High-Voltage Electron Tomography of Spindle Pole Bodies and Early Mitotic Spindles in the YeastSaccharomyces cerevisiae

Abstract

The spindle pole body (SPB) is the major microtubule-organizing center of budding yeast and is the functional equivalent of the centrosome in higher eukaryotic cells. We used fast-frozen, freeze-substituted cells in conjunction with high-voltage electron tomography to study the fine structure of the SPB and the events of early spindle formation. Individual structures were imaged at 5-10 nm resolution in three dimensions, significantly better than can be achieved by serial section electron microscopy. The SPB is organized in distinct but coupled layers, two of which show ordered two-dimensional packing. The SPB central plaque is anchored in the nuclear envelope with hook-like structures. The minus ends of nuclear microtubules (MTs) are capped and are tethered to the SPB inner plaque, whereas the majority of MT plus ends show a distinct flaring. Unbudded cells containing a single SPB retain 16 MTs, enough to attach to each of the expected 16 chromosomes. Their median length is ϳ150 nm. MTs growing from duplicated but not separated SPBs have a median length of ϳ130 nm and interdigitate over the bridge that connects the SPBs. As a bipolar spindle is formed, the median MT length increases to ϳ300 nm and then decreases to ϳ30 nm in late anaphase. Three-dimensional models confirm that there is no conventional metaphase and that anaphase A occurs. These studies complement and extend what is known about the three-dimensional structure of the yeast mitotic spindle and further our understanding of the organization of the SPB in intact cells. INTRODUCTIONMicrotubule-organizing centers (MTOCs) nucleate and anchor microtubules (MTs) during cell differentiation and division (for review, see Brinkley, 1985;Rose et al., 1993;Kellogg et al., 1994;Pereira and Schiebel, 1997). Although MTOCs vary widely in structure, their functions are largely conserved. In metazoa, the principal MTOC is the centrosome, which organizes both the interphase MTs and those of the mitotic spindle. In the yeast Saccharomyces cerevisiae, this role is served by the spindle pole body (SPB), a complex and dynamic organelle that undergoes significant structural changes during the yeast cell cycle (for review, see Winey and Byers, 1993;Kilmartin, 1994;Snyder, 1994). Nuclear MTs, which form the meiotic or mitotic spindles, attach to an inner plaque of the SPB, whereas cytoplasmic MTs, important for nuclear movements and position, are attached to an outer plaque (Moens and Rapport, 1971;Byers and Goetsch, 1974, 1975;Rabinow and Marak, 1966). Recent studies of isolated SPBs by cryomicroscopy and electron tomography have shown that SPBs are organized from six major layers, including a central crystalline core that contains the SPC42 gene product (Bullitt et al., 1997).Duplication of the SPB during the cell cycle begins with the formation of a "satellite" on the cytoplasmic face of the half-bridge, a specialized region of the nuclear envelope that lies immediately adjacent to the existing SPB. By processes not yet described in detail, the satellite develops into a...

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