![]() ![]() To test this hypothesis, single or double depletion of KIF4 and SMC2 was achieved by performing siRNA knockdowns on conditional knockout cells. In that case, cells doubly depleted of condensin and KIF4 would be expected to exhibit defects similar to those observed in cells depleted of condensin alone. The spatial and functional relationships between KIF4 and condensin on mitotic chromosomes demonstrated in previous sections could be explained if KIF4 and condensin work in a common pathway. We therefore hypothesized that other uncharacterized scaffold components, possibly including the little-studied kinesin superfamily member KIF4, might also have important roles in mitotic chromosome structure in vertebrate cells. Interestingly, cells depleted of topo IIα or condensin can form mitotic chromosomes, albeit with an imperfect morphology ( Steffensen et al., 2001 Chang et al., 2003 Hagstrom et al., 2002 Kaitna et al., 2002 Hudson et al., 2003 Carpenter and Porter, 2004 Sakaguchi and Kikuchi, 2004 Spence et al., 2007 Gonzalez et al., 2011). If topo II is depleted in budding yeast, circular chromosomes accumulate positive supercoils in a condensin-dependent manner ( Baxter et al., 2011). Depletion of either topo IIα or condensin impairs chromosome organization and segregation ( Holm et al., 1985 Uemura et al., 1987 Strunnikov et al., 1993 Saka et al., 1994 Bhat et al., 1996 Hirano et al., 1997 Hagstrom et al., 2002 Hudson et al., 2003, 2009 Vagnarelli et al., 2006 Csankovszki et al., 2009). The two localize on the chromatid axes ( Earnshaw and Heck, 1985 Saitoh et al., 1994) in an alternating manner ( Maeshima and Laemmli, 2003). Many studies have addressed the role of topo IIα and the condensin complexes in mitotic chromosome structure. Mitotic chromosomes isolated from chicken DT40 cells used in the present study have ∼10× less condensin II than condensin I ( Ohta et al., 2010a). Later it was shown that animals and plants have two condensin complexes that share core subunits (SMC2 and SMC4) but differ in their auxiliary non-SMC subunits: with condensin I containing CAP-D2/G/H and condensin II CAP-D3/G2/H2 ( Hirano et al., 1997 Ono et al., 2003). Together with the other condensin subunits, these proteins were also major components of mitotic chromosomes assembled in vitro in Xenopus egg extracts ( Hirano and Mitchison, 1994 Hirano et al., 1997). The first two scaffold proteins identified in purified chromosomes ( Lewis and Laemmli, 1982) were topoisomerase IIα (topo IIα) and condensin core subunit SMC2 ( Earnshaw et al., 1985 Gasser et al., 1986 Saitoh et al., 1994). Laemmli and co-workers first suggested that nonhistone proteins of the “chromosome scaffold” fraction have an important role in mitotic chromosome structure ( Adolph et al., 1977a, b). These three proteins are major determinants in shaping the characteristic mitotic chromosome morphology. ![]() In these experiments, topoisomerase IIα contributed to shaping mitotic chromosomes by promoting the shortening of the chromatid axes and apparently acting in opposition to the actions of KIF4 and condensins. Simultaneous depletion of KIF4 and condensin caused complete loss of chromosome morphology. Depletion of either caused sister chromatids to expand and compromised the “intrinsic structure” of the chromosomes (defined in an in vitro assay), with loss of condensin showing stronger effects. In this analysis, KIF4 and condensin were mutually dependent for their dynamic localization on the chromatid axes. Here we report results of a detailed morphological analysis, which revealed that chromokinesin KIF4 cooperated in a parallel pathway with condensin complexes to promote the lateral compaction of chromatid arms. Mitotic chromosome formation involves a relatively minor condensation of the chromatin volume coupled with a dramatic reorganization into the characteristic “X” shape. ![]()
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