Abstract
ABSTRACTThe requirement for condensin in chromosome formation in somatic cells remains unclear, as imperfectly condensed chromosomes do form in cells depleted of condensin by conventional methodologies. In order to dissect the roles of condensin at different stages of vertebrate mitosis, we have established a versatile cellular system that combines auxin-mediated rapid degradation with chemical genetics to obtain near-synchronous mitotic entry of chicken DT40 cells in the presence and absence of condensin. We analyzed the outcome by live- and fixed-cell microscopy methods, including serial block face scanning electron microscopy with digital reconstruction. Following rapid depletion of condensin, chromosomal defects were much more obvious than those seen after a slow depletion of condensin. The total mitotic chromatin volume was similar to that in control cells, but a single mass of mitotic chromosomes was clustered at one side of a bent mitotic spindle. Cultures arrest at prometaphase, eventually exiting mitosis without segregating chromosomes. Experiments where the auxin concentration was titrated showed that different condensin levels are required for anaphase chromosome segregation and formation of a normal chromosome architecture.This article has an associated First Person interview with the first author of the paper.
Highlights
As cells enter mitosis, the chromatin is compacted 2-3-fold in volume as it undergoes a dramatic re-organisation (Lleres et al, 2009; Martin and Cardoso, 2010; Vagnarelli and Earnshaw, 2012)
We find that mitotic chromatin is compacted to the normal extent though the intrinsic chromosome architecture is faulty in condensin-depleted cells
Mitotic index (MI), mitotic profile and cell cycle profile of the SMC2-Auxin Inducible Degron (AID)-GFP cells were similar to the ones of wild type cells without auxin (Figure 1-figure supplement 1A, Figure 2-figure supplement 1C)
Summary
The chromatin is compacted 2-3-fold in volume as it undergoes a dramatic re-organisation (Lleres et al, 2009; Martin and Cardoso, 2010; Vagnarelli and Earnshaw, 2012). Rod-shaped mitotic chromosomes form and chromosome scaffold proteins are localised along the sister chromatid axes (Paulson and Laemmli, 1977; Lewis and Laemmli, 1982; Earnshaw et al, 1985; Gasser et al., 1986; Saitoh et al, 1994; Samejima et al, 2012) These proteins include topoisomerase IIα, chromokinesin KIF4 and SMC2 (Structural Maintenance of Chromosomes 2), a core subunit of both condensin complexes (Earnshaw et al., 1985; Gasser and Laemmli, 1986; Saitoh et al, 1994; Hirano and Mitchison, 1994; Hirano et al, 1997; Ono et al, 2003; Samejima et al, 2012). Mitosis lasts about one hour in control cells after release from a 4 h 1NMPP1 block
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