Insights into Mitochondrial Dynamics from 3D Imaging of Stalled Fission Intermediates Using Cryo Scanning Transmission Electron Tomography (CSTET)

Mitochondria form a dynamic reticulum that undergoes fission and fusion¹. Organelles, e.g. the endoplasmic reticulum², the cytoskeleton³ and several proteins contribute to fission, including dynamin related protein 1 (Drp1) and mitochondrial fission factor (Mff)^4,5. DRP1 is a cytosolic protein that is recruited to the outer mitochondrial membrane (OMM) by Mff, an integral OMM protein ^4,5. Through GTP hydrolysis, the membrane-localized Drp1 constricts the mitochondrion, leading to fission ⁶. Mitochondrial fusion on the other hand is driven by dimerization of mitofusins (Mfn1 and Mfn2) ⁴. However, recent observations by the laboratory of David Chan from California Institute of Technology hint that mitofusions may also have a role in fission. Specifically, cells lacking Mff generate mitochondrial membrane constrictions under conditions that induce fission but, as expected, fail to complete fission, while cells lacking both Mff and the fusion factor Mfn1 appear not to form constrictions. At the resolution of fluorescent light microscopy, it is impossible to determine the details of the stalled fission intermediates, and therefore it is difficult to generate hypotheses regarding the role of mitofusins in fission. To solve this problem, we turned to CSTET, which is an ideal method to study organelle morphology deep inside the cell, as thicknesses up to 1 µm can be imaged. Tomographic reconstructions of mitochondria lacking Mff or both Mff and Mfn1 reveal in 3D the geometry of unproductive fission sites and the participation of surrounding organelles and cytoskeleton.