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T Pozzan

Publications and source records attributed to T Pozzan.

At least 73 records · Page 4Linked to original sources
PubMed1995

Overexpression of calreticulin increases the Ca2+ capacity of rapidly exchanging Ca2+ stores and reveals aspects of their lumenal microenvironment and function.

A molecularly tagged form of calreticulin (CR), a low affinity-high capacity Ca2+ binding protein that resides in the ER lumen, was transiently transfected into HeLa cells to specifically modify the Ca2+ buffering capacity of the intracellular Ca2+ stores. Fluorescence and confocal microscope immunocytochemistry revealed the tagged protein to be expressed by over 40% of the cells and to overlap in its distribution the endogenous CR yielding a delicate cytoplasmic network, i.e., the typical pattern of ER. In contrast, no signal was observed associated with the plasmalemma (marked by ConA) and within the nucleus. One- and two-dimensional Western blots revealed the transfected to exceed the endogenous CR of approximately 3.5-fold and to maintain its Ca2+ binding ability, whereas the expression of other ER proteins was unchanged. Ca2+ homeostasis in the transfected cells was investigated by three parallel approaches: (a) 45Ca equilibrium loading of cell populations; (b) [Ca2+]c measurement with fura-2 followed by quantitative immunocytochemistry of single cells and iii) [Ca2+]c measurement of cell population upon cotransfection with the Ca(2+)-sensitive photoprotein, aequorin. The three approaches revealed different aspects of Ca2+ homeostasis, yielding results which were largely complementary. In particular, the following conclusions were established: (a) both endogenous and transfected CR participate in Ca2+ buffering within the IP3-sensitive, rapidly exchanging, Ca2+ stores; the other pools of the cells were in contrast unaffected by CR transfection; (b) the Ca2+ capacity of the stores is not the main limiting factor of individual IP3-mediated Ca2+ release responses triggered by receptor agonists; (c) in control cells, the contribution of CR to Ca2+ buffering within the IP3-sensitive stores accounts for approximately 45% of the total, the rest being probably contributed by the other lumenal (and also membrane) Ca2+ binding proteins; (d) the free [Ca2+] within the lumen of the IP3-sensitive stores, revealed by the degree of Ca2+ binding to the transfected CR protein, amounts to values in (or approaching) the millimolar range; and (e) Ca2+ influx across the plasmalemma activated by depletion of the stores is directly dependent on the lumenal [Ca2+].

Amino Acid Sequence↗
PubMed1994

Modulation of Ca2+ influx dependent on store depletion by intracellular adenine-guanine nucleotide levels.

The effects of a number of metabolic inhibitors on the influx of Ca2+ activated by stimulation of receptors coupled to inositol 1,4,5-trisphosphate generation or by depletion of intracellular Ca2+ stores with thapsigargin were investigated in four different cell types: Ehrlich ascites tumor cells, Jurkat and HeLa cell lines, and rat hepatocytes. Independently of their chemical structure and site of inhibition, all of these metabolic poisons markedly inhibited Ca2+ influx without significantly affecting Ca2+ release. This inhibition was not due to membrane potential depolarization or to alteration in cytosolic pH but appeared correlated to a drop in the cellular concentration of ATP. The decreases in cellular [ATP] were paralleled by decreases in [GTP] and by increases in [ADP] and [GDP]. The reduction in ATP level necessary to drastically reduce Ca2+ influx was quite small, e.g. a 50% inhibition for a 5% reduction in [ATP], thus within the range of fluctuation presumably occurring under physiological conditions. We suggest that changes in the adenine or guanine nucleotide concentrations may represent an important modulatory mechanism of Ca2+ influx activated by store depletion.

Adenine Nucleotides↗
PubMed1994

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.

Aequorin↗
PubMed1994

Receptor-activated Ca2+ influx: how many mechanisms for how many channels?

Receptors that are coupled to the production of inositol (1,4,5)-trisphosphate cause an increase in cytosolic free Ca2+ concentration as a consequence of both Ca2+ mobilization from intracellular stores and Ca2+ influx through the plasma membrane. Although this latter phenomenon appears attributable to the activation of a number of Ca(2+)-permeable channels, the channels that are controlled by the Ca2+ content of the intracellular stores have recently received much attention. In this review, Cristina Fasolato, Barbara Innocenti and Tullio Pozzan summarize the characteristics of this Ca(2+)-influx pathway and discuss the hypotheses about its mechanism of activation and its relationship with other receptor-activated Ca2+ channels.

Animals↗
PubMed1994

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 defined cellular compartments was available. We have recently proposed the use of molecularly engineered Ca(2+)-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 InsP3 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.

Aequorin↗
PubMed1993

Microdomains with high Ca2+ close to IP3-sensitive channels that are sensed by neighboring mitochondria.

Microdomains of high intracellular calcium ion concentration, [Ca2+]i, have been hypothesized to occur in living cells exposed to stimuli that generate inositol 1,4,5-trisphosphate (IP3). Mitochondrially targeted recombinant aequorin was used to show that IP3-induced Ca2+ mobilization from intracellular stores caused increases of mitochondrial Ca2+ concentration, [Ca2+]m, the speed and amplitude of which are not accounted for by the relatively small increases in mean [Ca2+]i. A similar response was obtained by the addition of IP3 to permeabilized cells but not by perfusion of cells with Ca2+ at concentrations similar to those measured in intact cells. It is concluded that in vivo, domains of high [Ca2+]i are transiently generated close to IP3-gated channels and sensed by nearby mitochondria; this may provide an efficient mechanism for optimizing mitochondrial activity upon cell stimulation.

Aequorin↗
PubMed1993

Stimulated Ca2+ influx raises mitochondrial free Ca2+ to supramicromolar levels in a pancreatic beta-cell line. Possible role in glucose and agonist-induced insulin secretion.

The effects of stimulated Ca2+ influx on cytosolic ([Ca2+]c) or intramitochondrial free Ca2+ ([Ca2+]m) were examined in the new pancreatic beta-cell line, INS-1. [Ca2+]c was monitored by video imaging of single fura-2-loaded INS-1 cells, or in populations of cells transfected with non-targeted (cytosolic) aequorin. [Ca2+]m was measured after transfection with aequorin targeted to the mitochondria by fusion of the gene in frame with the signal peptide of cytochrome c oxidase subunit VIII. Two physiological stimuli of native beta-cells, glucose and ATP, raised [Ca2+]c in INS-1 cells largely by stimulating Ca2+ influx. Thus, glucose (20 mM) induced repetitive transient increases in [Ca2+]c (0.42 min-1, mean amplitude 229 nM above 102 nM basal). These transients were largely due to periodic stimulation of Ca2+ influx through voltage-sensitive Ca2+ channels, since they could be rapidly and reversibly blocked by chelation of external Ca2+, by addition of the hyperpolarizing agent diazoxide, or with the Ca2+ channel blocker SR 7037. ATP, by contrast, caused single transient [Ca2+]c increases, to about 300 nM above basal levels, which could be inhibited by > 90% upon external Ca2+ chelation. Challenge of aequorin-transfected cells with ATP increased [Ca2+]m to 4 microM or above, an effect blocked by EGTA. Furthermore, plasma membrane depolarization with high K+, used as a glucose surrogate to mimic, in a synchronized fashion, the influx-induced Ca2+ transients observed at the single-cell level, also increased [Ca2+]m to > 4 microM. Similar increases in [Ca2+]m were also measured in other aequorin-transfected insulin-secreting cells, RINm5F, during mobilization of internal Ca2+ with carbachol. In contrast, glucose-induced changes in [Ca2+]m were below the level of detection in INS-1 cell populations, consistent with the asynchrony of the [Ca2+]c transients induced by this nutrient at the single-cell level, and the consequent small average [Ca2+]c rise. These data are in line with the view that stimulated Ca2+ influx into excitable cells raises [Ca2+]m as efficiently as internal Ca2+ mobilization in nonexcitable cells. In the case of INS-1 and pancreatic beta-cells, this may be important both to enhance oxidative metabolism, hence fueling the secretory process, and also to maintain the production of metabolic signaling molecules.

Adenosine Triphosphate↗
PubMed1993

Nuclear Ca2+ concentration measured with specifically targeted recombinant aequorin.

Activation of nuclear transcription factors, breakdown of nuclear envelope and apoptosis represent a group of nuclear events thought to be modulated by changes in nucleoplasmic Ca2+ concentration, [Ca2+]n. Direct evidence for, or against, this possibility has been, however, difficult to obtain because measurements of [Ca2+]n are hampered by major technical problems. Here we describe a new approach for selectively monitoring Ca2+ concentrations inside the nucleus of living cells, which is based on the construction of a chimeric cDNA encoding a fusion protein composed of the photoprotein aequorin and a nuclear translocation signal derived from the rat glucocorticoid receptor. This modified aequorin (nuAEQ), stably expressed in HeLa cells, was largely confined to the nucleoplasm and thus utilized for monitoring [Ca2+]n in intact cells. No significant differences were observed between [Ca2+]n and cytosolic Ca2+ concentration ([Ca2+]i) under resting conditions. Upon stimulation of surface receptors linked to inositol-1,4,5-trisphosphate (InsP3) generation, and thus to intracellular Ca2+ signalling, the kinetics of [Ca2+]i and [Ca2+]n increases were indistinguishable. However, for the same rise in [Ca2+]i, the amplitude of [Ca2+]n increase was larger when evoked by Ca2+ mobilization from internal stores than when induced by Ca2+ influx across the plasma membrane. The functional significance of these transient nucleus-cytosol Ca2+ gradients is discussed.

Aequorin↗
PubMed1993

Intracellular targeting of the photoprotein aequorin: a new approach for measuring, in living cells, Ca2+ concentrations in defined cellular compartments.

We here present a novel method, based on the targeting of the photoprotein aequorin, for measuring the concentration of Ca2+ ions in defined cellular compartments of intact cells. In this contribution we will discuss the application to mitochondria. A chimaeric cDNA was constructed by fusing in frame the aequorin cDNA with that for a mitochondrial protein. The cDNA encoded a "mitochondrially-targeted" aequorin, composed of a typical mitochondrial targeting signal at the N-terminus and the photoprotein at the C-terminus. The cDNA, inserted in the expression vector pMT2, was co-transfected into bovine endothelial and HeLa cells together with the selectable plasmid pSV2-neo and stable transfectants, selected for high aequorin production, were analyzed. In subcellular fractionations, aequorin was shown to be localized in mitochondria; in intact cells, the first direct measurement of mitochondrial free Ca2+, [Ca2+]m, were obtained, which showed that [Ca2+]m is low at rest (< 0.5 microM), but rapidly increases to the micromolar range upon cell stimulation [1]. These data indicate that mitochondria "sense" very accurately the cytosolic Ca2+ concentration ([Ca2+]i), and after cell stimulation [Ca2+]m rises to values capable of activating the Ca(2+)-sensitive mitochondrial dehydrogenases.

Aequorin↗
PubMed1992

Identification of calreticulin isoforms in the central nervous system.

In the present paper we report the cloning and sequencing of the cDNA encoding two calreticulin isoforms from Xenopus laevis central nervous system. The two isoforms display 93% identity at the amino acid level. The predicted amino acid sequences of the amphibian calreticulins are very similar (76%) to those of mammalian liver and skeletal muscle. Xenopus laevis calreticulins are characterized by a very acidic c-terminal domain endowed with the endoplasmic-reticulum retention signal KDEL. The cDNAs of both clones encode an N-glycosylation consensus sequence. A third clone of calreticulin was also identified. The restriction map of this clone was clearly distinct from that of the two sequenced clones. These results indicate the existence of multiple calreticulin isoforms in the central nervous system and open questions about their functional role in different cells and/or subcellular compartments.

Amino Acid Sequence↗
PubMed1992

Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin.

Introduction of Ca2+ indicators (photoproteins, fluorescent dyes) that can be trapped in the cytosolic compartment of living cells has yielded major advances in our knowledge of Ca2+ homeostasis. Ca2+ however regulates functions not only in the cytosol but also within various organelles where indicators have not yet been specifically targeted. Here we present a novel procedure by which the free Ca2+ concentration of mitochondria, [Ca2+]m, can be monitored continuously at rest and during stimulation. The complementary DNA for the Ca2+ sensitive photoprotein aequorin was fused in frame with that encoding a mitochondrial presequence. The hybrid cDNA was transfected into bovine endothelial cells and stable clones were obtained expressing variable amounts of mitochondrially targeted apoaequorin. The functional photoprotein could be reconstituted in intact cells by incubation with purified coelenterazine and [Ca2+]m could thus be monitored in situ. This allowed the unprecedented direct demonstration that agonist-stimulated elevations of cytosolic free Ca2+, [Ca2+]i, (measured in parallel with Fura-2) evoke rapid and transient increases of [Ca2+]m, which can be prevented by pretreatment with a mitochondrial uncoupler. The possibility of targeting aequorin to cellular organelles not only offers a new and powerful method for studying aspects of Ca2+ homeostasis that up to now could not be directly approached, but might also be used in the future as a tool to report in situ a variety of apparently unrelated phenomena of wide biological interest.

Aequorin↗
PubMed1992

Receptor-activated Ca2+ influx. Two independently regulated mechanisms of influx stimulation coexist in neurosecretory PC12 cells.

Receptor-activated Ca2+ influx was investigated in PC12 cells clones loaded with fura-2. Cells were stimulated in a Ca(2+)-free medium and studied after reintroduction of the cation or addition of Mn2+ into the medium. A first influx component, independent of receptor activation and sustained by depletion of the intracellular inositol 1,4,5-trisphosphate sensitive Ca2+ store (store-dependent Ca2+ influx, SDCI), was identified by experiments with carbachol followed by atropine and with agents that induce store discharge without polyphosphoinositide hydrolysis: thapsigargin, an inhibitor of Ca(2+)-ATPase activity; ryanodine and caffeine, activators of the ryanodine receptor. A second component of Ca2+ influx, induced by carbachol and rapidly blocked by atropine, relies on receptor-effector coupling via G protein(s) different from that (those) involved in phospholipase C activation. SDCI and receptor-coupled influx are similar in their voltage dependence and insensitivity to forskolin and phorbol esters but they differ with respect to their Mn2+ permeability and their sensitivity to the SC 38249 imidazole blocker. The two components might play different roles. SDCI might act as a safety device to prevent Ca2+ store depletion whereas receptor-dependent influx might control physiological functions such as secretion and growth.

Animals↗
PubMed1992

The endoplasmic reticulum of Purkinje neuron body and dendrites: molecular identity and specializations for Ca2+ transport.

Immunofluorescence and immunogold labeling, together with sucrose gradient separation and Western blot analysis of microsomal subfractions, were employed in parallel to probe the endoplasmic reticulum in the cell body and dendrites of rat cerebellar Purkinje neurons. Two markers, previously investigated in non-nerve cells, the membrane protein p91 (calnexin) and the lumenal protein BiP, were found to be highly expressed and widely distributed to the various endoplasmic reticulum sections of Purkinje neurons, from the cell body to dendrites and dendritic spines. An antibody (denominated anti-rough-surfaced endoplasmic reticulum), which recognized two membrane proteins, p14 and p40, revealed a similar immunogold labeling pattern. However, centrifugation results consistent with a widespread distribution were obtained for p14 only, while p40 was concentrated in the rough microsome-enriched subfractions. The areas enriched in the inositol 1,4,5-triphosphate receptor and thus presumably specialized in Ca2+ transport (stacks of multiple smooth-surfaced cisternae; the dendritic spine apparatus) also exhibited labeling for BiP and p91, and were positive for the anti-rough-surfaced endoplasmic reticulum antibody (presumably via the p14 antigen). Additional antibodies, that yielded inadequate immunocytochemical signals, were employed only by Western blotting of the microsomal subfractions, while the ryanodine receptor was studied by specific binding. The latter receptor and the Ca2+ ATPase, known in other species to be concentrated in Purkinje neurons, exhibited bimodal distributions with a peak in the light and another in the heavy subfractions. A similar distribution was also observed with another lumenal protein, protein disulfide isomerase. Taken as a whole, the results that we have obtained suggest the existence in the endoplasmic reticulum of Purkinje neurons of two levels of organization; the first identified by widespread, probably general markers (BiP, p91, possibly p14 and others), the second by specialization markers, such as the inositol 1,4,5-triphosphate receptor and, possibly, p40, which appear restricted to areas where specific functions appear to be localized.

Animals↗