title: A new live-cell reporter strategy to simultaneously monitor mitochondrial biogenesis and morphology.
abstract: Changes in mitochondrial amount and shape are intimately linked to maintenance of cell homeostasis via adaptation of vital functions. Here, we developed a new live-cell reporter strategy to simultaneously monitor mitochondrial biogenesis and morphology. This was achieved by making a genetic reporter construct where a master regulator of mitochondrial biogenesis, nuclear respiratory factor 1 (NRF-1), controls expression of mitochondria targeted green fluorescent protein (mitoGFP). HeLa cells with the reporter construct demonstrated inducible expression of mitoGFP upon activation of AMP-dependent protein kinase (AMPK) with AICAR. We established stable reporter cells where the mitoGFP reporter activity corresponded with mitochondrial biogenesis both in magnitude and kinetics, as confirmed by biochemical markers and confocal microscopy. Quantitative 3D image analysis confirmed accordant increase in mitochondrial biomass, in addition to filament/network promoting and protecting effects on mitochondrial morphology, after treatment with AICAR. The level of mitoGFP reversed upon removal of AICAR, in parallel with decrease in mtDNA. In summary, we here present a new GFP-based genetic reporter strategy to study mitochondrial regulation and dynamics in living cells. This combinatorial reporter concept can readily be transferred to other cell models and contexts to address specific physiological mechanisms.
authors.affiliation: Department of Biomedicine, University of Bergen, Bergen, Norway.
authors.forename: Linn Iren
authors.initials: LI
authors.lastname: Hodneland Nilsson
authors.affiliation: Department of Biomedicine, University of Bergen, Bergen, Norway.
authors.forename: Ina Katrine
authors.initials: IK
authors.lastname: Nitschke Pettersen
authors.affiliation: Department of Biomedicine, University of Bergen, Bergen, Norway.
authors.forename: Julie
authors.initials: J
authors.lastname: Nikolaisen
authors.affiliation: BerGenBio AS, Bergen, Norway.
authors.forename: David
authors.initials: D
authors.lastname: Micklem
authors.affiliation: Molecular Imaging Center, Department of Biomedicine, University of Bergen, Bergen, Norway.
authors.forename: Hege
authors.initials: H
authors.lastname: Avsnes Dale
authors.affiliation: Department of Biomedicine, University of Bergen, Bergen, Norway.
authors.forename: Gro
authors.initials: G
authors.lastname: Vatne Røsland
authors.affiliation: Department of Biomedicine, University of Bergen, Bergen, Norway.
authors.forename: James
authors.initials: J
authors.lastname: Lorens
authors.affiliation: Department of Biomedicine, University of Bergen, Bergen, Norway.
authors.forename: Karl Johan
authors.initials: KJ
authors.lastname: Tronstad
mesh_terms: D000263:Adenylate Kinase / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008928:Mitochondria / Q000502:physiology* / Q000648:ultrastructure; D063154:Mitochondrial Dynamics; D051266:Nuclear Respiratory Factor 1 / Q000378:metabolism; D001678:Organelle Biogenesis; D059010:Single-Cell Analysis
chemical_list: D015415:Biomarkers; C495633:NRF1 protein, human; D051266:Nuclear Respiratory Factor 1; D049452:Green Fluorescent Proteins; D000263:Adenylate Kinase