle rupture.(a) An alignment of the CYK-4 C-terminal tail region, which corresponds to amino acids 644–675 of the C. elegans protein. The conserved motifs with the mutations assessed are highlighted. Ce, C. elegans; Cb, C. briggsae (nematode); Nv, N. vectensis (sea anemone); Hv, H. vulgaris (hydra); Ct, C. teleta (annelid); Cg, C. gigas (oyster); Sp, S. purpuratus (sea urchin); Dr, D. rerio (zebrafish); Xl, X. laevis (frog); Cl, C. livia (bird) and Hs, H. sapiens. (b) Yeast two-hybrid assay assessing the effects of mutations within the above conserved motifs. (c) An SPD-1 1-228 fragment was evaluated for in vitro binding with wild-type or mutant (AA and EAE) CYK-4 constructs immobilized on beads via a maltose-binding protein (MBP) tag (bottom panel, Coomassie staining) and detected with an anti-SPD-1 antibody (top panel). (d) Rescue of the embryonic lethality of cyk-4  embryos by the indicated cyk-4::gfp transgenes. For each strain (two independent strains each for wild-type, AA, EAE and ΔTail), at least 690 embryos produced by seven hermaphrodites homozygous for the cyk-4  allele and the cyk-4::gfp transgene were scored. (e,f) Spinning disk confocal time-lapse imaging of embryos expressing mCherry::tubulin and the indicated cyk-4::gfp transgene in the  background for the cyk-4 endogenous locus (n=25 and 23 for EAE and ΔTail, respectively). Mid-anaphase spindle rupture was observed in the mutants defective for the SPD-1–CYK-4 interaction (t=210 s). No recovery of the midzone accumulation of CYK-4 mutants was observed, although in some embryos, the transient association of mutant CYK-4 near the microtubule plus ends was detected (arrows). Scale bar, 10 μm. In f, the data for the mutants are displayed in colour, whereas those for the wild-type control (same as in Fig. 1; n=33) are plotted in grey.
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f3: Mutations in the C-terminal tail of CYK-4 affect the SPD-1–CYK-4 interaction and cause mid-anaphase spindle rupture.(a) An alignment of the CYK-4 C-terminal tail region, which corresponds to amino acids 644–675 of the C. elegans protein. The conserved motifs with the mutations assessed are highlighted. Ce, C. elegans; Cb, C. briggsae (nematode); Nv, N. vectensis (sea anemone); Hv, H. vulgaris (hydra); Ct, C. teleta (annelid); Cg, C. gigas (oyster); Sp, S. purpuratus (sea urchin); Dr, D. rerio (zebrafish); Xl, X. laevis (frog); Cl, C. livia (bird) and Hs, H. sapiens. (b) Yeast two-hybrid assay assessing the effects of mutations within the above conserved motifs. (c) An SPD-1 1-228 fragment was evaluated for in vitro binding with wild-type or mutant (AA and EAE) CYK-4 constructs immobilized on beads via a maltose-binding protein (MBP) tag (bottom panel, Coomassie staining) and detected with an anti-SPD-1 antibody (top panel). (d) Rescue of the embryonic lethality of cyk-4 embryos by the indicated cyk-4::gfp transgenes. For each strain (two independent strains each for wild-type, AA, EAE and ΔTail), at least 690 embryos produced by seven hermaphrodites homozygous for the cyk-4 allele and the cyk-4::gfp transgene were scored. (e,f) Spinning disk confocal time-lapse imaging of embryos expressing mCherry::tubulin and the indicated cyk-4::gfp transgene in the background for the cyk-4 endogenous locus (n=25 and 23 for EAE and ΔTail, respectively). Mid-anaphase spindle rupture was observed in the mutants defective for the SPD-1–CYK-4 interaction (t=210 s). No recovery of the midzone accumulation of CYK-4 mutants was observed, although in some embryos, the transient association of mutant CYK-4 near the microtubule plus ends was detected (arrows). Scale bar, 10 μm. In f, the data for the mutants are displayed in colour, whereas those for the wild-type control (same as in Fig. 1; n=33) are plotted in grey.
Mentions: To further assess the physiological importance of the interaction between SPD-1 and CYK-4, we introduced point mutations in CYK-4 that affected its interaction with SPD-1. Sequence comparison of the C-terminal tails of CYK-4 orthologues revealed the presence of two motifs, SILGPVTT and K/R-X-K/R, which have been widely conserved throughout metazoan evolution (Fig. 3a and Supplementary Fig. 5). Mutations in these motifs weakened the SPD-1–CYK-4 interaction, either moderately (IL to NN, IL to AA and SIL to AAA) or more severely (RAR to EAE; Fig. 3b). Similar effects were also observed in an in vitro pull-down assay (Fig. 3c). We generated transgenic animals carrying these mutations (AA and EAE) as well as a deletion of the tail domain (ΔTail) in a cyk-4::gfp transgene and expressed the transgenes in a strain in which endogenous cyk-4 had been deleted. Although the wild-type transgene efficiently suppressed the embryonic lethality caused by the deletion of endogenous cyk-4, embryos expressing the mutant transgenes showed different levels of lethality, according to the severity of the SPD-1-binding defect (Fig. 3d). Although the mild AA mutation caused mild lethality, the more severe EAE mutation caused much higher lethality. The ΔTail mutation resulted in the most severe phenotype (>90% lethality). This close correlation between the in vitro and in vivo phenotypes clearly shows that the SPD-1–CYK-4 interaction plays a crucial role in early C. elegans development.

Bottom Line: The central spindle should be flexible enough for efficient chromosome segregation while maintaining its structural integrity for reliable cytokinesis.How the cell balances these potentially conflicting requirements is poorly understood.This mechanism involves the direct interaction of two different types of conserved microtubule bundlers that are crucial for central spindle formation, PRC1 and centralspindlin.

View Article: PubMed Central - PubMed

Affiliation: 1] Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK [2] Division of Biomedical Cell Biology, Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK.

ABSTRACT
During animal cell division, the central spindle, an anti-parallel microtubule bundle structure formed between segregating chromosomes during anaphase, cooperates with astral microtubules to position the cleavage furrow. Because the central spindle is the only structure linking the two halves of the mitotic spindle, it is under mechanical tension from dynein-generated cortical pulling forces, which determine spindle positioning and drive chromosome segregation through spindle elongation. The central spindle should be flexible enough for efficient chromosome segregation while maintaining its structural integrity for reliable cytokinesis. How the cell balances these potentially conflicting requirements is poorly understood. Here, we demonstrate that the central spindle in C. elegans embryos has a resilient mechanism for recovery from perturbations by excess tension derived from cortical pulling forces. This mechanism involves the direct interaction of two different types of conserved microtubule bundlers that are crucial for central spindle formation, PRC1 and centralspindlin.

No MeSH data available.


Related in: MedlinePlus