Condensin phosphorylated by the Aurora-B-like kinase Ark1 is continuously required until telophase in a mode distinct from Top2

Abstract

SummaryCondensin is a conserved protein complex that functions in chromosome condensation and segregation. It has not been previously unequivocally determined whether condensin is required throughout mitosis. Here, we examined whether Schizosaccharomyces pombe condensin continuously acts on chromosomes during mitosis and compared its role with that of DNA topoisomerase II (Top2). Using double mutants containing a temperature-sensitive allele of the condensin SMC2 subunit cut14 (cut14-208) or of top2, together with the cold-sensitive nda3-KM311 mutation (in -tubulin), temperature-shift experiments were performed. These experiments allowed inactivation of condensin or Top2 at various stages throughout mitosis, even after late anaphase. The results established that mitotic chromosomes require condensin and Top2 throughout mitosis, even in telophase. We then showed that the Cnd2 subunit of condensin (also known as Barren) is the target subunit of Aurora-B-like kinase Ark1 and that Ark1-mediated phosphorylation of Cnd2 occurred throughout mitosis. The phosphorylation sites in Cnd2 were determined by mass spectrometry, and alanine and glutamate residue replacement mutant constructs for these sites were constructed. Alanine substitution mutants of Cnd2, which mimic the unphosphorylated protein, exhibited broad mitotic defects, including at telophase, and overexpression of these constructs caused a severe dominantnegative effect. By contrast, glutamate substitution mutants, which mimic the phosphorylated protein, alleviated the segregation defect in Ark1-inhibited cells. In telophase, the condensin subunits in cut14-208 mutant accumulated in lumps that contained telomeric DNA and proteins that failed to segregate. Condensin might thus serve to keep the segregated chromosomes apart during telophase.

Chromosome Dynamics during Mitosis

“…Thus, condensin I appears to help generate a signal that delays cytokinesis. Consistent with this view, a subfraction of condensin has also been localized to the spindle midzone in fission yeast (Nakazawa et al 2011). In retrospect, it is no mere coincidence that the original condensin mutants in fission yeast were isolated as those displaying the so-called "cut" phenotype, in which cytokinesis abnormally proceeds without proper segregation of chromosomes (Hirano et al 1986).…”

“…The molecular picture of how the localization and activities of condensin I might be regulated is far from complete, although numerous studies have identified involvement of multiple mitotic kinases, including Cdk1 (Kimura et al 1998;St-Pierre et al 2009), Aurora B (Lipp et al 2007;Collette et al 2011;Nakazawa et al 2011;Tada et al 2011), and Polo-like kinase (St-Pierre et al 2009). Sumoylation has also been implicated in the function of condensin in ribosomal DNA (rDNA) maintenance (Takahashi et al 2008) and kinetochore localization (BachellierBassi et al 2008) in budding yeast.…”

“…It is most likely that Sgo1 plays a role in retaining this population of cohesin along arms, which is eventually cleaved by separase Harrison et al 2009). Likewise, strong lines of evidence indicate that the final separation of sister arms requires continuous actions of condensin and topoisomerase II throughout anaphase (Renshaw et al 2010;Nakazawa et al 2011;Tada et al 2011). Comparison of biophysical properties between metaphase and anaphase chromatids by mechanical stretching experiments (Marko 2008) might be an interesting direction in the future.…”

Rational elicitation of cold-sensitive phenotypes

“…Thus, these conditional mutants have led to a greater understanding of phage genetics, protein folding, and macromolecular assembly (15)(16)(17). Similarly, cs and ts mutants of different genes essential for cell division, in combination with temperature-shift experiments, have been used to understand the cell cycle phases in which each of these genes act (18).Various changes at the amino acid level or gene regulation level can cause cold sensitivity. Some cs mutants have been suggested to result from altered feedback inhibition of certain metabolic pathways at lower temperatures (19,20).…”

“…Thus, these conditional mutants have led to a greater understanding of phage genetics, protein folding, and macromolecular assembly (15)(16)(17). Similarly, cs and ts mutants of different genes essential for cell division, in combination with temperature-shift experiments, have been used to understand the cell cycle phases in which each of these genes act (18).…”