NUCLEAR TARGETING OF AEQUORIN - A NEW APPROACH FOR MEASURING NUCLEAR CA2+ CONCENTRATION IN INTACT-CELLS

We here describe the measurement of nuclear Ca2+ concentration ([Ca2+]n) with targeted recombinant aequorin. Two aequorin chimeras have been constructed, composed of the Ca(2+)-sensitive photoprotein and two different portions of the glucocorticoid hormone receptor (GR). The shorter chimera (nuAEQ), which contains the nuclear localization signal (NLS) NL1 of GR, but lacks its hormone binding domain, HBD, is constitutively localized in the nucleus; the longer one (nu/cytAEQ), which contains both NLSs (NL1 + NL2) and the HBS of GR, is normally localized in the cytosol, but is translocated to the nucleus upon treatment with the hormone. When localized to the nucleus, both chimeras give the same estimates of [Ca2+]n, both at rest and upon stimulation with the InsP3 generating agonist histamine. The [Ca2+]n values appear very close, both at rest and upon stimulation, to those of the cytoplasm, measured with cytosolic recombinant aequorin, suggesting that, at least in this cell model, the nuclear membrane does not represent a major barrier to the diffusion of Ca2+ ions, and that the nucleus does not regulate its [Ca2+] independently from the cytosol

Mitochondrial Ca2+ homeostasis in intact cells

Ca2+ is a key regulator not only of multiple cytosolic enzymes, but also of a variety of metabolic pathways occurring within the lumen of intracellular organelles. Until recently, no technique to selectively monitor the Ca2+ concentration within denned cellular compartments was available. We have recently proposed the use of molecularly engineered Ca2+-sensitive photoproteins to obtain such a result and demonstrated the application of this methodology to the study of mitochondrial and nuclear Ca2+ dynamics. We here describe in more detail the use of chimeric recombinant aequorin targeted to the mitochondria. The technique can be applied with equivalent results to different cell models, transiently or permanently transfected. In all the cell types we analyzed, mitochondrial Ca2+ concentration ([Ca2+]m) increases rapidly and transiently upon stimulation with agonists coupled to InsP3 generation. We confirm that the high speed of mitochondrial Ca2+ accumulation with this type of stimuli depends on the generation of local gradients of Ca2+ in the cytosol, close to the channels sensitive to InsP3. In fact, only activation of these channels, but not the simple release from internal stores, as that elicited by blocking the intracellular Ca2+ ATPases, results in a fast mitochondrial Ca2+ accumulation. We also provide evidence in favor of a microheterogeneity among mitochondria of the same cells, about 30% of them apparently sensing the microdomains of high cytosolic Ca2+ concentration ([Ca2+]c). The changes in [Ca2+]m appear sufficiently large to induce a rapid activation of mitochondrial dehydrogenases, which can be followed by monitoring the level of NAD(P)H fluorescence. A general scheme can thus be envisaged by which the triggering of a plasma membrane receptor coupled to InsPS generation raises the Ca2+ concentration both in the cytoplasm (thereby triggering energy-consuming processes, such as cell proliferation, motility, secretion, etc.) and in the mitochondria, where it activates the metabolic activity according to the increased cell needs

Regulation of mitochondrial ATP synthesis by calcium: evidence for a long-term metabolic priming.

In recent years, mitochondria have emerged as important targets of agonist-dependent increases in cytosolic Ca(2+) concentration. Here, we analyzed the significance of Ca(2+) signals for the modulation of organelle function by directly measuring mitochondrial and cytosolic ATP levels ([ATP](m) and [ATP](c), respectively) with specifically targeted chimeras of the ATP-dependent photoprotein luciferase. In both HeLa cells and primary cultures of skeletal myotubes, stimulation with agonists evoking cytosolic and mitochondrial Ca(2+) signals caused increases in [ATP](m) and [ATP](c) that depended on two parameters: (i) the amplitude of the Ca(2+) rise in the mitochondrial matrix, and (ii) the availability of mitochondrial substrates. Moreover, the Ca(2+) elevation induced a long-lasting priming that persisted long after agonist washout and caused a major increase in [ATP](m) upon addition of oxidative substrates. These results demonstrate a direct role of mitochondrial Ca(2+) in driving ATP production and unravel a form of cellular memory that allows a prolonged metabolic activation in stimulated cells

Transfected aequorin in the measurement of cytosolic Ca2+ concentration ([Ca2+]c). A critical evaluation.

Targeted recombinant aequorins represent to date the most specific means of monitoring [Ca2+] in subcellular organelles (Rizzuto, R., Simpson, A. W. M., Brini, M., and Pozzan, T. (1992) Nature 358, 325-328; Brini, M., Murgia, M., Pasti, L., Picard, D., Pozzan, T., and Rizzuto, R. (1993) EMBO J. 12, 4813-4819; Kendall, J. M., Dormer, R. L., and Campbell, A. K. (1992) Biochem. Biophys. Res. Commun. 189, 1008-1016). Up until now, however, only limited attention has been paid to the use of recombinant photoproteins for measuring, in mammalian cells, the [Ca2+] in the cytoplasm, a compartment for which effective Ca2+ probes are already available. Here we describe this approach in detail, highlighting the advantages, under various experimental conditions, of using recombinant cytosolic aequorin (cytAEQ) instead of classical fluorescent indicators. We demonstrate that cytAEQ is expressed recombinantly at high levels in transiently transfected cell lines and primary cultures as well as in stably transfected clones, and we describe a simple algorithm for converting aequorin luminescence data into [Ca2+] values. We show that although fluorescent indicators at the usual intracellular concentrations (50-100 microM) are associated with a significant buffering of the [Ca2+]c transients, this problem is negligible with recombinantly expressed aequorin. The large dynamic range of the photoprotein also allows an accurate estimate of the large [Ca2+]c increases that are observed in some cell types such as neurons. Finally, cytAEQ appears to be an invaluable tool for measuring [Ca2+]c in cotransfection experiments. In particular, we show that when cotransfected with an alpha 1-adrenergic receptor (coupled to inositol 1,4,5-trisphosphate generation), cytAEQ faithfully monitors the subpopulation of cells expressing the receptor, whereas the signal of fura-2, at the population level, is dominated largely by that of the untransfected cells

A novel muscle specific enhancer identified within the deletion overlap region of two XLDC patients lacking muscle exon 1 of the human dystrophin gene

Previous studies point to the involvement of several discrete transcriptional enhancers in the modulation of dystrophin gene expression in skeletal and cardiac muscle. Analysis of deletion breakpoints in two X-linked dilated cardiomyopathy patients with mutations that remove muscle exon 1 identified a 3.2-kb deletion overlap region (XLDC3.2) between -1199 and +2057 bp predicted to contain regulatory elements essential for dystrophin gene expression in cardiac muscle. A novel-sequence-based search strategy was used to identify a 543-bp region downstream of muscle exon 1 rich in cardiac-specific transcriptional elements. Designated dystrophin muscle enhancer 2 (DME2), this candidate enhancer was seen to function in a position- and orientation-independent manner in muscle cell lines but not in fibroblasts. As only modest activity was observed in primary neonatal rat cardiomyocytes, DME2 is thought to play a role in dystrophin gene regulation at later stages of cardiac muscle development