X-ray tomography of Schizosaccharomyces pombe

Abstract

The genetic tractability of the unicellular yeast Schizosaccharomyces pombe has resulted in it becoming an important model organism for the study of many eukaryotic cellular processes, in particular cell division. Over the past few years much progress has been made toward understanding the mechanisms that regulate eukaryotic cell division and the cellular changes that occur—for example, the formation of the cytokinetic contractile ring. However, a full understanding requires both identification of the proteins involved and correlation of this information with images showing the location of molecules in context of the cell architecture. Electron microscopic analyses have revealed exquisite ultrastructural images of cell structure, but this technique typically requires extensive processing—procedures that are labor intensive and time consuming. Imaging techniques that can more rapidly obtain better resolution than light microscopy are needed to advance the use of this model system for precise molecular localization analyses. In this manuscript, we examined S. pombe using soft X-ray tomography, an imaging technique that generates three-dimensional (3-D) images of intact hydrated cells at better than 50 nm isotropic resolution. This technique uses X-rays in the “water window,” where organic material absorbs approximately an order of magnitude more strongly than water, producing high-contrast images of cellular structures. As cells are examined in the absence of any chemical fixatives, stains, or contrast enhancement reagents, the images reflect cellular structures in the near-native state. We conducted preliminary soft X-ray imaging of S. pombe cells before and during cell division that revealed subcellular organelles, the actomyosin ring, and the septum of dividing cells. These images reveal tantalizing details of the cytokinesis process and are the first steps in our goal of generating a portfolio of tomographic images that map the location of labeled molecules into high-resolution 3-D reconstructions of the cell.