Visualizing mitochondrial division machinery in situ

Mitochondria are dynamic organelles that serve a variety of metabolic roles for eukaryotic cells, including ATP generation and cell signaling. Unlike other organelles, mitochondria cannot be produced “de novo”, and instead rely on a tightly regulated division process called “fission”, which functions as a quality control mechanism to maintain a healthy population of mitochondria. However, several cellular stress pathways promote hyperactivation of the mitochondrial fission pathway that fragment the mitochondrial network and induce cell death (apoptosis). Despite increasing evidence that mitochondrial fragmentation is a hallmark feature of many neurodegenerative diseases, the molecular mechanisms that contribute to the mitochondrial fission process remain poorly defined. We recently developed a three-dimensional imaging approach using a combination of cryo-focused ion beam milling and cryo-electron tomography to visualize snapshots of mitochondrial constriction events in mammalian cells. Our three-dimensional reconstructions represent the highest resolution structures of the ultrastructural interactions between mitochondria and other subcellular components to date, enabling unprecedented analysis of these associations. Further analyses reveal that the endoplasmic reticulum and the cytoskeleton preferentially associate with mitochondrial membrane constrictions. Interestingly, we also observe the presence of a previously unidentified filamentous structure in association with dividing mitochondria, which we propose is a member of the septin family of intermediate cytoskeletal filaments. By mapping out the precise interactions of these components relative to mitochondrial membranes, our work describes the complete ultrastructural architecture of the mitochondrial fission machinery required for membrane constriction, and establishes cellular tomography as a valuable approach for studying snapshots of mitochondrial dynamics in situ.