SUBCELLULAR IMAGING OF INTRAMITOCHONDRIAL CA2- SIGNIFICANCE FOR THE REGULATION OF PYRUVATE-DEHYDROGENASE ACTIVITY( WITH RECOMBINANT TARGETED AEQUORIN )

Specific targeting of the recombinant, Ca2+-sensitive photoprotein, ae quorin to intracellular organelles has provided new insights into the mechanisms of intracellular Ca2+ homeostasis. When applied to small ma mmalian 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 des cribe the imaging in single mammalian cells (CHO.T) of [Ca2+] with rec ombinant chimeric aequorin targeted to mitochondria. This was achieved by optimizing expression of the protein through intranuclear injectio n of cDNA and through the use of a charge-coupled device camera fitted with a dual microchannel plate intensifier. This approach allows accu rate quantitation of the kinetics and extent of the large changes in m itochondrial matrix [Ca2+] ([Ca2+](m)) that follow receptor stimulatio n and reveal different behaviors of mitochondrial populations within i ndividual cells. The technique is compared with measurements of [Ca2+] (m) using the fluorescent indicator, rhod2. Comparison of [Ca2+](m) wi th the activity of the Ca2+-sensitive matrix enzyme, pyruvate dehydrog enase (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 w hich changes in the frequency of cytosolic (and hence mitochondrial) [ Ca2+] oscillations can be decoded by mitochondria.