The spindle pole body (SPB) of the yeast, Saccharomyces cerevisiae, is a multilayered microtubule organizing center. Cytoplasmic microtubules extend from the outer plaque to the cortex of the cell. The central plaque is anchored in the nuclear envelope, and spindle microtubules emanate into the nucleus from the inner plaque. Spc110p is the highly-regulated connector between the spindle microtubules and the central plaque of the SPB. A mutational analysis of Spc110 has identified three regions with different functions. Temperature-sensitive mutations affecting the C-terminus of Spc110p interfere with the assembly Spc110p into the SPB, resulting in the formation of aberrant structures in the nucleoplasm. We are using high pressure freezing and freeze substitution fixation to preserve strains that are mutant in the SPC110 gene. The resulting EM specimens will be used to analyze the 3-D fine structure of the SPB and mitotic spindles in these strains. In addition, specimens will be prepared for immunocytochemistry to localize structural components of the SPB in mutant strains and in wild-type. Preliminary results, using a strain with a defect in the C-terminus of Spc110p, show the presence of one normal SPB embedded in the nuclear envelope and an aster containing capped MT ends in the nucleoplasm. We will use HVEM tomography to look in detail at the fine structure of these asters in mutant strains. C4
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