Taken together with other cell biological and genetic studies during the past several years, it seems fair to conclude that condensins are the most important, central players in the assembly and structural maintenance of mitotic chromosomes ( Thadani et al., 2012 Frosi and Haering, 2015 Hirano, 2016 Kalitsis et al., 2017). A recent study has used the same cell-free extracts to demonstrate that chromosome-like structures can be assembled even in the near absence of nucleosomes ( Shintomi et al., 2017). Moreover, once assembly reactions are complete, removing topo IIα from mitotic chromosomes has little impact on their morphology in the Xenopus egg cell-free extracts ( Hirano and Mitchison, 1993). Equally important, a recent study has demonstrated that mitotic chromosome-like structures can be reconstituted in vitro by mixing sperm chromatin with only six purified components, which include core histones, topo IIα, and condensin I ( Shintomi et al., 2015). In fact, only two factors, topoisomerase IIα (topo IIα) and condensin I, have been demonstrated so far to be essential for mitotic chromatid assembly in the cell-free extracts ( Hirano and Mitchison, 1993 Hirano et al., 1997). It is therefore possible that the “core” components required for building the bulk part of mitotic chromosomes is much simpler, as had been shown in classical studies of metaphase chromosomes isolated from HeLa cells ( Gasser and Laemmli, 1987) or mitotic chromatids assembled in Xenopus egg cell-free extracts ( Hirano and Mitchison, 1994). It should be noted, however, that this number includes domain-specific components (e.g., centromere- and telomere-specific proteins) and contaminants that may artificially get associated with chromosomes during their isolation. In fact, a recent proteomics approach has identified ∼4000 proteins in mitotic chromosomes isolated from chicken DT40 cells ( Ohta et al., 2010). It is generally thought that the protein composition of mitotic chromosomes is highly complex, especially because they represent one of the largest structures observed within the cell. Despite enormous progress marked during the past two decades or so, its molecular mechanism remains not fully understood ( Belmont, 2006 Marko, 2008 Kinoshita and Hirano, 2017). This process, known as mitotic chromosome assembly or condensation, is an essential prerequisite for faithful segregation of genetic information into two daughter cells. When eukaryotic cells divide, chromatin residing within the interphase nucleus is converted into a discrete set of individual chromosomes, each composed of a pair of rod-shaped chromatids (sister chromatids). We propose that condensin II makes a primary contribution to mitotic chromosome architecture and maintenance in human cells. Furthermore, quantitative morphological analyses using the machine-learning algorithm wndchrm support the notion that chromosome shaping is tightly coupled to the reorganization of condensin II-based axes. This assay combined with small interfering RNA depletion demonstrates that the recovery of chromatin shapes and the reorganization of axes are highly sensitive to depletion of condensin II but less sensitive to depletion of condensin I or topoisomerase IIα. Both chromatin and condensin-positive chromosome axes are converted into near-original shapes at 100 mM NaCl. In this assay, mitotic chromosomes are first expanded in a hypotonic buffer containing a Mg 2+-chelating agent and then converted into different shapes in a NaCl concentration-dependent manner. To address these questions from a physico-chemical point of view, we have established a set of two-step protocols for inducing reversible assembly of chromosome structure in situ, namely within a whole cell. Although both complexes become concentrated along the axial region of each chromatid by metaphase, it remains unclear exactly how such axes might assemble and contribute to chromosome shaping. Condensins I and II are multisubunit complexes that play a central role in mitotic chromosome assembly.
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