We are using high pressure freezing and freeze substitution fixation to preserve strains of Schizosaccharomyces pombe that are mutant in genes required for normal mitosis. The resulting EM specimens are being used to analyze the 3-D fine structure of the mitotic spindle and spindle pole bodies, using HVEM tomography. Specimens will also be prepared for immunocytochemistry to localize components of the spindle pole bodies and kinetochores. Two strains, csp3 and csp6, are under study. These were obtained from a centromere silencing screen (centromere specific suppressor of silencing), and both strains exhibit chromosome-loss phenotypes at the permissive temperature. We have previously shown that Csp6p is a hsp70 chaperone protein, and immunoflourescence experiments have documented a spindle defect characteristic of prometaphase arrest. Preliminary studies in the Boulder 3D lab using high voltage electron tomography of semi-thick sections of cps6 cells grown at the restrictive temperature showed that there is an additional spindle pole body defect in these cells: the spindle pole body cannot insert properly into the nuclear membrane and a bipolar spindle is unable to form. The Csp3 gene product is a cation transporter with putative transmembrane domains. This strain exhibits an interesting extreme microtubule bundling phenotype and displays a high frequency of lagging chromosomes in anaphase. We hope to use high voltage tomography to look at the fine structure of the kinetochores and spindle pole bodies in these mutant strains. ?
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