HVEM TOMOGRAPHY OF YEAST SPINDLE POLE BODIES &EARLY MITOTIC SPINDLES

University Of Colorado At Boulder, Boulder CO

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Abstract

We are using cryofixed material and dual tilt-axis HVEM tomography to study the fine structure of the spindle pole body (SPB) and early spindle formation in the yeast, Saccharomyces cerevisiae. Yeast are high pressure frozen, freeze substituted and embedded in Lowicryl HM20. 3-D reconstructions of yeast cells at the appropriate stage of the cell cycle are obtained using dual-axis HVEM tomography. Portions of eight cells at different stages of the cell cycle have been reconstructed: 1 from a cell in G1, 2 cells that show the SPB in early duplication, 3 cells that have duplicated but unseparated SPBs, and 2 cells that SPBs separated by a ~ 1.2um spindle. Preliminary data from these cells show that during SPB duplication, the mother SPB is significantly larger than the daughter and initiates more microtubules. In addition, many of the microtubules show an outward flare at their plus ends. The increased resolution afforded by HVEM tomography of well-fixed material should allow us to study the 3D structure of both the SPB and the structure of forming mitotic spindles that would not be possible using standard serial section reconstruction methods. These studies should provide insight into how kinetochore microtubules are organized as the mitotic spindle is forming and provide a benchmark for comparison between wild-type and mutant strains.