Deciphering ’s actions during segregation

Sara Cuylen1 and Christian H. Haering1

1European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany Corresponding author: Haering C.H. ([email protected])

The correct segregation of eukaryotic genomes requires the resolution of sister DNA molecules and their movement into opposite halves of the cell prior to cell division. The dynamic changes need to undergo during these events depend on the action of a multi-subunit SMC (Structural Maintenance of Chromosomes) complex named condensin, yet its molecular function in chromosome segregation is still poorly understood. Recent studies suggest that condensin has a role in the removal of sister chromatid cohesion, in sister chromatid decatenation by topoisomerases, and in the structural reconfiguration of mitotic chromosomes. In this review, we discuss possible mechanisms that could explain the variety of condensin actions during chromosome segregation.

This is the accepted version of the manuscript published in final form in Trends in Cell Biology Volume 21, Issue 9, 552-559, 15 July 2011

The condensin complex - a key player for chromosome condensin function and in the end attempt to synthesize a segregation consensus model for how condensin’s action may allow the correct production of two daughter cells that inherit one - The sudden splitting of sister chromatids and their and precisely one - copy of the genome during every cell movement to the cell poles is one of the most dramatic division. events of the cell division cycle and has fascinated cell biologists for decades. The successful execution of this Scenario 1: Condensin is required for the complete segregation process requires the structural re-organization resolution of sister chromatid cohesion of a cell’s genetic material into defined mitotic chromosomes during prophase, the bi-orientation of the kinetochores of all A possible reason for the large number of unresolved sister sister chromatid pairs on the mitotic spindle during chromatids observed during anaphase in cells depleted of metaphase, and eventually the synchronous dissolution of condensin may be an inability to completely remove sister the connections between sisters to trigger their separation at chromatid cohesion. In metazoans, is released from anaphase onset. chromosomes in two waves, first during prophase through a pathway that is regulated by phosphorylation of cohesin’s Two related chromosomal protein complexes named SA-1/2 subunits and a cohesin-associated protein named cohesin and condensin play key roles in these steps (see Wapl [reviewed in 5], and second through cleavage of Textbox 1). While different models for the mechanisms cohesin’s α-kleisin subunit by separase at the transition from behind cohesin’s function in holding sister chromatids metaphase to anaphase. While the bulk of cohesin still together have been tested and discussed extensively [1-4], dissociates from incubated in condensin-depleted the molecular basis for condensin’s role in chromosome mitotic Xenopus egg extract [6], small amounts of cohesin segregation is less well defined. In this brief review, we can yet be detected on the arms of chromosomes isolated summarize the advances made in deciphering the actions of from nocodazole-arrested HeLa cells after depletion of eukaryotic condensin complexes by recent cell biological, condensin I (but not condensin II) [7]. As a result, the biochemical, and biophysical studies and try to also resolution of chromosome arms that is normally observed integrate novel insights gained from the investigation of under the conditions of the arrest is impaired. It therefore prokaryotic SMC complexes (see Textbox 2). We will seems that complete removal of cohesin from chromosome discuss three different scenarios that may explain the arms requires condensin. Whether the inability to release severe chromosome segregation failures in the absence of cohesin from chromosome arms in these cells is also the

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Box 1. Cohesin and condensin showed that chromosomes assembled after knock-down of condensin subunits are impaired in their structural integrity, even though such Cohesin and condensin are two of the three multi-subunit SMC protein chromosomes eventually seem to condense to almost normal degrees. complexes found in all eukaryotes. Both are built upon specific pairs of When cells attempt to undergo anaphase after condensin depletion, long coiled-coil subunits, which heterodimerize via the central half- chromosomes frequently fail to segregate, which is apparent by a doughnut shaped ‘hinge’ domain situated at one end of the coil. The significant number of lagging chromosomes and the formation of ATPase ‘head‘ domains formed by the N and C termini at the other end chromatin bridges [reviewed in 53 and 65]. In addition to its role during of the antiparallel coiled coil bind to different parts of a so-called kleisin cell division, condensin has been implicated in various interphase subunit, which recruits to the complex additional subunits that are processes that have been addressed in a recent review [66]. largely composed of HEAT repeat domains (see Figure I). Like all

ATPases of the ABC (ATP Binding Cassette) family, the SMC head domains can dimerize by sandwiching a pair of ATP molecules between them. Hydrolysis of the bound ATP molecules is thought to drive the heads apart again. While unicellular organisms express only a single isoform of cohesin and condensin during vegetative growth, metazoans express different variants of the non-SMC subunits that assemble in specific combinations with the SMC dimer [reviewed in 53]. Cohesin is responsible for holding together sister chromatids as soon as they are generated by the DNA replication machinery. A significant body of evidence suggests that cohesin does so by entrapping the two sister DNAs inside the tripartite ring structure formed by its Smc1/Smc3 and α-kleisin subunits [reviewed in 4]. Once all sister chromatid pairs have been bi-oriented on the mitotic spindle, proteolytic opening of cohesin rings by separase-mediated cleavage of the α-kleisin subunit triggers the segregation of sister chromatids at anaphase onset. Condensin is essential for the structural organization of mitotic and meiotic chromosomes. Pioneering work by Hirano and colleagues demonstrated that the transformation of sperm chromatin into compact mitotic-like chromosomes in Xenopus egg extract is impaired after condensin depletion [21, 64]. Studies in a number of cultured cell lines Figure I. Architecture of cohesin and condensin complexes. cause for the later anaphase segregation defects is however Yeast condensin mutants also seem to be incapable of not clear, since separase is evidently capable of removing efficiently removing all cohesin from chromosome arms excess arm cohesion when cohesin release during during mitotic and meiotic divisions [10, 11]. The fact that prophase is blocked by either preventing cohesin separase overexpression reduces the meiotic telomere phosphorylation or by depleting Wapl [8, 9]. segregation defects in a condensin mutant suggests that

Box 2. Prokaryotic SMC complexes overexpression on the other hand causes a two-fold over-compaction of the E. coli nucleoid [72]. These findings are consistent with a role of Most prokaryotic genomes encode only a single SMC protein, which prokaryotic SMC complexes in the structural organization, compaction, forms a complex with two additional subunits that have no apparent and/or disentanglement of the replicated bacterial chromosome during overall homology to their eukaryotic counterparts [reviewed in 67]. its segregation. Studies of the prokaryotic SMC complexes from Bacillus subtilis or Escherichia coli showed that prokaryotic SMCs homodimerize via their ‘hinge’ domains (see Figure II). Recent crystal structures of MukB, MukE, and MukF subcomplexes demonstrated that two MukF protamers, each bound by a dimer of MukE molecules, dimerize via their N-terminal domains and bind to the MukB ATPase head domains via their C-terminal winged helix domains (WHDs) [50]. Similarly, the ScpA subunit of the B. subtilis complex binds to the SMC ATPase head and recruits the ScpB subunit [68, 69]. The MukF and ScpA ‘kleisin’ subunits [70] therefore connect the head domains of V-shaped SMC dimers in an overall arrangement similar to eukaryotic cohesin and condensin complexes. The stoichiometry of the non-SMC subunits in the complexes may vary, potentially as a consequence of ATP-mediated SMC head dimerization (see Figure 3c) [50]. Although prokaryotic SMC complexes are - in contrast to their eukaryotic equivalents - not essential for cell viability under conditions of slow growth, null mutations in encoding any of their subunits nevertheless cause severe chromosome segregation defects in fast proliferating cells. Such defects are evident by the appearance of anucleate cells, cells whose DNA mass has been split by the septum (‘cut’ phenotype), and cells with a mispositioned, extended, or Figure II. Architecture of prokaryotic SMC complexes irregularly shaped nucleoid mass [reviewed in 71]. MukBEF

properties of mitotic chromosomes [7]. A recent study of the anaphase movement dynamics of different fluorescently labeled yeast chromosome loci supports this notion [11]. When sister centromeres separate and move towards the poles, regions along the chromosome arm split with delays that increase towards the telomere. Additional cohesin removal (by inducing α-kleisin degradation) upon anaphase onset abolishes this delay, suggesting that cohesin- dependent linkages along chromosome arms that have escaped cleavage by separase cause a sequential stretching of chromosome arm regions, followed by ‘recoiling’ when these links are broken. Strikingly, recoiling is impaired in condensin mutants. Mathematic modeling suggests that the condensin-dependent recoiling activity is an active process [11], and it is therefore conceivable that condensin-mediated conformational changes along the chromatin fiber generate the force to break or otherwise remove leftover cohesin bridges (Figure 1).

Scenario 2: Condensin promotes the decatenation of sister DNA molecules

A second reason for why sister chromatids lacking condensin frequently fail to resolve may be the persistence of DNA catenations beyond anaphase onset. Such DNA intertwinings result from the collision of replication forks [17] Figure 1. Removal of “leftover” cohesin bridges by chromosome and are normally removed by type-II topoisomerases. One recoiling. Condensin could break cohesin rings at chromosome arms function of condensin could hence be to drive the DNA that had escaped removal by the prophase pathway or separase decatenation activity of topoisomerases during chromosome cleavage by either actively promoting the contraction of chromosome arms, or by stiffening the chromatid fiber and thereby allowing the segregation. transmission of mitotic spindle pulling forces along the chromatid axis. Is there any evidence for a direct interplay between topoisomerases and an SMC protein complex? Two recent some unresolved arm cohesion may persist beyond studies report that the E. coli SMC protein MukB directly anaphase onset, which requires resolution by separase in a binds to and stimulates the activity of the type-II condensin-dependent fashion. While this effect may not only topoisomerase topo IV [18, 19]. Addition of excess MukB to be due to the enhanced cohesin removal but also possibly topo IV promotes the relaxation of DNA supercoiling and - to due to other consequences of separase overexpression (like a lesser extent - the disentangling of concatenated DNA over-stimulation of the FEAR network), an obvious circles in vitro. Mutations in the hinge domain of MukB or in explanation could be that condensin might increase the the C-terminal domain of the topo IV subunit ParC that susceptibility of leftover cohesin to separase cleavage. It prevent their association abolish this stimulatory effect. A has been previously shown that phosphorylation of simple explanation for these observations may be that the cohesin’s α-kleisin subunit by PLK1 renders it a better interaction with MukB renders topo IV more active. substrate for separase in yeast [12]. The finding that Alternatively, MukB may recognize sites of DNA catenation chromosome localization of PLK1 and consequently and help to recruit topoisomerase to them (Figure 2a). The phosphorylation of cohesin’s α-kleisin subunit are reduced in formation of chiral knots into circular plasmid DNA by topo II condensin mutants undergoing the first meiotic division are in the presence of MukB in vitro (see Textbox 3) could consistent with this possibility [10]. It is, however, unlikely indeed be the result of a specific binding of MukB to DNA that condensin recruits PLK1 directly to cohesin, since for crossovers. the most part the two SMC complexes localize to different sites on yeast chromosome arms [13-16]. Does a similar interplay exist between condensin and topoisomerases in eukaryotic cells? While such a link was An alternative possibility is that condensin indirectly first suggested by the finding that mutations in the fission destabilizes cohesin binding by altering the structural yeast genes encoding condensin’s Smc4 subunit and topo II

28] support the hypothesis that they might have an affinity Box 3. Reconfiguration of DNA topology by condensin and for DNA crossover sites. If this hypothesis were true, it could prokaryotic SMC complexes in vitro explain the reduction in topo II staining on mitotic Several biochemical activities have been found for condensin and chromosome spreads in a condensin yeast mutant [29] and prokaryotic SMC complexes in vitro that are presumably important for their in vivo functions. Condensin isolated from mitotic Xenopus the massive enrichment of condensin at highly repetitive extracts has the ability to promote the formation of positive chromosome regions such as the yeast ribosomal DNA supercoils in closed circular DNAs in the presence of type I cluster [15, 16, 29, 30], which may be particularly difficult to topoisomerase (topo I) [27]. Such supercoiling may be the consequence of wrapping DNA around the condensin complex in disentangle due to a large number of catenations that two gyres, as electron spectroscopic images of condensin bound to remain at this region through metaphase [31]. Condensin’s small DNA circles would suggest [42]. Since this activity is both function might, however, not be limited to topo II recruitment ATP-dependent and stimulated by phosphorylation of condensin by mitotic kinases such as cyclin-dependent kinase 1 (CDK1) and at these sites, since topo II function is no longer required polo-like kinase 1 (PLK1) [73, 74], it may be conceivable that during anaphase for rDNA segregation under certain condensin-mediated reconfiguration of chromosome topology conditions, while condensin function still is [32]. Another during mitosis is essential to drive chromosome segregation. Similarly, the MukB subunit of the E. coli SMC complex can support argument against a role of condensin in topo II recruitment the formation of supercoils in circular plasmids in the presence of are the facts that condensin and topo II do not appear to co- topo I. This reaction is ATP-independent and the supercoils localize on mitotic chromosomes [33] and that chromosomal produced in this assay are of opposite sign to the ones produced by eukaryotic condensin [75]. topo II levels are not significantly affected by mutation or A different change in DNA topology can be observed when depletion of different condensin subunits in metazoans [7, condensin holocomplexes immunopurified from Xenopus egg 22-24, 34]. The finding that topo II localization is no longer extracts or isolated yeast Smc2/Smc4 dimers are incubated with restricted to a central chromosome axis after Smc2 or Smc4 nicked circular DNA in the presence of ATP and topoisomerase II depletion [7, 23, 34] might be the consequence of a loss in (topo II). In this case the DNA circles are converted into trefoil knots, which are the result of a topo II-catalyzed DNA strand overall chromosome organization in the absence of passage [37, 76]. Surprisingly, a mutant Smc2/Smc4 dimer condensin function rather than a defect in topoisomerase defective in ATP hydrolysis shows a similar if not identical knotting recruitment. activity [38]. In analogy to its eukaryotic counterpart, the E. coli MukB dimer promotes the formation of right-handed DNA knots in the presence of topo II [75]. Scenario 3: Condensin reconfigures the topology of Yet another ATPase-independent activity is the promotion of single- mitotic chromosomes stranded DNA annealing by the fission yeast Smc2/Smc4 heterodimer [28, 77]. It was recently speculated that this activity Even if condensin does not directly interact with or recruit may be required to remove ‘leftover’ products of interphase topoisomerase, it may still promote disentangling sister processes, for example RNA-DNA hybrids, from mitotic chromatids by shifting the reaction equilibrium of topo II chromosomes to allow their correct segregation [78]. The B. subtilis SMC dimer was shown to promote DNA re-annealing in a similar towards DNA decatenation through an action that alters yet ATP-stimulated manner [79]. properties of the sister chromatid fibers. Condensin may, for are synthetic lethal [20], there is very little evidence for a example, contract the sister DNAs to pull them apart once direct interaction between the two [21, 22]. they had been decatenated by topo II, making a reversal of Furthermore, the evidence that condensin could stimulate the reaction improbable (Figure 2b). Since folding up the the activity of topo II is limited. While extracts prepared from chromatin fiber is entropically unfavorable, such an activity Drosophila cells depleted of Smc4 fail to decatenate DNA would presumably be an energy-dependent process. The circles [23], mitotic frog extracts depleted of condensin show presence of ATPase domains in condensin’s SMC subunits in contrast no reduction in decatenation activity [24]. The suggests that they could in principle act as engines that absence of condensin has apparently also no strong effect drive active chromatin contraction. While purified condensin on topo II activity in vivo, since the enzyme still efficiently complexes only display weak ATPase activities (5-20 cleaves the DXZ1 α-satellite array in cells lacking Smc2 molecules ATP hydrolyzed per minute, per complex), the [25], and the fraction of concatenated forms of a 14 kb ATP turnover rates are stimulated two to five-fold by the circular minichromosome is not increased in a yeast addition of DNA [27, 35, 36]. This stimulation is probably condensin mutant [26]. mediated by the non-SMC subunits of the complex, since ATP hydrolysis of isolated Smc2/Smc4 dimers is not Even though condensin doesn’t seem to directly promote influenced by the presence of DNA [35, 37]. One can hence the enzymatic activity of topo II, it might still be important for imagine that upon chromosome binding, condensin is topoisomerase recruitment to catenated sister chromatids. converted into a motor that actively reconfigures The findings that or Smc2/Smc4 dimers chromosomes. As expected if such an activity were promote knotting of plasmids (see Textbox 3) and essential for chromosome segregation, mutations that preferentially bind to structured DNA substrates in vitro [27,

prevent ATP binding or hydrolysis eliminate condensin [40]. While alternative causes for the changes in DNA function [38, 39]. superhelicity, which may for example result from overstretching of small catenated DNA circles under the What could be the mechanistic basis for re-shaping of tension of the mitotic spindle, still need to be ruled out, this chromosome fibers by condensins? The finding that model is in line with the previous idea that changes in DNA condensin complexes are able to affect the superhelicity of coiling by condensin may be translated into a global re- DNA circles in vitro (see Textbox 3) suggests one organization of the chromosome fiber [27, 41]. possibility. A recent report describes an increase in positive supercoiling of circular yeast minichromosomes preceding It has been suggested that DNA supercoiling could be the their segregation, which depends on both the presence of result of wrapping DNA around the SMC head domains (and mitotic spindle microtubules and condensin function [40]. presumably also the non-SMC subunits) in two positive Surprisingly, positively supercoiled minichromosome dimers turns [42]. In budding and fission yeasts, where condensin isolated from topo II-deficient cells arrested in mitosis are binding sites have been mapped genome-wide, individual more efficiently decatenated in vitro by recombinant topo II binding sites are spaced in average by 10 or 40 kb DNA, enzyme than negatively supercoiled dimers. This leads to respectively [15, 16]. If condensin binding to DNA were to the suggestion that a condensin-dependent change in DNA change the global superhelicity of the DNA fiber in a topology imposes a geometry on (mini)chromosomes that chromatin context, it would first need to overcome the large promotes the decatenation of inter-sister DNA crossovers number of negative turns introduced by the binding of ~50 to 250 nucleosomes to DNA regions of these lengths [43, 44]. While future experiments need to test whether the density of condensin binding may be higher on vertebrate chromosomes, which could account for their stronger compaction during mitosis, the results from the yeast studies suggest that condensin would rather need to use a catalytic mechanism (probably by directing topoisomerases; see above) if its mechanism lay in altering of the overall superhelicity of a chromosomal DNA helix.

Alternatively, condensin may alter the structural properties of sister chromatids by acting as a molecular linker that fastens together different regions of a chromatid. Evidence that condensin can indeed connect different segments within a single DNA strand comes from single molecule experiments. In a magnetic tweezers setup, a linear DNA fragment is stretched between a glass surface and a paramagnetic bead. Addition of condensin I immunopurified from frog egg extract and ATP induces a rapid movement of the bead towards the glass surface [45], suggesting that condensin can support the contraction of linear DNA by bringing together two segments of the DNA and looping the DNA in-between. Importantly, this reaction depends on ATP and can only be measured when condensin is isolated from mitotic extract and not when it is isolated from interphase extract.

How might condensin link two segments of a chromatin fiber? This requires either the presence of (at least) two Figure 2. Two possibilities for how prokaryotic SMC complexes and condensin may promote DNA decatenation by type II chromosome binding sites in one condensin complex, or the topoisomerases. (a) Prokaryotic SMC complexes could recruit association of two (or more) condensin complexes that each topoisomerase (green) to catenated DNA molecules by directly bind to a different chromosome segment. Atomic force binding the enzyme and its DNA substrate. (b) Alternatively, microscopy (AFM) of fission yeast condensin bound to linear condensin may shift the equilibrium between sister chromatid decatenation and catenation towards decatenation by modifying the DNA fragments show individual rod-shaped structures that structural organization of the chromatid fibers. appear to associate with the DNA via their SMC hinge

domains [36]. Further evidence for an interaction of the binding of SMC proteins to DNA (e.g. during loading onto hinge domains with DNA comes from the findings that chromosomes), but may not be required for stably holding addition of DNA protects the Smc2 hinge domain from together segments of a chromatin fiber. Is there another proteolytic cleavage in vitro [46] and that isolated possibility how condensin could act as a molecular linker? Smc2/Smc4 hinge domains shift DNA during gel Since condensin’s kleisin and Smc2/Smc4 subunits form a electrophoresis [47]. It could therefore be possible that a triangular ring structure similar to cohesin rings [46], it is condensin complex binds to one chromatin segment via its conceivable that condensin rings could encircle two hinge domain while making direct contacts to a second chromosomal DNA segments in an analogous manner as chromatin segment via another part of the complex (Figure cohesin rings entrap two sister chromatids (Figure 3b) [41, 3a). 51]. However, the coiled-coil arms of eukaryotic condensin complexes appear closely attached in most electron or Is there any evidence for the existence of a DNA binding atomic force micrographs [36, 52], and proteolytic cleavage site in SMC protein complexes besides in their hinge of Smc2’s coiled coils does not release condensin from domains? Studies of prokaryotic SMCs identified a positively isolated chromosomes in vitro [39]. Both findings argue charged patch not only in their hinge domains [48] but also against the idea that DNA could pass through condensin in the structures of dimerized head domains [49, 50]. rings, yet the first may be a consequence of attaching Mutations that reverse the charges in either domain reduce condensin complexes onto mica surfaces for EM or AFM the electrophoretic mobility shift of plasmid DNA. It is imaging and the second may be hampered by the possibility therefore possible that prokaryotic SMC proteins link two that cleavage of the two strands of the Smc2 coiled coil in DNA segments by binding one via their head and the other offset positions may not break ring integrity. Entrapment via their hinge domains. Finding out whether a DNA binding within rings would not rule out any additional direct contacts site also exists in the associated head domains of between condensin subunits and the chromatin fiber condensin’s Smc2/Smc4 heterodimer might presumably discussed above. need to await the solution their structure at atomic resolution. Irrespective of how condensin complexes contact DNAs, they might not act in isolation. Multiple condensin One caveat of the in vitro DNA binding experiments is that complexes, each holding on to a single chromosome site, detection of electrophoretic mobility shifts so far always may interact as dimers or higher order assemblies and required an excess of protein over DNA. It may hence be thereby generate a network of chromosomal linkages [53]. possible that the observed interactions reflect only transient Condensin’s localization Figure 3. Three different possibilities for how the chromatin fiber may be organized through linkages by SMC protein complexes. (a) Condensin may bridge two chromosome segments by binding one segment via its SMC hinge and the other via its SMC head domain or the non-SMC subunits. (b) Alternatively, different chromosome segments may be linked by their entrapment within the same condensin ring structure in an analogous manner to the entrapment of sister chromatids within cohesin rings. (c) Biochemical and structural data

suggests that one of the MukE2F protamers dissociates from the MukB head domain upon ATP- dependent head dimerization. The free protamer may bind to the head domain of another MukB dimer, thereby forming multimers of SMC complexes that could arrange into rosette or spiral structures. Such networks might form a central scaffold for loops of DNA.

along the inner axes of mitotic chromosomes is indeed Finding out how condensin can reinforce mitotic consistent with the formation of a condensin chromosome chromosomes on a mechanistic level is a challenge for ‘scaffold’ [7, 33, 54]. Such condensin networks may not be future studies. While drawing parallels between the work on static structures but could at least in part be quite dynamic, prokaryotic SMCs and condensin may be speculative at this given the rapid turnover of condensin I on chromosomes stage, it is conceivable that some of the insights gained from measured in FRAP experiments [55, 56]. How condensin the in vitro studies of the latter may also help to understand complexes might form such networks is not known. It may condensin’s action. A key step forward would be the be possible that condensin multimerization could follow a correlation of the various biochemical activities observed for similar principle as the formation of linear or rosette-like SMC complexes on naked DNA substrates in vitro (see aggregates observed for prokaryotic SMC complexes in Textbox 3) with condensin’s binding to a chromatin electron and atomic force micrographs [57, 58]. Two recent substrate in vivo. This may require first the isola crystal structures of MukBEF complexes suggest a tion and characterization of condensin-bound chromatin molecular mechanism for the formation of such multimers using a combination of molecular biology, biochemistry, and [50]. In the first structure, two MukB head domains dimerize biophysical techniques, and then the reconstitution of this by sandwiching a pair of ATPγS molecules between them, interaction in vitro with defined components. Central to and each head binds to the C-terminal winged helix domain understanding condensin’s molecular machinery will be to (WHD) of one MukF kleisin molecule. In the second explain the dynamic changes the complex undergoes structure, only one MukF WHD is bound to the MukB through of cycles of ATP binding and hydrolysis by its SMC homodimer, while the second MukF has been displaced by head domains, and how these changes may be controlled the central region of the bound MukF. The WHD of the by post-translational modifications such as phosphorylation displaced MukF subunit would therefore be free to bind [reviewed in 62]. Selective inhibition of condensin’s ATPase another MukB dimer (Figure 3c). activity in vivo may prove a powerful approach towards this goal. Deciphering the interplay of condensin with other A consensus model for condensin function? components of mitotic chromosomes, like INCENP, Which of the three scenarios we discussed - complete KIF4A/chromokinesin [34, 39], or yet unknown partners, is cohesin removal, resolution of sister catenations, and another priority. Finally, high resolution optical imaging reconfiguration of chromosome topology - represents technologies that allow the localization of individual condensin’s major role during chromosome segregation? It condensin molecules on mitotic chromosomes [63] will be is obvious that all three scenarios are interconnected. A essential for understanding the formation and maintenance condensin-driven reconfiguration of chromosome topology of a structure that is without a doubt one of the cell’s most may, for example, be required to expose cohesin binding fascinating molecules, the mitotic chromosome. sites that had previously been inaccessible to the separase Acknowledgements protease [7]. Stiffening of the chromatid fiber resulting from linkage of different chromatin segments by condensin could We are grateful to Jan Ellenberg, Marko Kaksonen, and allow the transmission of mechanical forces generated by Ilaria Piazza for suggestions and comments on the the pulling spindle at centromeres to chromosome arms and manuscript. Work in the Haering lab is supported by EMBL thereby tear apart cohesin linkages that had not been and the German Research Foundation (DFG) Priority removed by separase cleavage (Figure 1) [11]. 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