Aurora A promotes chromosome congression by activating the condensin-dependent pool of KIF4A.

Aurora kinases create phosphorylation gradients within the spindle during prometaphase and anaphase, thereby locally regulating factors that promote spindle organization, chromosome condensation and movement, and cytokinesis. We show that one such factor is the kinesin KIF4A, which is present along the chromosome axes throughout mitosis and the central spindle in anaphase. These two pools of KIF4A depend on condensin I and PRC1, respectively. Previous work has shown KIF4A is activated by Aurora B at the anaphase central spindle. However, whether or not chromosome-associated KIF4A bound to condensin I is regulated by Aurora kinases remain unclear. To determine the roles of the two different pools of KIF4A, we generated specific point mutants that are unable to interact with either condensin I or PRC1 or are deficient for Aurora kinase regulation. By analyzing these mutants, we show that Aurora A phosphorylates the condensin I-dependent pool of KIF4A and thus actively promotes chromosome congression from the spindle poles to the metaphase plate.

Dynamic SUMO remodeling drives a series of critical events during the meiotic divisions in Caenorhabditis elegans.

Chromosome congression and segregation in C. elegans oocytes depend on a complex of conserved proteins that forms a ring around the center of each bivalent during prometaphase; these complexes are then removed from chromosomes at anaphase onset and disassemble as anaphase proceeds. Here, we uncover mechanisms underlying the dynamic regulation of these ring complexes (RCs), revealing a strategy by which protein complexes can be progressively remodeled during cellular processes. We find that the assembly, maintenance, and stability of RCs is regulated by a balance between SUMO conjugating and deconjugating activity. During prometaphase, the SUMO protease ULP-1 is targeted to the RCs but is counteracted by SUMO E2/E3 enzymes; then in early anaphase the E2/E3 enzymes are removed, enabling ULP-1 to trigger RC disassembly and completion of the meiotic divisions. Moreover, we found that SUMO regulation is essential to properly connect the RCs to the chromosomes and then also to fully release them in anaphase. Altogether, our work demonstrates that dynamic remodeling of SUMO modifications facilitates key meiotic events and highlights how competition between conjugation and deconjugation activity can modulate SUMO homeostasis, protein complex stability, and ultimately, progressive processes such as cell division.

NOD is a plus end-directed motor that binds EB1 via a new microtubule tip localization sequence.

Chromosome congression, the process of positioning chromosomes in the midspindle, promotes the stable transmission of the genome to daughter cells during cell division. Congression is typically facilitated by DNA-associated, microtubule (MT) plus end-directed motors called chromokinesins. The Drosophila melanogaster chromokinesin NOD contributes to congression, but the means by which it does so are unknown in large part because NOD has been classified as a nonmotile, orphan kinesin. It has been postulated that NOD promotes congression, not by conventional plus end-directed motility, but by harnessing polymerization forces by end-tracking on growing MT plus ends via a mechanism that is also uncertain. Here, for the first time, it is demonstrated that NOD possesses MT plus end-directed motility. Furthermore, NOD directly binds EB1 through unconventional EB1-interaction motifs that are similar to a newly characterized MT tip localization sequence. We propose NOD produces congression forces by MT plus end-directed motility and tip-tracking on polymerizing MT plus ends via association with EB1.

Orientation and repositioning of chromosomes correlate with cell geometry-dependent gene expression.

Extracellular matrix signals from the microenvironment regulate gene expression patterns and cell behavior. Using a combination of experiments and geometric models, we demonstrate correlations between cell geometry, three-dimensional (3D) organization of chromosome territories, and gene expression. Fluorescence in situ hybridization experiments showed that micropatterned fibroblasts cultured on anisotropic versus isotropic substrates resulted in repositioning of specific chromosomes, which contained genes that were differentially regulated by cell geometries. Experiments combined with ellipsoid packing models revealed that the mechanosensitivity of chromosomes was correlated with their orientation in the nucleus. Transcription inhibition experiments suggested that the intermingling degree was more sensitive to global changes in transcription than to chromosome radial positioning and its orientations. These results suggested that cell geometry modulated 3D chromosome arrangement, and their neighborhoods correlated with gene expression patterns in a predictable manner. This is central to understanding geometric control of genetic programs involved in cellular homeostasis and the associated diseases.

An engineered minimal chromosomal passenger complex reveals a role for INCENP/Sli15 spindle association in chromosome biorientation.

The four-subunit chromosomal passenger complex (CPC), whose enzymatic subunit is Aurora B kinase, promotes chromosome biorientation by detaching incorrect kinetochore-microtubule attachments. In this study, we use a combination of truncations and artificial dimerization in budding yeast to define the minimal CPC elements essential for its biorientation function. We engineered a minimal CPC comprised of the dimerized last third of the kinase-activating Sli15/INCENP scaffold and the catalytic subunit Ipl1/Aurora B. Although native Sli15 is not oligomeric, artificial dimerization suppressed the biorientation defect and lethality associated with deletion of a majority of its microtubule-binding domain. Dimerization did not act through a physical clustering-based kinase activation mechanism but instead promoted spindle association, likely via a putative helical domain in Sli15 that is essential even when dimerized and is required to target kinetochore substrates. Based on the engineering and characterization of a minimal CPC, we suggest that spindle association is important for active Ipl1/Aurora B complexes to preferentially destabilize misattached kinetochores.

Interphase chromosome positioning in in vitro porcine cells and ex vivo porcine tissues.

BACKGROUND: In interphase nuclei of a wide range of species chromosomes are organised into their own specific locations termed territories. These chromosome territories are non-randomly positioned in nuclei which is believed to be related to a spatial aspect of regulatory control over gene expression. In this study we have adopted the pig as a model in which to study interphase chromosome positioning and follows on from other studies from our group of using pig cells and tissues to study interphase genome re-positioning during differentiation. The pig is an important model organism both economically and as a closely related species to study human disease models. This is why great efforts have been made to accomplish the full genome sequence in the last decade. RESULTS: This study has positioned most of the porcine chromosomes in in vitro cultured adult and embryonic fibroblasts, early passage stromal derived mesenchymal stem cells and lymphocytes. The study is further expanded to position four chromosomes in ex vivo tissue derived from pig kidney, lung and brain. CONCLUSIONS: It was concluded that porcine chromosomes are also non-randomly positioned within interphase nuclei with few major differences in chromosome position in interphase nuclei between different cell and tissue types. There were also no differences between preferred nuclear location of chromosomes in in vitro cultured cells as compared to cells in tissue sections. Using a number of analyses to ascertain by what criteria porcine chromosomes were positioned in interphase nuclei; we found a correlation with DNA content.

Modeling and experimental methods to probe the link between global transcription and spatial organization of chromosomes.

Genomes are spatially assembled into chromosome territories (CT) within the nucleus of living cells. Recent evidences have suggested associations between three-dimensional organization of CTs and the active gene clusters within neighboring CTs. These gene clusters are part of signaling networks sharing similar transcription factor or other downstream transcription machineries. Hence, presence of such gene clusters of active signaling networks in a cell type may regulate the spatial organization of chromosomes in the nucleus. However, given the probabilistic nature of chromosome positions and complex transcription factor networks (TFNs), quantitative methods to establish their correlation is lacking. In this paper, we use chromosome positions and gene expression profiles in interphase fibroblasts and describe methods to capture the correspondence between their spatial position and expression. In addition, numerical simulations designed to incorporate the interacting TFNs, reveal that the chromosome positions are also optimized for the activity of these networks. These methods were validated for specific chromosome pairs mapped in two distinct transcriptional states of T-Cells (naïve and activated). Taken together, our methods highlight the functional coupling between topology of chromosomes and their respective gene expression patterns.

Kif18A and chromokinesins confine centromere movements via microtubule growth suppression and spatial control of kinetochore tension.

Alignment of chromosomes at the metaphase plate is a signature of cell division in metazoan cells, yet the mechanisms controlling this process remain ambiguous. Here we use a combination of quantitative live-cell imaging and reconstituted dynamic microtubule assays to investigate the molecular control of mitotic centromere movements. We establish that Kif18A (kinesin-8) attenuates centromere movement by directly promoting microtubule pausing in a concentration-dependent manner. This activity provides the dominant mechanism for restricting centromere movement to the spindle midzone. Furthermore, polar ejection forces spatially confine chromosomes via position-dependent regulation of kinetochore tension and centromere switch rates. We demonstrate that polar ejection forces are antagonistically modulated by chromokinesins. These pushing forces depend on Kid (kinesin-10) activity and are antagonized by Kif4A (kinesin-4), which functions to directly suppress microtubule growth. These data support a model in which Kif18A and polar ejection forces synergistically promote centromere alignment via spatial control of kinetochore-microtubule dynamics.

A three-dimensional model of the yeast genome.

Layered on top of information conveyed by DNA sequence and chromatin are higher order structures that encompass portions of chromosomes, entire chromosomes, and even whole genomes. Interphase chromosomes are not positioned randomly within the nucleus, but instead adopt preferred conformations. Disparate DNA elements co-localize into functionally defined aggregates or 'factories' for transcription and DNA replication. In budding yeast, Drosophila and many other eukaryotes, chromosomes adopt a Rabl configuration, with arms extending from centromeres adjacent to the spindle pole body to telomeres that abut the nuclear envelope. Nonetheless, the topologies and spatial relationships of chromosomes remain poorly understood. Here we developed a method to globally capture intra- and inter-chromosomal interactions, and applied it to generate a map at kilobase resolution of the haploid genome of Saccharomyces cerevisiae. The map recapitulates known features of genome organization, thereby validating the method, and identifies new features. Extensive regional and higher order folding of individual chromosomes is observed. Chromosome XII exhibits a striking conformation that implicates the nucleolus as a formidable barrier to interaction between DNA sequences at either end. Inter-chromosomal contacts are anchored by centromeres and include interactions among transfer RNA genes, among origins of early DNA replication and among sites where chromosomal breakpoints occur. Finally, we constructed a three-dimensional model of the yeast genome. Our findings provide a glimpse of the interface between the form and function of a eukaryotic genome.

The kinesin-8 Kif18A dampens microtubule plus-end dynamics.

Motility is a fundamentally important property of most members of the kinesin superfamily, but a rare subset of kinesins are also able to alter microtubule dynamics. At kinetochore-microtubule plus ends, the kinesin-8 family member Kif18A is essential to align mitotic chromosomes at the spindle equator during cell division, but how it accomplishes this function is unclear. We report here that Kif18A is a plus-end-directed motor that inhibits the polymerization dynamics of microtubule plus ends without destabilizing them, distinguishing Kif18A from the budding yeast ortholog Kip3. In interphase cells, Kif18A uses this activity to reduce the overall dynamicity of microtubule plus ends and effectively constrains the distance over which plus ends grow and shrink. Our findings suggest that kinesin-8 family members have developed biochemically distinct activities throughout evolution and have implications for how Kif18A affects kinetochore-microtubule plus-end dynamics during mitosis in animal cells.

Both actin and myosin inhibitors affect spindle architecture in PtK1 cells: does an actomyosin system contribute to mitotic spindle forces by regulating attachment and movements of chromosomes in mammalian cells?

Immunocytochemical techniques are used to analyze the effects of both an actin and myosin inhibitor on spindle architecture in PtK(1) cells to understand why both these inhibitors slow or block chromosome motion and detach chromosomes. Cytochalasin J, an actin inhibitor and a myosin inhibitor, 2, 3 butanedione 2-monoxime, have similar effects on changes in spindle organization. Using primary antibodies and stains, changes are studied in microtubule (MT), actin, myosin, and chromatin localization. Treatment of mitotic cells with both inhibitors results in detachment or misalignment of chromosomes from the spindle and a prominent buckling of MTs within the spindle, particularly evident in kinetochore fibers. Evidence is presented to suggest that an actomyosin system may help to regulate the initial and continued attachment of chromosomes to the mammalian spindle and could also influence spindle checkpoint(s).

Histone H3 lysine 56 acetylation by Rtt109 is crucial for chromosome positioning.

Correct intranuclear organization of chromosomes is crucial for many genome functions, but the mechanisms that position chromatin are not well understood. We used a layered screen to identify Saccharomyces cerevisiae mutants defective in telomere localization to the nuclear periphery. We find that events in S phase are crucial for correct telomere localization. In particular, the histone chaperone Asf1 functions in telomere peripheral positioning. Asf1 stimulates acetylation of histone H3 lysine 56 (H3K56) by the histone acetyltransferase Rtt109. Analysis of rtt109Delta and H3K56 mutants suggests that the acetylation/deacetylation cycle of the H3K56 residue is required for proper telomere localization. The function of H3K56 acetylation in localizing chromosome domains is not confined to telomeres because deletion of RTT109 also prevents the correct peripheral localization of a newly identified S. cerevisiae "chromosome-organizing clamp" locus. Because chromosome positioning is subject to epigenetic inheritance, H3K56 acetylation may mediate correct chromosome localization by facilitating accurate transmission of chromatin status during DNA replication.

Positioning of human chromosomes in murine cell hybrids according to synteny.

Chromosomes occupy non-random spatial positions in interphase nuclei. It remains unclear what orchestrates this high level of organisation. To determine how the nuclear environment influences the spatial positioning of chromosomes, we utilised a panel of stable mouse hybrid cell lines carrying a single, intact human chromosome. Eleven of 22 human chromosomes revealed an alternative location in hybrid nuclei compared to that of human fibroblasts, with the majority becoming more internally localised. Human chromosomes in mouse nuclei position according to neither their gene density nor size, but rather the position of human chromosomes in hybrid nuclei appears to mimic that of syntenic mouse chromosomes. These results suggest that chromosomes adopt the behaviour of their host species chromosomes and that the nuclear environment is an important determinant of the interphase positioning of chromosomes.

Where shall we meet? A role for genome organisation and nuclear sub-compartments in mediating interchromosomal interactions.

A recent spate of examples of specific interactions between loci on separate chromosomes in mammalian nuclei has illuminated another layer of complexity in gene regulation. As the specifics of the cross-talk between interacting loci are worked out, it is also important to consider exactly how, when and where loci can ever reliably find each other within such an intricate environment. Answers may lie in how the genome is organised in relation to itself and to specialised nuclear sub-compartments. Here, we discuss how such specialised nuclear bodies may have the potential to specifically sequester loci and provide a context where interchromosomal communications can occur.

Interphase chromosome positioning affects the spectrum of radiation-induced chromosomal aberrations.

In interphase, chromosomes occupy defined nuclear volumes known as chromosome territories. To probe the biological consequences of the described nonrandom spatial positioning of chromosome territories in human lymphocytes, we performed an extensive FISH-based analysis of ionizing radiation-induced interchanges involving chromosomes 1, 4, 18 and 19. Since the probability of exchange formation depends strongly on the spatial distance between the damage sites in the genome, a preferential formation of exchanges between proximally positioned chromosomes is expected. Here we show that the spectrum of interchanges deviates significantly from one expected based on random chromosome positioning. Moreover, the observed exchange interactions between specific chromosome pairs as well as the interactions between homologous chromosomes are consistent with the proposed gene density-related radial distribution of chromosome territories. The differences between expected and observed exchange frequencies are more pronounced after exposure to densely ionizing neutrons than after exposure to sparsely ionizing X rays. These experiments demonstrate that the spatial positioning of interphase chromosomes affects the spectrum of chromosome rearrangements.

Postmeiotic sex chromatin in the male germline of mice.

In mammals, the X and Y chromosomes are subject to meiotic sex chromosome inactivation (MSCI) during prophase I in the male germline, but their status thereafter is currently unclear. An abundance of X-linked spermatogenesis genes has spawned the view that the X must be active . On the other hand, the idea that the imprinted paternal X of the early embryo may be preinactivated by MSCI suggests that silencing may persist longer . To clarify this issue, we establish a comprehensive X-expression profile during mouse spermatogenesis. Here, we discover that the X and Y occupy a novel compartment in the postmeiotic spermatid and adopt a non-Rabl configuration. We demonstrate that this postmeiotic sex chromatin (PMSC) persists throughout spermiogenesis into mature sperm and exhibits epigenetic similarity to the XY body. In the spermatid, 87% of X-linked genes remain suppressed postmeiotically, while autosomes are largely active. We conclude that chromosome-wide X silencing continues from meiosis to the end of spermiogenesis, and we discuss implications for proposed mechanisms of imprinted X-inactivation.

Long-range directional movement of an interphase chromosome site.

Increasing evidence suggests functional compartmentalization of interphase nuclei. This includes preferential interior localization of gene-rich and early replicating chromosome regions versus peripheral localization of gene-poor and late replicating chromosome regions , association of some active genes with nuclear speckles or transcription "factories", and association of transcriptionally repressed genes with heterochromatic regions. Dynamic changes in chromosome compartmentalization imply mechanisms for long-range interphase chromatin movements. However, live cell imaging in mammalian cells has revealed limited chromatin mobility, described as "constrained diffusion". None of these studies, though, have examined a chromosome locus undergoing an inducible repositioning between two different nuclear compartments. Here we demonstrate migration of an interphase chromosome site from the nuclear periphery to the interior 1-2 hr after targeting a transcriptional activator to this site. Spot redistribution is perturbed by specific actin or nuclear myosin I mutants. Extended periods of chromosome immobility are interspersed with several minute periods in which chromosomes move unidirectionally along curvilinear paths oriented roughly perpendicular to the nuclear envelope at velocities of 0.1-0.9 microm/min over distances of 1-5 microm. Our results suggest an active mechanism for fast and directed long-range interphase chromosome movements dependent directly or indirectly on actin/myosin.

APC and EB1 function together in mitosis to regulate spindle dynamics and chromosome alignment.

Recently, we have shown that a cancer causing truncation in adenomatous polyposis coli (APC) (APC(1-1450)) dominantly interferes with mitotic spindle function, suggesting APC regulates microtubule dynamics during mitosis. Here, we examine the possibility that APC mutants interfere with the function of EB1, a plus-end microtubule-binding protein that interacts with APC and is required for normal microtubule dynamics. We show that siRNA-mediated inhibition of APC, EB1, or APC and EB1 together give rise to similar defects in mitotic spindles and chromosome alignment without arresting cells in mitosis; in contrast inhibition of CLIP170 or LIS1 cause distinct spindle defects and mitotic arrest. We show that APC(1-1450) acts as a dominant negative by forming a hetero-oligomer with the full-length APC and preventing it from interacting with EB1, which is consistent with a functional relationship between APC and EB1. Live-imaging of mitotic cells expressing EB1-GFP demonstrates that APC(1-1450) compromises the dynamics of EB1-comets, increasing the frequency of EB1-GFP pausing. Together these data provide novel insight into how APC may regulate mitotic spindle function and how errors in chromosome segregation are tolerated in tumor cells.

Dynamic positioning of the fission yeast cell division plane.

A key question in cytokinesis is how the cell division plane is positioned. Whereas microtubules of the mitotic apparatus specify the division site in animal cells, we show here that the nucleus plays this role in the fission yeast Schizosaccharomyces pombe. By centrifuging cells to move the nucleus, we find that the nucleus (or a nuclear-associated structure) actively influences the position of contractile ring assembly during early mitosis. Displacement of the nucleus during this induction period can lead to formation of multiple rings. The nucleus signals its position in a microtubule-independent manner by emitting the protein mid1p. Furthermore, movement of ring fragments together minimizes formation of multiple division sites. These dynamic mechanisms of ring positioning provide a robust coordination of nuclear and cell division.