Proper chromosome segregation in mitosis relies on correct kinetochore interaction with spindle microtubules. In early mitosis, each kinetochore usually interacts with the lateral side of each microtubule and is subsequently tethered at the microtubule end. However, since eukaryotic cells carry multiple chromosomes, multiple kinetochores could occasionally interact with a single microtubule.
View Article and Find Full Text PDFThe initial kinetochore (KT) encounter with a spindle microtubule (MT; KT capture) is one of the rate-limiting steps in establishing proper KT-MT interaction during mitosis. KT capture is facilitated by multiple factors, such as MT extension in various directions, KT diffusion, and MT pivoting. In addition, KTs generate short MTs, which subsequently interact with a spindle MT.
View Article and Find Full Text PDFBackground: Genomes of eukaryotes exist as chromatin, and it is known that different chromatin states can influence gene regulation. Chromatin is not a static structure, but is known to be dynamic and vary between cells. In order to monitor the organisation of chromatin in live cells we have engineered fluorescent fusion proteins which recognize specific operator sequences to tag pairs of syntenic gene loci.
View Article and Find Full Text PDFFor proper chromosome segregation, sister kinetochores must interact with microtubules from opposite spindle poles (bi-orientation). To establish bi-orientation, aberrant kinetochore-microtubule attachments are disrupted (error correction) by aurora B kinase (Ipl1 in budding yeast). Paradoxically, during this disruption, new attachments are still formed efficiently to enable fresh attempts at bi-orientation.
View Article and Find Full Text PDFHow kinetochores regulate microtubule dynamics to ensure proper kinetochore-microtubule interactions is unknown. Here, we studied this during early mitosis in Saccharomyces cerevisiae. When a microtubule shrinks and its plus end reaches a kinetochore bound to its lateral surface, the microtubule end attempts to tether the kinetochore.
View Article and Find Full Text PDFIn early mitosis, microtubules can be generated at kinetochores as well as at spindle poles. However, the role and regulation of kinetochore-derived microtubules have been unclear. In general, metaphase spindle microtubules are oriented such that their plus ends bind to kinetochores.
View Article and Find Full Text PDFKinetochore capture and transport by spindle microtubules plays a crucial role in high-fidelity chromosome segregation, although its detailed mechanism has remained elusive. It has been difficult to observe individual kinetochore-microtubule interactions because multiple kinetochores are captured by microtubules during a short period within a small space. We have developed a method to visualize individual kinetochore-microtubule interactions in Saccharomyces cerevisiae, by isolating one of the kinetochores from others through regulation of the activity of a centromere.
View Article and Find Full Text PDFIn the budding yeast Saccharomyces cerevisiae, microtubule-organizing centers called spindle pole bodies (SPBs) are embedded in the nuclear envelope, which remains intact throughout the cell cycle (closed mitosis). Kinetochores are tethered to SPBs by microtubules during most of the cell cycle, including G1 and M phases; however, it has been a topic of debate whether microtubule interaction is constantly maintained or transiently disrupted during chromosome duplication. Here, we show that centromeres are detached from microtubules for 1-2 min and displaced away from a spindle pole in early S phase.
View Article and Find Full Text PDFSegregation of sister chromatids to opposite spindle poles during anaphase is dependent on the prior capture of sister kinetochores by microtubules extending from opposite spindle poles (bi-orientation). If sister kinetochores attach to microtubules from the same pole (syntelic attachment), the kinetochore-spindle pole connections must be re-oriented to be converted to proper bi-orientation. This re-orientation is facilitated by Aurora B kinase (Ipl1 in budding yeast), which eliminates kinetochore-spindle pole connections that do not generate tension.
View Article and Find Full Text PDFIn mitosis, kinetochores are initially captured by the lateral sides of single microtubules and are subsequently transported toward spindle poles. Mechanisms for kinetochore transport are not yet known. We present two mechanisms involved in microtubule-dependent poleward kinetochore transport in Saccharomyces cerevisiae.
View Article and Find Full Text PDFFaithful DNA replication ensures genetic integrity in eukaryotic cells, but it is still obscure how replication is organized in space and time within the nucleus. Using timelapse microscopy, we have developed a new assay to analyze the dynamics of DNA replication both spatially and temporally in individual Saccharomyces cerevisiae cells. This allowed us to visualize replication factories, nuclear foci consisting of replication proteins where the bulk of DNA synthesis occurs.
View Article and Find Full Text PDFThe beta-1,3(4)-glucanase A (GluA)-encoding gene named gluA was cloned from the genomic library of a marine bacterium Pseudomonas sp. PE2 by expression in Escherichia coli, and the complete DNA sequence was determined. The recombinant enzyme from Pseudomonas sp.
View Article and Find Full Text PDFA novel antifungal protein (SAP) was found in the culture supernatant of a marine bacterium, Streptomyces sp. strain AP77, and was purified. This protein was characterized by chemical, biochemical, and biological analyses.
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