RESINLESS SECTION ELECTRON-MICROSCOPY REVEALS THE YEAST CYTOSKELETON

The cytoskeleton of Saccharomyces cerevisiae is essentially invisible using conventional microscopy techniques. A similar problem was solved for the mammalian cell cytoskeleton using resinless section electron microscopy, a technique applied here to yeast. In the resinless image, soluble proteins are no longer cloaked by embedding medium and must b e removed by selective detergent extraction. In yeast, this requires b reaching the cell wall by digesting with Zymolyase sufficiently to all ow detergent extraction of the plasma membrane lipids. Gel electropher ograms show that the extracted or ''soluble'' proteins are distinct fr om the retained or ''structural'' proteins that presumably comprise th e cytoskeleton. These putative cytoskeleton proteins include the major portions of a 43-kDa protein, which is presumably actin, and of prote ins in a band appearing at 55 kDa, as well as numerous less abundant, nonactin proteins. Resinless section electron micrographs show a dense , three-dimensional web of anastomosing, polymorphic filaments bounded by the remnant cell wall. Although the filament network is very heter ogenous, there appear to be two principal classes of filament diameter s-5 nm and 15-20 nm-which may correspond to actin and intermediate fil aments, respectively. A large oval region of lower filament density pr obably corresponds to the vacuole, and an electron dense spheroidal bo dy, 300-500 nm in diameter, is likely the nucleus. The techniques deta iled in this report afford new approaches to the study of yeast cytoar chitecture.