Electron tomography of yeast cells

Abstract

Electron microscopy (EM) is a useful method for the study of yeast cells and is complementary to a genetic analysis. EM provides high-resolution structure data about wild type cells and can document structural defects in mutant strains1,2. A number of EM techniques have been used to study the complex three-dimensional (3-D) arrangements of organelles in yeast. Examples include the use of freeze fracture replicas to reveal the surface topology of nuclear envelopes and cytoplasmic membrane systems3,4,5, as well as the use of serial sections to reconstruct entire mating factor-arrested cells6, to describe the 3-D geometry of the mitotic spindle7,8, and to document the 3-D distribution of nuclear pore complexes in wild type9 and mutant strains10. More recently, electron tomography has been used to describe the 3-D architecture of the yeast spindle pole body (SPB)11 and forming mitotic spindles12. Electron tomography has proven to be a useful method for obtaining 3-D structure data to describe complex cellular substructures13. Tomography is based on a series of tilted images, usually collected from a comparatively thick (0.2-1μm) section or from isolated organelles, to generate a 3-D reconstruction using back projection algorithms14. This method is like a ‘CAT’ scan of a cell, where the result is a computer-generated reconstruction that can be computationally sectioned and viewed in any orientation at 5-10 nm spatial resolution. In contrast to serial section reconstruction where multiple thin (50-70nm) sections have been used to reconstruct cellular objects that extend over many micrometers (e.g. an anaphase mitotic spindle15,16) tomography is best suited for structures whose dimensions change significantly, such that standard thin sections cannot reconstruct them accurately. Tomographic reconstruction permits the viewing of computer-generated ‘slices’ that are much thinner than could ever be cut physically with a microtome. This approach is therefore ideal for structures that have a complex 3-D geometry, such as arrays of cytoskeletal elements or convoluted membrane systems. In this chapter, we describe the steps necessary to prepare yeast cells for tomography and provide examples that illustrate the procedures for calculating tomographic reconstructions of organelles in situ and of whole cell profiles.