title: Probing of protein localization and shuttling in mitochondrial microcompartments by FLIM with sub-diffraction resolution.
abstract: The cell is metabolically highly compartmentalized. Especially, mitochondria host many vital reactions in their different microcompartments. However, due to their small size, these microcompartments are not accessible by conventional microscopy. Here, we demonstrate that time-correlated single-photon counting (TCSPC) fluorescence lifetime-imaging microscopy (FLIM) classifies not only mitochondria, but different microcompartments inside mitochondria. Sensor proteins in the matrix had a different lifetime than probes at membrane proteins. Localization in the outer and inner mitochondrial membrane could be distinguished by significant differences in the lifetime. The method was sensitive enough to monitor shifts in protein location within mitochondrial microcompartments. Macromolecular crowding induced by changes in the protein content significantly affected the lifetime, while oxidizing conditions or physiological pH changes had only marginal effects. We suggest that FLIM is a versatile and completive method to monitor spatiotemporal events in mitochondria. The sensitivity in the time domain allows for gaining substantial information about sub-mitochondrial localization overcoming diffraction limitation. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.
authors.affiliation: Mitochondrial Dynamics, School of Biology, University of Osnabrück, D-49076 Osnabrück, Germany; Institute of Molecular Cell Biology, Department of Biology, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany.
authors.forename: Anna-Carina
authors.initials: AC
authors.lastname: Söhnel
authors.affiliation: Mitochondrial Dynamics, School of Biology, University of Osnabrück, D-49076 Osnabrück, Germany.
authors.forename: Wladislaw
authors.initials: W
authors.lastname: Kohl
authors.affiliation: Physics Department III, University of Göttingen, D-37077 Göttingen, Germany.
authors.forename: Ingo
authors.initials: I
authors.lastname: Gregor
authors.affiliation: Physics Department III, University of Göttingen, D-37077 Göttingen, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Enderlein
authors.affiliation: Mitochondrial Dynamics, School of Biology, University of Osnabrück, D-49076 Osnabrück, Germany; Institute of Molecular Cell Biology, Department of Biology, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany.
authors.forename: Bettina
authors.initials: B
authors.lastname: Rieger
authors.affiliation: Mitochondrial Dynamics, School of Biology, University of Osnabrück, D-49076 Osnabrück, Germany; Institute of Molecular Cell Biology, Department of Biology, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany. Electronic address: buschkar@uni-muenster.de.
authors.forename: Karin B
authors.initials: KB
authors.lastname: Busch
mesh_terms: D054758:A Kinase Anchor Proteins / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D014450:Electron Transport Complex III / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D005990:Glycerol / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D061848:Optical Imaging / Q000295:instrumentation / Q000379:methods*; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D054758:A Kinase Anchor Proteins; C105137:AKAP1 protein, human; D001426:Bacterial Proteins; D008164:Luminescent Proteins; D026901:Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C084008:TOMM20 protein, human; C054562:yellow fluorescent protein, Bacteria; D006861:Hydrogen Peroxide; D014450:Electron Transport Complex III; D005990:Glycerol
keywords: Fluorescence lifetime imaging microscopy; Live cell imaging; Mitochondrial compartments; Nano-environment sensing; Protein localization; Sub-diffraction localization analysis