The laboratory continues to study the spindle pole body of the budding yeast, Saccharomyces cerevisiae. The spindle pole body is the microtubule-organizing center in this cell, organizing all of the microtubules of the mitotic and meiotic spindles, and is therefore the yeast equivalent of the centrosome found in vertebrate cells. We have been studying several genes implicated in the cell cycle duplication of the spindle pole body. Our analysis has always included electron microscopy to visualize the spindle pole body. In collaboration with Eileen O'Toole of the Boulder Laboratory for 3D Fine Structure, University of Colorado - Boulder (J. Richard McIntosh, P.I.), we have begun to use dual-axis electron tomography to create 3-dimensional tomograms of spindle pole bodies in situ. Similar to our pervious work, cells are frozen under high-pressure, freeze substituted in 3% glutaraldehyde and then low-temperature embedded in Lowicryl HM20. However, for electron tomography, 300-400 nM thick sections are imaged in a JEOL-JEM 1000 HVEM operating at 1MeV. Serial tilts were taken every 1.5o over a + 60o range about two perpendicular axes and tomograms computed. This method, which allows resampling of the same material in slices of 2.7 nM at any orientation, reveals tremendous detail of the spindle pole body. For instance, at least six separate SPB layers are seen in the tomographic slices. We have also observed connections between all spindle pole body layers and these probably help define rigorous order in the spindle pole body along the spindle axis. Furthermore, we have identified a new hook-like structure attached to the spindle pole body's central plaque potentially anchoring it the nuclear envelope. We have also examined the ends of microtubules and find that the minus ends at the spindle pole body are capped, whereas the majority of nuclear plus ends show a distinct flaring. Electron tomography has provided new structural insights into the spindle pole body and its behavior in during the budding yeast cell cycle. Electron tomography is clearly a powerful tool in cell biology and we believe that it will be widely used and that it will make major contributions to the study of cellular structures.

Recent Publications:

O'Toole, E. T., Winey, M. and McIntosh, J. R. (1999) High voltage electron tomography of spindle pole bodies and early mitotic spindles in the yeast Saccharomyces cerevisiae. Mol. Biol. Cell, 10:2017-2031.

Munoz-Centeno, M., McBratney, S., Monterrosa, A., Byers, B., Mann, C. and Winey M. (1999) Saccharomyces cerevisiae MPS2 encodes a membrane protein localized at the spindle pole body and the nuclear envelope. Mol. Biol. Cell 10:2393-2406.

Jones, M. H., Bachant, J. B., Castillo, A. R., Giddings Jr., T. H. and Winey, M. (1999) Yeast Dam1p is required to maintain spindle integrity during mitosis and interacts with the Mps1p kinase. Mol. Biol. Cell 10:2377-2391.

Winey, M. (1999) Cell cycle: Driving the centrosome cycle. Current Biol. 9:R449-R452.

Chial, H. J., Giddings Jr., T. H., Siewert, E. A., Hoyt, M. A. and Winey, M. (1999) Altered dosage of the S. cerevisiae spindle pole body duplication gene, NDC1, leads to aneuploidy and polyploidy. Proc. Natl. Acad. Sci. 96:10200-10205.

Chial, H. J. and Winey, M. (1999) Mechanisms of genetic instability revealed by analysis of yeast spindle pole body duplication. Biol. Cell 91:439-450.