The reversible condensation of chromosomes during cell division remains a classic problem in cell biology. Condensation requires the condensin complex 1 in certain experimental systems 2-8, but not in many others 9-15. Anaphase chromosome segregation almost always fails in condensin-depleted cells, leading to the formation of prominent chromatin bridges and cytokinesis failure 4, 9-17. Here, live cell analysis of chicken DT40 cells bearing a conditional knockout of condensin subunit SMC2 reveals 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 phosphatase targeting subunit RepoMan from recruiting 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, RCA (regulator of chromosome architecture), cooperates with condensin to preserve the characteristic chromosome architecture during mitosis.Mitosis is normal in SMC2 conditional knockout (SMC2 ON/OFF ) chicken DT40 cells grown without doxycycline (SMC2 ON ) 12. By 30 hours after addition of doxycycline to the culture medium (SMC2 OFF ) SMC2 mRNA levels drop at least 160-fold (QRT-PCR, Supplementary Figure 1a) and the protein becomes undetectable in immunoblots. The cells begin to die within 24-48 hours as anaphase chromosome segregation fails and massive chromatin bridges block cytokinesis (Figure 1a-d). The loss of SMC2 is accompanied by loss of other condensin subunits (e.g. CAP-H) from mitotic chromosomes (Supplementary Figure 1b- d).While this anaphase failure is unlikely to be due to defects in cohesin dynamics (see 18 , our unpublished results), it could reflect a loss of DNA topoisomerase II (topo II) function, because topo II localisation is altered in condensin-depleted chromosomes 12,18 , and the activity of extracted Drosophila topo II against an exogenous substrate is decreased following condensin RNAi 18. We therefore examined topo II activity in vivo at a physiological site by quantitating in situ topo II cleavage within the highly characterized 2.1 Mb centromeric α-satellite DXZ1 array of the human X chromosome 19 in four independent SMC2 ON/OFF DT40 hybrid cell lines. No significant differences in topo II activity at this site were found in the presence or absence of condensin (Supplementary Figure 2). Therefore, Correspondence should be addressed to WCE. telephone -44-(0)131-650-7101, fax -44-(0)131-650-7100, Bill.Earnshaw@ed.ac.uk.

Mitotic chromosome formation involves a relatively minor condensation of the chromatin volume coupled with a dramatic reorganization into the characteristic “X” shape. 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. In this analysis, KIF4 and condensin were mutually dependent for their dynamic localization on the chromatid axes. 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. Simultaneous depletion of KIF4 and condensin caused complete loss of 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. These three proteins are major determinants in shaping the characteristic mitotic chromosome morphology.

When chromosomes are aligned and bioriented at metaphase, the elastic stretch of centromeric chromatin opposes pulling forces exerted on sister kinetochores by the mitotic spindle. Here we show that condensin ATPase activity is an important regulator of centromere stiffness and function. Condensin depletion decreases the stiffness of centromeric chromatin by 50% when pulling forces are applied to kinetochores. However, condensin is dispensable for the normal level of compaction (rest length) of centromeres, which probably depends on other factors that control higher-order chromatin folding. Kinetochores also do not require condensin for their structure or motility. Loss of stiffness caused by condensindepletion produces abnormal uncoordinated sister kinetochore movements, leads to an increase in Mad2(؉) kinetochores near the metaphase plate and delays anaphase onset. INTRODUCTIONCentromeric chromatin is a special region of chromosomes that has important mechanical and signaling functions in mitosis (Pidoux and Allshire, 2005;Ekwall, 2007;Cheeseman and Desai, 2008;Vagnarelli et al., 2008). In metaphase, pulling forces generated by interactions between spindle microtubules (MTs) and kinetochores are opposed by tension produced by centromeric chromatin stretch. Centromere and kinetochore tension and stretch are important for maintaining chromosome alignment (McIntosh et al., 2002), stabilizing kinetochore microtubule (kMT) attachments (Nicklas and Koch, 1969), spindle checkpoint signaling (Musacchio and Salmon, 2007;McEwen and Dong, 2009), and also for the back-to-back orientation of sister kinetochores (Loncarek et al., 2007). At least three independent factors have roles in the establishment of centromeric tension in metaphase: sister chromatid cohesion (Yeh et al., 2008), the elastic properties of chromatin (Houchmandzadeh et al., 1997;Almagro et al., 2004;Marko, 2008), and the higher order structure of the centromeric chromatin.Condensin is important for the architecture of mitotic chromosome arms (Coelho et al., 2003;Hudson et al., 2003;Hirota et al., 2004;Hirano, 2006), but it also localizes to centromeres (Saitoh et al., 1994;Gerlich et al., 2006), where condensin I, but not condensin II was reported to have a role in stabilizing the structure (Gerlich et al., 2006). It has recently been suggested that condensin could have a role in regulating the elastic behavior of centromeric chromatin. One study found that condensin I-depleted Drosophila chromosomes were unable to align at a metaphase plate, had distorted kinetochore structures, and lost elasticity of their centromeric chromatin (Oliveira et al., 2005). However a similar study in human cells reported that although loss of condensin I caused kinetochores to undergo abnormal movements, these movements were bidirectional (e.g., reversible; Gerlich et al., 2006).Even after the publication of those results, the regulation and functional significance of centromere stretch remained unknown. An elegant study in budding yeast went on to find that chromatin struct...

Summary Pili are proteinaceous polymers of linked pilins that protrude from the cell surface of many bacteria and often mediate adherence and virulence. We investigated a set of 20 Bacteroidia pilins from the human microbiome whose structures and mechanism of assembly were unknown. Crystal structures and biochemical data revealed a diverse protein superfamily with a common Greek-key β-sandwich fold with two transthyretin-like repeats that polymerize into a pilus through a strand-exchange mechanism. The assembly mechanism of the central, structural pilins involves proteinase-assisted removal of their N-terminal β-strand, creating an extended hydrophobic groove that binds the C-terminal donor strands of the incoming pilin. Accessory pilins at the tip and base have unique structural features specific to their location, allowing initiation or termination of the assembly. The bacteroidia pilus therefore has a biogenesis mechanism that is distinct from other known pili and likely represents a different type of bacterial pilus.

Enveloped viruses enter cells by binding to their entry receptors and fusing with the membrane at the cell surface or after trafficking through acidic endosomal compartments. Species-specific virus tropism is usually determined by these entry receptors. Because mouse mammary tumor virus (MMTV) is unable to infect Chinese hamster cells, we used phenotypic screening of the T31 mouse͞ hamster radiation hybrid panel to map the MMTV cell entry receptor gene and subsequently found that it is transferrin receptor 1. MMTV-resistant human cells that expressed mouse transferrin receptor 1 became susceptible to MMTV infection, and treatment of mouse cells with a monoclonal antibody that downregulated cell surface expression of the receptor blocked infection. MMTV, like vesicular stomatitis virus, depended on acid pH for infection. MMTV may use transferrin receptor 1, a membrane protein that is endocytosed via clathrin-coated pits and traffics through the acidic endosomes, to rapidly get to a compartment where acid pH triggers the conformational changes in envelope protein required for membrane fusion.

The nucleotide sequence of the regions flanking the A+T region of Drosophila melanogaster mitochondrial DNA (mtDNA) has been determined. Included are the genes encoding the transfer RNAs for valine, isoleucine, glutamine and methionine, the small ribosomal RNA and the 5'-coding sequences of the large ribosomal RNA and NADH dehydrogenase subunit II. This completes the nucleotide sequence of the D. melanogaster mitochondrial genome. The circular mtDNA of D. melanogaster varies in size among different populations largely due to length differences in the control region (Fauron & Wolstenholme, 1976; Fauron & Wolstenholme, 1980a, b); the mtDNA region we have sequenced, combined with those sequenced by others, yields a composite genome that is 19,517 bp in length as compared to 16,019 bp for the mtDNA of D. yakuba. D. melanogaster mtDNA exhibits an extreme bias in base composition; it comprises 82.2% deoxyadenylate and thymidylate residues as compared to 78.6% in D. yakuba mtDNA. All genes encoded in the mtDNA of both species are in identical locations and orientations. Nucleotide substitution analysis reveals that tRNA and rRNA genes evolve at less than half the rate of protein coding genes.

Telomeric sequences of eukaryotes consist of short tandem repeats organized in arrays of variable length in which the guanine-rich strand runs 5' -* 3' toward the chromosomal end. The terminal repeats in yeast are the only elements necessary for telomere function in this organism. To test whether mammalian terminal repeats can function after reintroduction into a mammalian cell, a repeat-containing terminal fragment from a human chromosome was electroporated into a hamster-human hybrid cell line. In 6 of 27 independent transformants analyzed, the introduced sequences were found at the ends of chromosomes, based on all available criteria. Terminal restriction-fragment heterogeneity and the survival of these chromosomes demonstrate that these telomeres are functional. Cytogenetic evidence from one of these cell lines suggests that chromosome breakage with healing at the integration site is the mechanism responsible for the terminal location.

Defects of mitochondrial polymerase gamma (POLG) underlie neurological diseases ranging from myopathies to parkinsonism and infantile Alpers syndrome. The most severe manifestations have been associated with mutations of the 'spacer' region of POLG, the function of which has remained unstudied in humans. We identified a family, segregating three POLG amino acid variants, A467T, R627Q and Q1236H. The first two affect the spacer region and the third is a polymorphism, allelic with R627Q. Three grades of disease severity appeared to correlate with the genotypes. The patient with the most severe outcome, cerebellar ataxia syndrome, had all three variants, those with R627Q and Q1236H had juvenile-onset ptosis and gait disturbance and those with a single A467T allele had late-onset ptosis. To evaluate the molecular pathogenesis of these spacer defects, we expressed and purified the mutant proteins and studied their catalytic properties in vitro. The A467T substitution resulted in clearly decreased activity, DNA binding and processivity of the polymerase. Our biochemical data, the dominant manifestation of A467T and its previously reported high frequency in the Belgian population (0.6%), emphasize the role of this mutation as a common cause of neurological disease. Further, biochemical evidence that a polymorphic variant may modify the function of a mutant POLG, if occurring in the same polypeptide, is shown here. Finally, and surprisingly, other pathogenic spacer mutants showed DNA-binding affinities and processivities similar to or higher than the controls, suggesting that the disease-causing mechanisms of spacer mutations extend beyond the basic catalytic functions of POLG.