High-voltage electron tomography of spindle pole bodies and early mitotic spindles in the yeast Saccharomyces cerevisiae

The spindle pole body (SPB) is the major microtubule-organizing center of b udding yeast and is the functional equivalent of the centrosome in higher e ukaryotic cells. We used fast-frozen, freeze-substituted cells in conjuncti on 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 organize d in distinct but coupled layers, two of which show ordered two-dimensional packing. The SPB central plaque is anchored in the nuclear envelope with h ook-like structures. The minus ends of nuclear microtubules (MTs) are cappe d 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 retai n 16 MTs, enough to attach to each of the expected 16 chromosomes. Their me dian length is similar to 150 nm. MTs growing from duplicated but not separ ated SPBs have a median length of similar to 130 nm and interdigitate over the bridge that connects the SPBs. As a bipolar spindle is formed, the medi an MT length increases to similar to 300 nm and then decreases to similar t o 30 nm in late anaphase. Three-dimensional models confirm that there is no conventional metaphase and that anaphase A occurs. These studies complemen t and extend what is known about the three-dimensional structure of the yea st mitotic spindle and further our understanding of the organization of the SPB in intact cells.