Abstract
Reminiscent of their evolutionary origin, mitochondria contain their own genome (mtDNA) compacted into the mitochondrial chromosome or nucleoid (mt-nucleoid). Many mitochondrial disorders are characterized by disruption of mt-nucleoids, either by direct mutation of genes involved in mtDNA organization or by interfering with other vital proteins for mitochondrial function. Thus, changes in mt-nucleoid morphology, distribution, and structure are a common feature in many human diseases and can be exploited as an indicator of cellular fitness. Electron microscopy provides the highest possible resolution that can be achieved, delivering spatial and structural information about all cellular structures. Recently, the ascorbate peroxidase APEX2 has been used to increase transmission electron microscopy (TEM) contrast by inducing diaminobenzidine (DAB) precipitation. DAB has the ability to accumulate osmium during classical EM sample preparation and, due to its high electron density, provides strong contrast for TEM. Among the nucleoid proteins, the mitochondrial helicase Twinkle fused with APEX2 has been successfully used to target mt-nucleoids, providing a tool to visualize these subcellular structures with high contrast and with the resolution of an electron microscope. In the presence of H2O2, APEX2 catalyzes the polymerization of DAB, generating a brown precipitate that can be visualized in specific regions of the mitochondrial matrix. Here, we provide a detailed protocol to generate murine cell lines expressing a transgenic variant of Twinkle, suitable to target and visualize mt-nucleoids. We also describe all the necessary steps to validate the cell lines prior to electron microscopy imaging and offer examples of anticipated results.