title: Subcellular imaging of intramitochondrial Ca2+ with recombinant targeted aequorin: significance for the regulation of pyruvate dehydrogenase activity.
abstract: Specific targeting of the recombinant, Ca2+ -sensitive photoprotein, aequorin to intracellular organelles has provided new insights into the mechanisms of intracellular Ca2+ homeostasis. When applied to small mammalian cells, a major limitation of this technique has been the need to average the signal over a large number of cells. This prevents the identification of inter- or intracellular heterogeneities. Here we describe the imaging in single mammalian cells (CHO.T) of [Ca2+] with recombinant chimeric aequorin targeted to mitochondria. This was achieved by optimizing expression of the protein through intranuclear injection of cDNA and through the use of a charge-coupled device camera fitted with a dual microchannel plate intensifier. This approach allows accurate quantitation of the kinetics and extent of the large changes in mitochondrial matrix [Ca2+] ([Ca2+](m)) that follow receptor stimulation and reveal different behaviors of mitochondrial populations within individual cells. The technique is compared with measurements of [Ca2+](m) using the fluorescent indicator, rhod2. Comparison of [Ca2+](m) with the activity of the Ca2+ -sensitive matrix enzyme, pyruvate dehydrogenase (PDH), reveals that this enzyme is a target of the matrix [Ca2+] changes. Peak [Ca2+](m) values following receptor stimulation are in excess of those necessary for full activation of PDH in situ, but may be necessary for the activation of other mitochondrial dehydrogenases. Finally, the data suggest that the complex regulation of PDH activity by a phosphorylation-dephosphorylation cycle may provide a means by which changes in the frequency of cytosolic (and hence mitochondrial) [Ca2+] oscillations can be decoded by mitochondria.
authors.affiliation: Department of Biochemistry, University of Bristol, School of Medical Sciences, Bristol, United Kingdom.
authors.forename: G A
authors.initials: GA
authors.lastname: Rutter
authors.forename: P
authors.initials: P
authors.lastname: Burnett
authors.forename: R
authors.initials: R
authors.lastname: Rizzuto
authors.forename: M
authors.initials: M
authors.lastname: Brini
authors.forename: M
authors.initials: M
authors.lastname: Murgia
authors.forename: T
authors.initials: T
authors.lastname: Pozzan
authors.forename: J M
authors.initials: JM
authors.lastname: Tavaré
authors.forename: R M
authors.initials: RM
authors.lastname: Denton
mesh_terms: D000331:Aequorin / Q000096:biosynthesis*; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D006388:Hemagglutinins / Q000096:biosynthesis; D006575:Heterocyclic Compounds, 3-Ring; D006706:Homeostasis; D007700:Kinetics; D008164:Luminescent Proteins / Q000096:biosynthesis; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism*; D015388:Organelles / Q000378:metabolism; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012680:Sensitivity and Specificity; D014162:Transfection
chemical_list: D005456:Fluorescent Dyes; D006388:Hemagglutinins; D006575:Heterocyclic Compounds, 3-Ring; D008164:Luminescent Proteins; D011768:Pyruvate Dehydrogenase Complex; D011993:Recombinant Fusion Proteins; C068483:rhod-2; D049452:Green Fluorescent Proteins; D000331:Aequorin; D002118:Calcium