Mitotic Chromosome Structure Research Articles

Visualization by atomic force microscopy and FISH of the 45S rDNA gaps in mitotic chromosomes of Lolium perenne

The mitotic chromosome structure of 45S rDNA site gaps in Lolium perenne was studied by atomic force microscope (AFM) combining with fluorescence in situ hybridization (FISH) analysis in the present study. FISH on the mitotic chromosomes showed that 45S rDNA gaps were completely broken or local despiralizations of the chromatid which had the appearance of one or a few thin DNA fiber threads. Topography imaging using AFM confirmed these observations. In addition, AFM imaging showed that the broken end of the chromosome fragment lacking the 45S rDNA was sharper, suggesting high condensation. In contrast, the broken ends containing the 45S rDNA or thin 45S rDNA fibers exhibited lower density and were uncompacted. Higher magnification visualization by AFM of the terminals of decondensed 45S rDNA chromatin indicated that both ends containing the 45S rDNA also exhibited lower density zones. The measured height of a decondensed 45S rDNA chromatin as obtained from the AFM image was about 55-65 nm, composed of just two 30-nm single fibers of chromatin. FISH in flow-sorted G2 interphase nuclei showed that 45S rDNA was highly decondensed in more than 90% of the G2/M nuclei. Our results suggested that a failure of the complex folding of the chromatin fibers occurred at 45S rDNA sites, resulting in gap formation or break.

Mitotic Chromosome Structure: Reproducibility of Folding and Symmetry between Sister Chromatids

Mitotic chromosome structure and pathways of mitotic condensation remain unknown. The limited amount of structural data on mitotic chromosome structure makes it impossible to distinguish between several mutually conflicting models. Here we used a Chinese hamster ovary cell line with three different lac operator-tagged vector insertions distributed over an ∼1 μm chromosome arm region to determine positioning reproducibility, long-range correlation in large-scale chromatin folding, and sister chromatid symmetry in minimally perturbed, metaphase chromosomes. The three-dimensional positions of these lac operator-tagged spots, stained with lac repressor, were measured in isolated metaphase chromosomes relative to the central chromatid axes labeled with antibodies to topoisomerase II. Longitudinal, but not axial, positioning of spots was reproducible but showed intrinsic variability, up to ∼300 nm, between sister chromatids. Spot positions on the same chromatid were uncorrelated, and no correlation or symmetry between the positions of corresponding spots on sister chromatids was detectable, showing the absence of highly ordered, long-range chromatin folding over tens of mega-basepairs. Our observations are in agreement with the absence of any regular, reproducible helical, last level of chromosome folding, but remain consistent with any hierarchical folding model in which irregularity in folding exists at one or multiple levels.

The Kleisin Subunit of Cohesin Dictates Damage-Induced Cohesion

Cohesin, the protein complex that mediates sister chromatid cohesion, is required for faithful chromosome segregation and efficient repair of double-strand breaks (DSBs). Cohesion generation is normally restricted to S phase. However, in G2/M, a DSB activates cohesion generation near the DSB and genome-wide. Here, using budding yeast, we show that DSB-induced cohesion occurs when cohesin contains the kleisin subunit, Mcd1 (Scc1), but not when Mcd1 is replaced by its meiotic isoform, Rec8. We exploit this divergence to demonstrate that serine 83 of Mcd1 and the Chk1 kinase are critical determinants for DSB-induced cohesion. We propose that a DSB in G2/M activates Mec1 (ATR), which in turn stimulates Chk1-dependent phosphorylation of Mcd1 at serine 83. Serine 83 phosphorylation promotes chromatin-bound cohesin to become cohesive.

Emerging roles of the SUMO pathway in mitosis

SUMO proteins are small ubiquitin-like modifiers found in all eukaryotes that become covalently conjugated to other cellular proteins. The SUMO conjugation pathway is biochemically similar to ubiquitin conjugation, although the enzymes within the pathway act exclusively on SUMO proteins. This post-translational modification controls many processes. Here, I will focus on evidence that SUMOylation plays a critical role(s) in mitosis: Early studies showed a genetic requirement for SUMO pathway components in the process of cell division, while later findings implicated SUMOylation in the control of mitotic chromosome structure, cell cycle progression, kinetochore function and cytokinesis. Recent insights into the targets of SUMOylation are likely to be extremely helpful in understanding each of these aspects. Finally, growing evidence suggests that SUMOylation is a downstream target of regulation through Ran, a small GTPase with important functions in both interphase nuclear trafficking and mitotic spindle assembly.

Packaging the Genome: the Structure of Mitotic Chromosomes

Mitotic chromosomes are essential structures for the faithful transmission of duplicated genomic DNA into two daughter cells during cell division. Although more than 100 years have passed since chromosomes were first observed, it remains unclear how a long string of genomic DNA is packaged into compact mitotic chromosomes. Although the classical view is that human chromosomes consist of radial 30 nm chromatin loops that are somehow tethered centrally by scaffold proteins, called condensins, cryo-electron microscopy observation of frozen hydrated native chromosomes reveals a homogeneous, grainy texture and neither higher-order nor periodic structures including 30 nm chromatin fibres were observed. As a compromise to fill this huge gap, we propose a model in which the radial chromatin loop structures in the classic view are folded irregularly toward the chromosome centre with the increase in intracellular cations during mitosis. Consequently, compact native chromosomes are made up primarily of irregular chromatin networks cross-linked by self-assembled condensins forming the chromosome scaffold.

Sororin Is Required for Stable Binding of Cohesin to Chromatin and for Sister Chromatid Cohesion in Interphase

Sister chromatid cohesion depends on cohesin [1-3]. Cohesin associates with chromatin dynamically throughout interphase [4]. During DNA replication, cohesin establishes cohesion [5], and this process coincides with the generation of a cohesin subpopulation that is more stably bound to chromatin [4]. In mitosis, cohesin is removed from chromosomes, enabling sister chromatid separation [6]. How cohesin associates with chromatin and establishes cohesion is poorly understood. By searching for proteins that are associated with chromatin-bound cohesin, we have identified sororin, a protein that was known to be required for cohesion [7]. To obtain further insight into sororin's function, we have addressed when during the cell cycle sororin is required for cohesion. We show that sororin is dispensable for the association of cohesin with chromatin but that sororin is essential for proper cohesion during G2 phase. Like cohesin, sororin is also needed for efficient repair of DNA double-strand breaks in G2. Finally, sororin is required for the presence of normal amounts of the stably chromatin-bound cohesin population in G2. Our data indicate that sororin interacts with chromatin-bound cohesin and functions during the establishment or maintenance of cohesion in S or G2 phase, respectively.

Condensin and Repo-Man–PP1 co-operate in the regulation of chromosome architecture during mitosis

The reversible condensation of chromosomes during cell division remains a classic problem in cell biology. Condensation requires the condensin complex in certain experimental systems, but not in many others. Anaphase chromosome segregation almost always fails in condensin-depleted cells, leading to the formation of prominent chromatin bridges and cytokinesis failure. Here, live-cell analysis of chicken DT40 cells bearing a conditional knockout of condensin subunit SMC2 revealed that condensin-depleted chromosomes abruptly lose their compact architecture during anaphase and form massive chromatin bridges. The compact chromosome structure can be preserved and anaphase chromosome segregation rescued by preventing the targeting subunit Repo-Man from recruiting protein phosphatase 1 (PP1) to chromatin at anaphase onset. This study identifies an activity critical for mitotic chromosome structure that is inactivated by Repo-Man-PP1 during anaphase. This activity, provisionally termed 'regulator of chromosome architecture' (RCA), cooperates with condensin to preserve the characteristic chromosome architecture during mitosis.

Establishment of Sister Chromatid Cohesion at the S. cerevisiae Replication Fork

Two identical sister copies of eukaryotic chromosomes are synthesized during S phase. To facilitate their recognition as pairs for segregation in mitosis, sister chromatids are held together from their synthesis onward by the chromosomal cohesin complex. Replication fork progression is thought to be coupled to establishment of sister chromatid cohesion, facilitating identification of replication products, but evidence for this has remained circumstantial. Here we show that three proteins required for sister chromatid cohesion, Eco1, Ctf4, and Ctf18, are found at, and Ctf4 travels along chromosomes with, replication forks. The ring-shaped cohesin complex is loaded onto chromosomes before S phase in an ATP hydrolysis-dependent reaction. Cohesion establishment during DNA replication follows without further cohesin recruitment and without need for cohesin to re-engage an ATP hydrolysis motif that is critical for its initial DNA binding. This provides evidence for cohesion establishment in the context of replication forks and imposes constraints on the mechanism involved.

Condensin I Interacts with the PARP-1-XRCC1 Complex and Functions in DNA Single-Strand Break Repair

Condensins are essential protein complexes critical for mitotic chromosome organization. Little is known about the function of condensins during interphase, particularly in mammalian cells. Here we report the interphase-specific interaction between condensin I and the DNA nick-sensor poly(ADP-ribose) polymerase 1 (PARP-1). We show that the association between condensin I, PARP-1, and the base excision repair (BER) factor XRCC1 increases dramatically upon single-strand break damage (SSB) induction. Damage-specific association of condensin I with the BER factors flap endonuclease 1 (FEN-1) and DNA polymerase delta/epsilon was also observed, suggesting that condensin I is recruited to interact with BER factors at damage sites. Consistent with this, DNA damage rapidly stimulates the chromatin association of PARP-1, condensin I, and XRCC1. Furthermore, depletion of condensin in vivo compromises SSB but not double-strand break (DSB) repair. Our results identify a SSB-specific response of condensin I through PARP-1 and demonstrate a role for condensin in SSB repair.

Proteolysis of Mitotic Chromosomes Induces Gradual and Anisotropic Decondensation Correlated with a Reduction of Elastic Modulus and Structural Sensitivity to Rarely Cutting Restriction Enzymes

The effect of nonspecific proteolysis on the structure of single isolated mitotic newt chromosomes was studied using chromosome elastic response as an assay. Exposure to either trypsin or proteinase K gradually decondensed and softened chromosomes but without entirely eliminating their elastic response. Analysis of chromosome morphology revealed anisotropic decondensation upon digestion, with length increasing more than width. Prolonged protease treatment resulted only in further swelling of the chromosome without complete dissolution. Mild trypsinization induced sensitivity of chromosome elasticity to five- and six-base-specific restriction enzymes. These results, combined with previous studies of effects of nucleases on mitotic chromosome structure, indicate that mild proteolysis gradually reduces the density of chromatin-constraining elements in the mitotic chromosome, providing evidence consistent with an anisotropically folded "chromatin network" model of mitotic chromosome architecture.

Experimental Visualization of Chromoneme as a Higher Level of Chromatin Compactization in the Mitotic Chromosome

We succeeded to visualize the chromoneme or a filamentous chromatin structure, with the mean thickness 0.1-0.2 microm, as a higher level of chromatin compactization in animal and plant cells at different stages of chromosome condensation at mitotic prophase and during chromatid decondensation at telophase. Under the natural conditions, chromoneme elements are not detected in the most condensed chromatin of metaphase chromosomes on ultrathin sections. We studied the ultrastructure and behavior of the chromatin of mitotic chromosomes in situ in cultured mouse L-197 cells under the conditions selectively demonstrating the chromoeneme structure of the mitotic chromosomes in the presence of Ca2+. Loosely packaged dense chromatin bands, ca. 100 nm in diameter, chromonemes, were detected in chromosome arms in a solution containing 3 mM CaCl2. When transferred in a hypotonic solution containing 10 mM tris-HCl, these chromosome swelled, lost the chromoneme level of structure, and rapidly transformed in loose aggregates of elementary DNP fibrils, 30 nm in diameter. After this decondensation in the low ionic strength solution, the chromoneme structure of mitotic chromosomes was restored when they were transferred in a Ca2+ containing solution. The morphological characteristics of the chromoneme and pattern of its packaging in the chromosome were preserved. However, when the mitotic cells with chromosomes, in which the chromoneme structure was visualized with the help of 3 mM CaCl2, were treated with a photosensbilizer, ethidium bromide, and illuminate with a light with the wavelength 460 nm, chromatic decondensation under the hypotonic solution was not observed. The chromoneme elements in a stabilized chromatin of the mitotic chromosome preserved specific interconnection and their general pattern of packaging in in the chromatic was also preserved. The chromoneme elements in the chromosomes stabilized by light preserved their density and diameter even in a 0.6 M NaCl solution, which normally leads to chromoneme destruction. An even more rigid treatment of the stabilized chromosomes with a 2 M NaCl solution, which normally fully decondenses the chromosomes, made it possible to detect a 3D reticular skeleton devoid of any axial structures.

Ctf7p/Eco1p exhibits acetyltransferase activity–but does it matter?

In eukaryotic cells, faithful chromosome segregation depends upon the physical pairing, or cohesion, between sister chromatids. Budding yeast CTF7/ECO1 (herein termed CTF7) encodes an essential protein required to establish cohesion during S-phase and associates with DNA replication factors [1.Skibbens R.V. Corson L.B. Koshland D. Hieter P. Ctf7p is essential for sister chromatid cohesion and links mitotic chromosome structure to the DNA replication machinery.Genes Dev. 1999; 13: 307-319Crossref PubMed Scopus (372) Google Scholar, 2.Toth A. Ciosk R. Uhlmann F. Galova M. Schleiffer A. Nasmyth K. Yeast cohesin complex requires a conserved protein, Eco1p(Ctf7), to establish cohesion between sister chromatids during DNA replication.Genes Dev. 1999; 13: 320-333Crossref PubMed Scopus (486) Google Scholar, 3.Mayer M.L. Gygi S.P. Aebersold R. Hieter P. Identification of RFC (Ctf18p, Ctf8p, Dcc1p): An alternate RFC complex required for sister chromatid cohesion in S. cerevisiae.Mol. Cell. 2001; 7: 959-970Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 4.Hanna J.S. Kroll E.S. Lundblad V. Spencer F.A. Saccharomyces cerevisiae CTF18 and CTF4 are required for sister chromatid cohesion.Mol. Cell. Biol. 2001; 21: 3144-3158Crossref PubMed Scopus (251) Google Scholar, 5.Wang Z. Castano I.B. De Las Penas A. Adams C. Christman M.F. Pol kappa: A DNA polymerase required for sister chromatid cohesion.Science. 2000; 289: 774-779Crossref PubMed Scopus (160) Google Scholar, 6.Krause S.A. Loupart M.L. Vass S. Schoenfelder S. Harrison S. Heck M.M. Loss of cell cycle checkpoint control in Drosophila Rfc4 mutants.Mol. Cell. Biol. 2001; 21: 5156-5168Crossref PubMed Scopus (40) Google Scholar, 7.Kenna M.A. Skibbens R.V. Mechanical link between cohesion establishment and DNA replication: Ctf7p/Eco1p, a cohesion establishment factor, associates with three different Replication Factor C Complexes.Mol. Cell. Biol. 2003; 23: 2999-3007Crossref PubMed Scopus (90) Google Scholar, 8.Edwards S. Li C.M. Levy D.L. Brown J. Snow P.M. Campbell J. Saccharomyces cerevisiae DNA polymerase varepsilon and polymerase sigma interact physically and functionally, suggesting a role for polymerase varepsilon in sister chromatid cohesion.Mol. Cell. Biol. 2003; 23: 2733-2748Crossref PubMed Scopus (75) Google Scholar, 9.Skibbens R.V. Chl1p, a DNA helicase-like protein in budding yeast, functions in sister chromatid cohesion.Genetics. 2004; 166: 32-42Crossref Scopus (123) Google Scholar, 10.Petronczki M. Chwalla B. Siomos M. Yokobayashi S. Helmhart W. Deutschbauer A. et al.Sister-chromatid cohesion mediated by the alternative RF-CCtf18/Dcc1/Ctf8, the helicase Chl1 and the polymerase-alpha-associated protein Ctf4 is essential for chromatid disjunction during meiosis II.J. Cell Sci. 2004; 117: 3547-3559Crossref PubMed Scopus (115) Google Scholar]. However, the molecular mechanism by which Ctf7p establishes cohesion remains unknown. In vitro characterization of Ctf7p as an acetyltransferase led to the model that this activity provides for Ctf7p's essential function [11.Ivanov D. Schleiffer A. Eisenhaber F. Mechtler K. Haering C.H. Nasmyth K. Eco1 is a novel acetyltransferase that can acetylate proteins involved in cohesion.Curr. Biol. 2002; 12: 323-328Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar]. However, in vivo Ctf7p substrates have yet to be documented, nor has an in vivo acetyltransferase activity been demonstrated even when Ctf7p is overexpressed [11.Ivanov D. Schleiffer A. Eisenhaber F. Mechtler K. Haering C.H. Nasmyth K. Eco1 is a novel acetyltransferase that can acetylate proteins involved in cohesion.Curr. Biol. 2002; 12: 323-328Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar] (A. Brands and R.V. Skibbens, unpublished data). In fact, the effects of acetylation-defective Ctf7p (ctf7ack-) in yeast remain to be rigorously tested, leaving unanswered the critical questions of whether Ctf7p acetyltransferase activity is essential for cell viability and to what extent this activity is required for the establishment of cohesion. Here, we show that yeast strains harboring acetyltransferase-defective alleles [11.Ivanov D. Schleiffer A. Eisenhaber F. Mechtler K. Haering C.H. Nasmyth K. Eco1 is a novel acetyltransferase that can acetylate proteins involved in cohesion.Curr. Biol. 2002; 12: 323-328Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar] as the sole source of Ctf7p function exhibit robust growth and high fidelity chromosome transmission.

The Mitotic Chromosome Is an Assembly of Rigid Elastic Axes Organized by Structural Maintenance of Chromosomes (SMC) Proteins and Surrounded by a Soft Chromatin Envelope

The structure of mitotic chromosomes is still poorly understood. Here we describe the use of a novel approach based on elasticity measurements of a single chromosome for studying the organization of these objects. The data reveal that mitotic chromosomes exhibit a non-homogenous structure consisting of rigid elastic axes surrounded by a soft chromatin envelope. The chemical continuity of DNA, but not RNA, was required for the maintenance of these axes. The axes show a modular structure, and the structural maintenance of chromosomes (SMC) proteins participate in their organization. Topoisomerase II was not involved in either the organization of the axes or the maintenance of the mitotic chromosomes. A model for the assembly and the structure of the mitotic chromosome is proposed. According this model, the chromosome axes are dynamic structures that assemble at the onset and disassemble the end of mitosis, respectively. The SMC proteins, in addition to maintaining axis elasticity, are essential for the determination of the rod-like chromosome shape. The extreme compaction of mitotic chromosomes is determined mainly by the high amount of bivalent ions bound to DNA at mitosis.

Meiotic condensin is required for proper chromosome compaction, SC assembly, and resolution of recombination-dependent chromosome linkages.

Condensin is an evolutionarily conserved protein complex that helps mediate chromosome condensation and segregation in mitotic cells. Here, we show that condensin has two activities that contribute to meiotic chromosome condensation in Saccharomyces cerevisiae. One activity, common to mitosis, helps mediate axial length compaction. A second activity promotes chromosome individualization with the help of Red1 and Hop1, two meiotic specific components of axial elements. Like Red1 and Hop1, condensin is also required for efficient homologue pairing and proper processing of double strand breaks. Consistent with these functional links condensin is necessary for proper chromosomal localization of Red1 and Hop1 and the subsequent assembly of the synaptonemal complex. Finally, condensin has a Red1/Hop1-independent role in the resolution of recombination-dependent linkages between homologues in meiosis I. The existence of distinct meiotic activities of condensin (axial compaction, individualization, and resolution of recombination-dependent links) provides an important framework to understand condensin's role in both meiotic and mitotic chromosome structure and function.

A Distinction with a Difference

CELL BIOLOGY In the transition from interphase to mitosis, chromosomal material changes from a fuzzy indistinct mass into compact and well-defined pairs of chromosomes. Condensin complexes contain proteins of the structural maintenance of chromosomes (SMC) family and function in the assembly and segregation of mitotic chromosomes. Ono et al. suggest that two types of condensin complexes are present in vertebrate cells. While sharing SMC subunits, condensin I and II differ in their other subunits. Depleting either or both of the condensin complexes disrupted the normal ordered structure of mitotic chromosomes in slightly different ways and obscured the demarcation of the two chromosomal chromatids. Furthermore, the two condensin complexes localized to different sites along the condensed chromosomes and hence appear to make separate contributions to the generation and maintenance of condensed chromosome structure. — SMH Cell 115 , 109 (2003).

Engineered chromosome regions with altered sequence composition demonstrate hierarchical large-scale folding within metaphase chromosomes.

Mitotic chromosome structure and DNA sequence requirements for normal chromosomal condensation remain unknown. We engineered labeled chromosome regions with altered scaffold-associated region (SAR) sequence composition as a formal test of the radial loop and other chromosome models. Chinese hamster ovary cells were isolated containing high density insertions of a transgene containing lac operator repeats and a dihydrofolate reductase gene, with or without flanking SAR sequences. Lac repressor staining provided high resolution labeling with good preservation of chromosome ultrastructure. No evidence emerged for differential targeting of SAR sequences to a chromosome axis within native chromosomes. SAR sequences distributed uniformly throughout the native chromosome cross section and chromosome regions containing a high density of SAR transgene insertions showed normal diameter and folding. Ultrastructural analysis of two different transgene insertion sites, both spanning less than the full chromatin width, clearly contradicted predictions of simple radial loop models while providing strong support for hierarchical models of chromosome architecture. Specifically, an ∼250-nm-diam folding subunit was visualized directly within fully condensed metaphase chromosomes. Our results contradict predictions of simple radial loop models and provide the first unambiguous demonstration of a hierarchical folding subunit above the level of the 30-nm fiber within normally condensed metaphase chromosomes.

Male Mouse Recombination Maps for Each Autosome Identified by Chromosome Painting

Linkage maps constructed from genetic analysis of gene order and crossover frequency provide few clues to the basis of genomewide distribution of meiotic recombination, such as chromosome structure, that influences meiotic recombination. To bridge this gap, we have generated the first cytological recombination map that identifies individual autosomes in the male mouse. We prepared meiotic chromosome (synaptonemal complex [SC]) spreads from 110 mouse spermatocytes, identified each autosome by multicolor fluorescence in situ hybridization of chromosome-specific DNA libraries, and mapped >2,000 sites of recombination along individual autosomes, using immunolocalization of MLH1, a mismatch repair protein that marks crossover sites. We show that SC length is strongly correlated with crossover frequency and distribution. Although the length of most SCs corresponds to that predicted from their mitotic chromosome length rank, several SCs are longer or shorter than expected, with corresponding increases and decreases in MLH1 frequency. Although all bivalents share certain general recombination features, such as few crossovers near the centromeres and a high rate of distal recombination, individual bivalents have unique patterns of crossover distribution along their length. In addition to SC length, other, as-yet-unidentified, factors influence crossover distribution leading to hot regions on individual chromosomes, with recombination frequencies as much as six times higher than average, as well as cold spots with no recombination. By reprobing the SC spreads with genetically mapped BACs, we demonstrate a robust strategy for integrating genetic linkage and physical contig maps with mitotic and meiotic chromosome structure.

The bending rigidity of mitotic chromosomes.

The bending rigidities of mitotic chromosomes isolated from cultured N. viridescens (newt) and Xenopus epithelial cells were measured by observing their spontaneous thermal bending fluctuations. When combined with simultaneous measurement of stretching elasticity, these measurements constrain models for higher order mitotic chromosome structure. We measured bending rigidities of B approximately 10(-22) N. m(2) for newt and approximately 10(-23) N. m(2) for Xenopus chromosomes extracted from cells. A similar bending rigidity was measured for newt chromosomes in vivo by observing bending fluctuations in metaphase-arrested cells. Following each bending rigidity measurement, a stretching (Young's) modulus of the same chromosome was measured in the range of 10(2) to 10(3) Pa for newt and Xenopus chromosomes. For each chromosome, these values of B and Y are consistent with those expected for a simple elastic rod, B approximately YR(4), where R is the chromosome cross-section radius. Our measurements rule out the possibility that chromosome stretching and bending elasticity are principally due to a stiff central core region and are instead indicative of an internal structure, which is essentially homogeneous in its connectivity across the chromosome cross-section.

C. elegans condensin promotes mitotic chromosome architecture, centromere organization, and sister chromatid segregation during mitosis and meiosis.

Chromosome segregation and X-chromosome gene regulation in Caenorhabditis elegans share the component MIX-1, a mitotic protein that also represses X-linked genes during dosage compensation. MIX-1 achieves its dual roles through interactions with different protein partners. To repress gene expression, MIX-1 acts in an X-chromosome complex that resembles the mitotic condensin complex yet lacks chromosome segregation function. Here we show that MIX-1 interacts with a mitotic condensin subunit, SMC-4, to achieve chromosome segregation. The SMC-4/MIX-1 complex positively supercoils DNA in vitro and is required for mitotic chromosome structure and segregation in vivo. Thus, C. elegans has two condensin complexes, one conserved for mitosis and another specialized for gene regulation. SMC-4 and MIX-1 colocalize with centromere proteins on condensed mitotic chromosomes and are required for the restricted orientation of centromeres toward spindle poles. This cell cycle-dependent localization requires AIR-2/AuroraB kinase. Depletion of SMC-4/MIX-1 causes aberrant mitotic chromosome structure and segregation, but not dramatic decondensation at metaphase. Moreover, SMC-4/MIX-1 depletion disrupts sister chromatid segregation during meiosis II but not homologous chromosome segregation during meiosis I, although both processes require chromosome condensation. These results imply that condensin is not simply required for compaction, but plays a more complex role in chromosome architecture that is essential for mitotic and meiotic sister chromatid segregation.

Reversible hypercondensation and decondensation of mitotic chromosomes studied using combined chemical-micromechanical techniques.

We show that the chromatin in mitotic chromosomes can be drastically overcompacted or unfolded by temporary shifts in ion concentrations. By locally 'microspraying' reactants from micron-size pipettes, while simultaneously monitoring the size of and tension in single chromosomes, we are able to quantitatively study the dynamics of these reactions. The tension in a chromosome is monitored through observation and calibration of bending of the glass pipettes used to manipulate the chromosomes. For concentrations > 500 mM of NaCl and > 200 mM of MgCl2, we find that the initially applied tensions of approximately 500 pN relax to zero and that mitotic chromatin temporarily disperses in agreement with previous work (Maniotis et al. [1997] J. Cell. Biochem. 65:114-130). This unfolding occurs in about 1 s, and is reversible once the charge density is returned to physiological levels, if the exposure is not longer than approximately 1 min. Low concentrations of NaCl (< 30 mM) also induces a decrease in tension and increase in size. We observe this swelling to be isotropic in experiments on chromosomes under zero tension, a behavior inconsistent with the existence of a well-defined central chromosome 'scaffold'. By contrast 10 mM of divalent cations (MgCl2 and CaCl2) induces an extremely rapid and reversible increase in tension and a reduction in the size of mitotic chromosomes. Hexaminecobalt trichloride (trivalent cation) has the same effect as MgCl2 and CaCl2, except the magnitude of force increase and size change are much larger. Hexaminecobalt trichloride reduces mitotic chromosomes to 65% of their original volume, indicating that at least 1/3 of their apparent volume is aqueous solution. These results indicate that chromatin inside mitotic chromatids has a large amount of conformational freedom allowing dynamic unfolding and refolding and that charge interactions play a central role in maintaining mitotic chromosome structure.