Satoru Uzawa

Condensin-driven remodelling of X chromosome topology during dosage compensation

The three-dimensional organization of a genome plays a critical role in regulating gene expression, yet little is known about the machinery and mechanisms that determine higher-order chromosome structure. Here we perform genome-wide chromosome conformation capture analysis, fluorescent in situ hybridization (FISH), and RNA-seq to obtain comprehensive three-dimensional (3D) maps of the Caenorhabditis elegans genome and to dissect X chromosome dosage compensation, which balances gene expression between XX hermaphrodites and XO males. The dosage compensation complex (DCC), a condensin complex, binds to both hermaphrodite X chromosomes via sequence-specific recruitment elements on X (rex sites) to reduce chromosome-wide gene expression by half. Most DCC condensin subunits also act in other condensin complexes to control the compaction and resolution of all mitotic and meiotic chromosomes. By comparing chromosome structure in wild-type and DCC-defective embryos, we show that the DCC remodels hermaphrodite X chromosomes into a sex-specific spatial conformation distinct from autosomes. Dosage-compensated X chromosomes consist of self-interacting domains (∼1 Mb) resembling mammalian topologically associating domains (TADs). TADs on X chromosomes have stronger boundaries and more regular spacing than on autosomes. Many TAD boundaries on X chromosomes coincide with the highest-affinity rex sites and become diminished or lost in DCC-defective mutants, thereby converting the topology of X to a conformation resembling autosomes. rex sites engage in DCC-dependent long-range interactions, with the most frequent interactions occurring between rex sites at DCC-dependent TAD boundaries. These results imply that the DCC reshapes the topology of X chromosomes by forming new TAD boundaries and reinforcing weak boundaries through interactions between its highest-affinity binding sites. As this model predicts, deletion of an endogenous rex site at a DCC-dependent TAD boundary using CRISPR/Cas9 greatly diminished the boundary. Thus, the DCC imposes a distinct higher-order structure onto X chromosomes while regulating gene expression chromosome-wide.

The fission yeast cut1+ gene regulates spindle pole body duplication and has homology to the budding yeast ESP1 gene

Mutations in the fission yeast cut1+, cut2+, and cut10+ genes uncouple normally coordinated mitotic events and deregulate, rather than arrest, mitosis. DNA synthesis continues, making polyploid nuclei with several spindles. Multiple, aberrant spindle pole bodies (SPBs) are produced in cut1 mutant cells. The cut1+ and cut2+ genes are cloned by transformation. High gene dosage of cut1+ also complements cut2 and cut10 mutants. The cut2+ gene, however, complements only cut2. The 210 kd cut1+ gene product contains putative ATP binding and helical coil regions followed by a COOH-terminal domain homologous to the S. cerevisiae gene ESP1. Mutations in the ESP1 gene also result in many SPBs. The cut1+ product is shown by anti-cut1 antibody to be a rare component of the insoluble nuclear fraction. It may play a key role in coupling chromosome disjunction with other cell cycle events and is potentially a component, regulator, or motor for the SPB and/or kinetochores.

I5S: Wide-Field Light Microscopy with 100-nm-Scale Resolution in Three Dimensions

A new type of wide-field fluorescence microscopy is described, which produces 100-nm-scale spatial resolution in all three dimensions, by using structured illumination in a microscope that has two opposing objective lenses. Illumination light is split by a grating and a beam splitter into six mutually coherent beams, three of which enter the specimen through each objective lens. The resulting illumination intensity pattern contains high spatial frequency components both axially and laterally. In addition, the emission is collected by both objective lenses coherently, and combined interferometrically on a single camera, resulting in a detection transfer function with axially extended support. These two effects combine to produce near-isotropic resolution. Experimental images of test samples and biological specimens confirm the theoretical predictions.

Fast live simultaneous multiwavelength four-dimensional optical microscopy.

Live fluorescence microscopy has the unique capability to probe dynamic processes, linking molecular components and their localization with function. A key goal of microscopy is to increase spatial and temporal resolution while simultaneously permitting identification of multiple specific components. We demonstrate a new microscope platform, OMX, that enables subsecond, multicolor four-dimensional data acquisition and also provides access to subdiffraction structured illumination imaging. Using this platform to image chromosome movement during a complete yeast cell cycle at one 3D image stack per second reveals an unexpected degree of photosensitivity of fluorophore-containing cells. To avoid perturbation of cell division, excitation levels had to be attenuated between 100 and 10,000× below the level normally used for imaging. We show that an image denoising algorithm that exploits redundancy in the image sequence over space and time allows recovery of biological information from the low light level noisy images while maintaining full cell viability with no fading.

Wsh3/Tea4 Is a Novel Cell-End Factor Essential for Bipolar Distribution of Tea1 and Protects Cell Polarity under Environmental Stress in S. pombe

BACKGROUND The fission yeast Schizosaccharomyces pombe has a cylindrical cell shape, for which growth is strictly limited to both ends, and serves as an excellent model system for genetic analysis of cell-polarity determination. Previous studies identified a cell-end marker protein, Tea1, that is transported by cytoplasmic microtubules to cell tips and recruits other cell-end factors, including the Dyrk-family Pom1 kinase. The deltatea1 mutant cells cannot grow in a bipolar fashion and show T-shaped morphology after heat shock. RESULTS We identified Wsh3/Tea4 as a novel protein that interacts with Win1 MAP kinase kinase kinase (MAPKKK) of the stress-activated MAP kinase cascade. Wsh3 forms a complex with Tea1 and is transported to cell tips by growing microtubules. The deltawsh3 mutant shows monopolar growth with abnormal Tea1 aggregate at the non-growing cell end; this abnormal aggregate fails to recruit Pom1 kinase. Consistent with the observed interaction between Win1 and Wsh3, cells lacking Wsh3 or Tea1 show more severe cell-polarity defects under osmolarity and heat-stress stimuli that are known to activate the stress MAPK cascade. Furthermore, mutants of the stress MAPK also exhibit cell-polarity defects when exposed to the same stress. CONCLUSIONS Wsh3/Tea4 is an essential component of the Tea1 cell-end complex. In addition to its role in bipolar growth during the normal cell cycle, the Wsh3-Tea1 complex, together with the stress-signaling MAPK cascade, contributes to cell-polarity maintenance under stress conditions.

Individual microtubule dynamics contribute to the function of mitotic and cytoplasmic arrays in fission yeast

Schizosaccharomyces pombe is an excellent organism for studying microtubule dynamics owing to the presence of well-defined microtubule arrays that undergo dramatic rearrangements during various stages of the cell cycle. Using sensitive time-lapse video microscopy and kymographic analysis, we have determined the polymerization/depolymerization kinetics of individual microtubules within these arrays throughout the fission yeast cell cycle. Interphase bundles are composed of 4-7 microtubules that act autonomously, demonstrating that individual microtubules are responsible for mediating the functions ascribed to these arrays. The nucleation and growth of cytoplasmic microtubules is inhibited upon cellular transition into mitosis, leading to their gradual disappearance. At the onset of mitosis, microtubules form on the nuclear face of the spindle pole body and exhibit dramatically increased dynamics. The presence of these intra-nuclear astral microtubules (INA) is reminiscent of spindle assembly and the search and chromosome capture mechanism observed in metazoan cells. Consistent with other in vivo studies, we do not observe microtubule flux in the anaphase B spindle. Finally, the depolymerization of individual microtubules alternates between each half-spindle, resulting in spindle collapse during telophase. On the basis of these observations, we conclude that microtubules in these diverse cytoskeletal arrays have autonomous behaviors that are an essential component of any model describing cell-cycle-dependent changes in the behavior and function of microtubule arrays.

Meiotic chromosome structures constrain and respond to designation of crossover sites

Crossover recombination events between homologous chromosomes are required to form chiasmata, temporary connections between homologues that ensure their proper segregation at meiosis I. Despite this requirement for crossovers and an excess of the double-strand DNA breaks that are the initiating events for meiotic recombination, most organisms make very few crossovers per chromosome pair. Moreover, crossovers tend to inhibit the formation of other crossovers nearby on the same chromosome pair, a poorly understood phenomenon known as crossover interference. Here we show that the synaptonemal complex, a meiosis-specific structure that assembles between aligned homologous chromosomes, both constrains and is altered by crossover recombination events. Using a cytological marker of crossover sites in Caenorhabditis elegans, we show that partial depletion of the synaptonemal complex central region proteins attenuates crossover interference, increasing crossovers and reducing the effective distance over which interference operates, indicating that synaptonemal complex proteins limit crossovers. Moreover, we show that crossovers are associated with a local 0.4–0.5-micrometre increase in chromosome axis length. We propose that meiotic crossover regulation operates as a self-limiting system in which meiotic chromosome structures establish an environment that promotes crossover formation, which in turn alters chromosome structure to inhibit other crossovers at additional sites.

Characterization of a Schizosaccharomyces pombeStrain Deleted for a Sequence Homologue of the Human Damaged DNA Binding 1 (DDB1) Gene

Human damaged DNA-binding protein (DDB) is a heterodimer of p48/DDB2 and p127/DDB1 subunits. Mutations in DDB2 are responsible for Xeroderma Pigmentosum group E, but no mutants of mammalian DDB1 have been described. To study DDB1, theSchizosaccharomyces pombe DDB1 sequence homologue (ddb1+ ) was cloned, and a ddb1deletion strain was constructed. The gene is not essential; however, mutant cells showed a 37% impairment in colony-forming ability, an elongated phenotype, and abnormal nuclei. The ddb1Δstrain was sensitive to UV irradiation, X-rays, methylmethane sulfonate, and thiabendazole, and these sensitivities were compared with those of the well characterized rad13Δ,rhp51Δ, and cds1Δ mutant strains. Ddb1p showed nuclear and nucleolar localization, and the aberrant nuclear structures observed in the ddb1Δ strain suggest a role for Ddb1p in chromosome segregation.