Volume electron microscopy analysis reveals a new type of membrane junction required for mixing of parental genomes after fertilization.

Imaging cells by light or fluorescence microscopy often provides great insights into mechanisms and dynamics of cellular processes. However, to gain information regarding the cellular structures that underlie these processes, nm-scale resolution is often required. This type of resolution can be obtain using electron microscopy (EM). Traditionally, investigators imaged slices of chemically fixed cells or tissue in order to glimpse at relevant structures in 2-dimensions. While powerful, this approach can potentially miss important information due to the somewhat random location of the planes analyzed, especially if a small number of images is a acquired. More recently, methodologies of volume EM have been developed that allow for sampling of a continuous volume at high resolution. Here we will describe how one such methodology, Focused Ion Beam- Scanning Electron Microscopy (FIB-SEM), has been used to understand the process by which parental genomes mix after fertilization in C. elegans embryos. One-cell embryos were collected during different stages of interphase and mitosis, fixed using high-pressure freezing followed by freeze substitution, and stained to maximize membrane contrast. Images were acquired at 9 nm lateral (x,y) and axial (z) resolution, for volumes of approximately 10 X 10 X 25 µm . These analyses revealed that for parental chromosomes to mix, the membranes of the two pronuclei must fused via a membrane structure that we termed "3-way sheet junction". The steps leading to the formation of 3-way sheet junctions, and the more recent discovery of a novel membrane structure around centrosomes using these datasets, will be discussed.