title: Single Molecule Tracking and Localization of Mitochondrial Protein Complexes in Live Cells.
abstract: Mitochondria are the power plant of most non-green eukaryotic cells. An understanding of their function and regulation is only possible with the knowledge of the spatiotemporal dynamics of their proteins. Mitochondrial membrane proteins involved in diverse functions like protein import, cell respiration, metabolite transport, and mitochondrial morphology are mobile within membranes. Here, we provide a protocol for a superresolution fluorescence microscopy technique named tracking and localization microscopy (TALM) that allows for localization and diffusion analysis of single mitochondrial membrane proteins in situ in cell cultures. This noninvasive imaging technique is a useful tool to reveal the spatiotemporal organization of proteins in diverse mitochondrial membrane compartments in living cells. Proteins of interest are tagged with the HaloTag®and specifically labeled with functionalized rhodamine dyes. The method profits from low abundance of proteins and therefore works better with substoichiometric labeling of HaloTag®-tagged proteins. In particular, the use of photostable bright rhodamine dyes enables the specific tagging and localization of single molecules with a calculated precision below 20 nm and the recording of single trajectories.
authors.affiliation: Mitochondrial Dynamics Group, School of Biology, University of Osnabrück, Osnabrück, Germany.
authors.forename: Timo
authors.initials: T
authors.lastname: Appelhans
authors.affiliation: Mitochondrial Dynamics Group, School of Biology, University of Osnabrück, Osnabrück, Germany. buschkar@uni-muenster.de.|Department of Biology, Institute of Molecular Cell Biology, Westphalian Wilhelms-University Münster, Schloßplatz 5, 48149, Münster, Germany. buschkar@uni-muenster.de.
authors.forename: Karin
authors.initials: K
authors.lastname: Busch
mesh_terms: D005456:Fluorescent Dyes; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008856:Microscopy, Fluorescence / Q000379:methods; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000378:metabolism*; D000072760:Single Molecule Imaging / Q000295:instrumentation / Q000379:methods*; D013223:Statistics as Topic
chemical_list: D005456:Fluorescent Dyes; D024101:Mitochondrial Proteins; D046912:Multiprotein Complexes
keywords: ATP synthase; Live cell imaging; Membrane protein dynamics; Mitochondria; Single molecule localization and tracking; Superresolution microscopy; Tom20