Overexpression of CALNUC (Nucleobindin) Increases Agonist and Thapsigargin Releasable Ca2+ Storage in the Golgi

We previously demonstrated that CALNUC, a Ca2+-binding protein with two EF-hands, is the major Ca2+-binding protein in the Golgi by 45Ca2+ overlay (Lin, P., H. Le-Niculescu, R. Hofmeister, J.M. McCaffery, M. Jin, H. Henneman, T. McQuistan, L. De Vries, and M. Farquhar. 1998. J. Cell Biol. 141:1515–1527). In this study we investigated CALNUC's properties and the Golgi Ca2+ storage pool in vivo. CALNUC was found to be a highly abundant Golgi protein (3.8 μg CALNUC/mg Golgi protein, 2.5 × 105 CALNUC molecules/NRK cell) and to have a single high affinity, low capacity Ca2+-binding site (Kd = 6.6 μM, binding capacity = 1.1 μmol Ca2+/μmol CALNUC). 45Ca2+ storage was increased by 2.5- and 3-fold, respectively, in HeLa cells transiently overexpressing CALNUC-GFP and in EcR-CHO cells stably overexpressing CALNUC. Deletion of the first EF-hand α helix from CALNUC completely abolished its Ca2+-binding capability. CALNUC was correctly targeted to the Golgi in transfected cells as it colocalized and cosedimented with the Golgi marker, α-mannosidase II (Man II). Approximately 70% of the 45Ca2+ taken up by HeLa and CHO cells overexpressing CALNUC was released by treatment with thapsigargin, a sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) (Ca2+ pump) blocker. Stimulation of transfected cells with the agonist ATP or IP3 alone (permeabilized cells) also resulted in a significant increase in Ca2+ release from Golgi stores. By immunofluorescence, the IP3 receptor type 1 (IP3R-1) was distributed over the endoplasmic reticulum and codistributed with CALNUC in the Golgi. These results provide direct evidence that CALNUC binds Ca2+ in vivo and together with SERCA and IP3R is involved in establishment of the agonist-mobilizable Golgi Ca2+ store

Dynamic Properties of an Inositol 1,4,5-Trisphosphate– and Thapsigargin-insensitive Calcium Pool in Mammalian Cell Lines

The functional characteristics of a nonacidic, inositol 1,4,5-trisphosphate– and thapsigargin-insensitive Ca2+ pool have been characterized in mammalian cells derived from the rat pituitary gland (GH3, GC, and GH3B6), the adrenal tissue (PC12), and mast cells (RBL-1). This Ca2+ pool is released into the cytoplasm by the Ca2+ ionophores ionomycin or A23187 after the discharge of the inositol 1,4,5-trisphosphate–sensitive store with an agonist coupled to phospholipase C activation and/or thapsigargin. The amount of Ca2+ trapped within this pool increased significantly after a prolonged elevation of intracellular Ca2+ concentration elicited by activation of Ca2+ influx. This pool was affected neither by caffeine-ryanodine nor by mitochondrial uncouplers. Probing mitochondrial Ca2+ with recombinant aequorin confirmed that this pool did not coincide with mitochondria, whereas its homogeneous distribution across the cytosol, as revealed by confocal microscopy, and its insensitivity to brefeldin A make localization within the Golgi complex unlikely. A proton gradient as the driving mechanism for Ca2+ uptake was excluded since ionomycin is inefficient in releasing Ca2+ from acidic pools and Ca2+ accumulation/release in/from this store was unaffected by monensin or NH4Cl, drugs known to collapse organelle acidic pH gradients. Ca2+ sequestration inside this pool, thus, may occur through a low-affinity, high-capacity Ca2+–ATPase system, which is, however, distinct from classical endosarcoplasmic reticulum Ca2+–ATPases. The cytological nature and functional role of this Ca2+ storage compartment are discussed

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