Drosophila CAP-D2 Is Required for Condensin Complex Stability and Resolution of Sister Chromatids

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Drosophila CAP-D2 Is Required for Condensin Complex Stability and Resolution of Sister Chromatids Research Article 2529 Drosophila CAP-D2 is required for condensin complex stability and resolution of sister chromatids Ellada Savvidou1, Neville Cobbe1, Søren Steffensen2, Sue Cotterill3 and Margarete M. S. Heck1,* 1Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Michael Swann Building, King’s Buildings, Mayfield Road, Edinburgh, EH9 3JR, UK 2Instituto de Histologia, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisboa, Portugal 3St Georges Hospital Medical School, Cranmer Terrace, London, SW17 0RE, UK *Author for correspondence (e-mail: margarete.heck@ed.ac.uk) Accepted 21 March 2005 Journal of Cell Science 118, 2529-2543 Published by The Company of Biologists 2005 doi:10.1242/jcs.02392 Summary The precise mechanism of chromosome condensation and chromosome arm and centromere resolution. The loss decondensation remains a mystery, despite progress over of CAP-D2 after RNAi has additional downstream the last 20 years aimed at identifying components essential consequences on the stability of CAP-H, the localization of to the mitotic compaction of the genome. In this study, we DNA topoisomerase II and other condensin subunits, and analyse the localization and role of the CAP-D2 non-SMC chromosome segregation. Finally, we discovered that even condensin subunit and its effect on the stability of the after interfering with two components important for condensin complex. We demonstrate that a condensin chromosome architecture (DNA topoisomerase II and complex exists in Drosophila embryos, containing CAP-D2, condensin), chromosomes were still able to compact, paving the anticipated SMC2 and SMC4 proteins, the CAP- the way for the identification of further components or H/Barren and CAP-G (non-SMC) subunits. We show that activities required for this essential process. CAP-D2 is a nuclear protein throughout interphase, increasing in level during S phase, present on chromosome axes in mitosis, and still present on chromosomes as they Supplementary material available online at start to decondense late in mitosis. We analysed the http://jcs.biologists.org/cgi/content/full/118/11/2529/DC1 consequences of CAP-D2 loss after dsRNA-mediated interference, and discovered that the protein is essential for Key words: Chromosome, Chromatid, Mitosis, CAP-D2 Journal of Cell Science Introduction to associate with chromatin in a mitosis-specific manner Chromosomal DNA, if stretched end to end, measures about 2 (Kimura and Hirano, 2000). Each of the condensin subunits is metres in any one cell of the human body and therefore must essential and required for proper organisation and segregation be highly folded to fit into a nucleus of only 5 µm diameter. of mitotic chromosomes, but exactly how the complex DNA has to condense even further prior to cell division in contributes to these processes is still unclear. Recently a second order to form the highly compact metaphase chromosome condensin complex has been identified, with a different comprising two sister chromatids, capable of withstanding complement of non-SMC subunits (Ono et al., 2003). anaphase segregation (Heck, 1997; McHugh, 2003; Swedlow Studies of condensin function have resulted in conflicting and Hirano, 2003). Thus, mitotic chromosome condensation hypotheses as to the role of this complex. Cells of involves a process in which interphase chromatin is (1) Schizosaccharomyces pombe mutant in Cut3 and Cut14 compacted, and (2) resolved into two distinct rod-shaped sister (the SMC4 and SMC2 subunits, respectively) exhibited chromatids. Recent studies have contributed to the unravelling chromosome condensation defects and displayed a ‘cut’ of this process. A five subunit ‘condensin’ complex was phenotype where septation (cell division) occurred with initially identified biochemically in Xenopus (Hirano et al., incompletely separated chromosomes (Saka et al., 1994). 1997; Hirano and Mitchison, 1994), and comprises two SMC Fluorescence in situ hybridization in these mutants (structural maintenance of chromosomes) proteins (SMC2 and demonstrated that the length of mitotic chromosome arms SMC4) and three non-SMC proteins [CAP-D2, CAP-G or increased while the centromeric DNA could separate and move CAP-H (Barren in Drosophila)]. All five subunits have been to the opposite poles normally. In S. pombe and Saccharomyces identified in the eukaryotes examined to date (Cobbe and Heck, cerevisiae, studies have shown that all members of the 2000). The SMC proteins belong to a superfamily of highly condensin complex are required for proper chromosome conserved and ubiquitous chromosomal ATPases (Cobbe and condensation and segregation (Lavoie et al., 2000; Ouspenski Heck, 2004). The non-SMC subunits have been proposed to et al., 2000; Strunnikov et al., 1995; Sutani et al., 1999). In have dual roles in the regulation of condensin function: one is Xenopus, when condensin was depleted from mitotic extracts to activate the SMC ATPases to perform ATP-dependent before or after condensation, unreplicated chromatin failed to supercoiling activity, and the other is to allow the holocomplex assemble into individual chromatids or became completely 2530 Journal of Cell Science 118 (11) disorganised, suggesting that the complex was required both Laemmli, 2003; Steffensen et al., 2001). The localisation for assembly and maintenance of mitotic chromosomes pattern and the phenotype after the depletion of either the (Hirano et al., 1997; Hirano and Mitchison, 1994). In condensin complex or topo II suggested that these two contrast, genetic studies in Drosophila melanogaster and components of the chromosome scaffold may collaborate to Caenorhabditis elegans showed that resolution between the bring about chromosome compaction and organisation. sister chromatids rather than condensation of the chromosomes Biochemical and localisation studies in Drosophila showed was compromised when the condensin complex was disrupted that CAP-H/Barren associated with topo II throughout mitosis, (Bhat et al., 1996; Hagstrom et al., 2002; Steffensen et al., while SMC4 appeared to be responsible for the axial 2001). In both organisms, a high degree of compaction relative localisation of topo II on the chromosomes and for its to interphase and a bipolar metaphase plate formed. However, decatenation activity (Bhat et al., 1996; Coelho et al., 2003). severe chromosome segregation defects and chromosome However, in S. pombe, the localisation but not the activity of breakage were observed in anaphase and telophase. DmSMC4 topo II was affected in condensin mutants, while topo II RNAi in Drosophila cultured cells confirmed the requirement localization to well-defined axial structures in Xenopus was of this protein for chromosome organisation and segregation independent of condensin (Bhalla et al., 2002; Cuvier and (Coelho et al., 2003). In both flies and worms, the absence of Hirano, 2003). Given that chromosomes experience different condensin subunits results in the formation of chromosome constraints depending on the cell, organism, or time in bridges because of incomplete resolution and ensuing failure development, it is perhaps not surprising that a ‘unified’ of sister chromatids to separate completely during anaphase hypothesis regarding these two components has not emerged. (Hagstrom and Meyer, 2003). Particular insight was gleaned In this study, we demonstrate that a condensin I complex when the robustness of chromosome architecture was assessed exists in Drosophila. We subsequently examined the cell cycle after SMC2 knock-out in chicken DT40 cells: while localization and role of the Drosophila CAP-D2 subunit in chromosomes still reached normal levels of condensation, clear chromosome dynamics during mitosis. We show that depletion defects in association of other non-histone chromosomal of CAP-D2 by RNAi in Drosophila cultured cells results in a proteins was observed (Hudson et al., 2003). compromised mitotic chromosome architecture with defective Recently, a second condensin complex, called condensin II, sister chromatid resolution and subsequent chromosome was identified in vertebrates (Ono et al., 2003). This complex bridges in anaphase. We demonstrate here for the first time, shares the same two SMC subunits as the original complex, that the fate of other members of the condensin complex now named condensin I, but appears to contain different non- and other chromosomal proteins essential for chromosome SMC subunits (named CAP-D3, CAP-G2 and CAP-H2). The dynamics during mitosis is altered when a condensin subunit two complexes appear to alternate along the axis of metaphase is disrupted either by depletion or mutation. We additionally chromatids but are both present at the centromere, albeit in examine Drosophila mutations of condensin subunits to assess distinct regions (Ono et al., 2004). Depletion of these the stability of the complex in the context of the organism. We complexes in Xenopus and HeLa cells produced distinct also show that mitotic coordination with regard to chromosome defects in chromosome morphology, suggesting that the passengers is compromised when condensin function is complexes may contribute differently to mitotic chromosome affected. Finally we demonstrate that when both condensin and Journal of Cell Science architecture (Ono et al., 2003). Furthermore, the topo II are disrupted by RNAi, chromosomes are surprisingly centromere/kinetochore regions were structurally disorganised still able to compact, intimating the existence of additional,
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