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R Rizzuto

Publications and source records attributed to R Rizzuto.

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

Gene transfer into satellite cell from regenerating muscle: bupivacaine allows beta-Gal transfection and expression in vitro and in vivo.

A large bulk of experimental evidence (15) suggests that myogenic cell transfer can be regarded as a promising therapeutic approach in the cure of inherited pathologies. In particular, it has been shown that primary myoblasts obtained from embryonic or neonatal muscles allows the recovery of the normal phenotype in defective muscle tissues. The utilization of this approach in clinical settings still bears heavy limitations. Apart from the legal and ethical difficulties, the use of muscles obtained from aborted fetus is challenged by a large risk of rejection, due to the incompatibility between donor and recipient. In this context based on the genetic alteration and reimplanting of the patient's own satellite cells, appears an approach attractive. Myoblasts derived from satellite cells are the obligate candidates for experiments, but the production of sufficient cell numbers is a major problem. Local anesthetics [Bupivacaine (1-n-butyl-DL-piperidine-2-carboxylic acid-2, 6-dimethyl anilide hydrochloride) and related molecules] had been used to induce myofiber damage (and thus satellite cells proliferation) and thereby may represent a tool for increasing the yield of myoblasts from adult muscles (1,9,17). We will show that satellite cells obtained from adult muscles after bupivacaine injection can be transfected in vitro and that the transfected gene is expressed in vitro and in vivo, after reimplantation of the modified myoblasts in recipient muscles.

Animals↗
PubMed1994

Cytosolic free calcium concentration in the mitogenic stimulation of T lymphocytes by anti-CD3 monoclonal antibodies.

The effects of anti-CD3 monoclonal antibodies on cytosolic free Ca2+ concentration, [Ca2+]i, were investigated in freshly isolated lymphocytes, T cell lines, T clones and the leukemic T cell line Jurkat with three different methodologies, i.e. classical cuvette experiments, cytofluorimetry and videoimaging. With any technique, concentrations of anti-CD3 antibodies optimal for stimulation of DNA synthesis were completely ineffective at inducing early increases of [Ca2+]i in freshly isolated lymphocytes. At supraoptimal mitogenic concentrations: (i) anti-CD3 mAb induced negligible increases of [Ca2+]i when tested in suspensions of freshly isolated lymphocytes, but the response increased progressively during in vitro culturing with IL2; (ii) most, but not all, T clones, when tested in suspension, were responsive to these concentrations of anti-CD3 antibodies in terms of [Ca2+]i; (iii) using the videoimaging technique at the single cell level, it was demonstrated that the anti-CD3 antibodies induced large increases of [Ca2+]i in lymphocytes only under conditions which allowed adherence of the antibodies (and of the cells) to the glass surface. In all T cell types investigated, the [Ca2+]i increases were most often composed by multiple, asynchronous oscillations. The buffering of [Ca2+]i increases, obtained by loading the cells with membrane permeant esters of Quin-2 and Fura-2, inhibited anti-CD3 mAb induced DNA synthesis, but this appeared entirely attributable to a toxic side effect of the ester hydrolysis. The relevance of these data is discussed in terms of their methodological and functional implications for the understanding of the role of Ca2+ in mitogenic stimulation of T cells.

Aminoquinolines↗
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

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

Structure of the promoter region of the gene encoding cytochrome c oxidase subunit V in Dictyostelium.

A cDNA for the nuclear-encoded subunit V of Dictyostelium discoideum cytochrome-c oxidase was used as a probe to screen a genomic library and isolate the complete gene. Primer-extension analysis revealed two transcription start sites located 32 and 39 nucleotides upstream of the translation initiation codon. The chloramphenicol acetyltransferase assay in transient and stable Dictyostelium transformants indicated that the 400-bp dT-rich segment 5' to the transcription start sites retained promoter activity. This region contains an octanucleotide sequence similar to the yeast HAP2/3/4 responsive element.

Animals↗
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

Differential expression of genes specifying two isoforms of subunit VIa of human cytochrome c oxidase.

Subunit VIa of mammalian cytochrome c oxidase (COX; EC 1.9.3.1) exists in two isoforms, one present ubiquitously ('liver' isoform; COX VIa-L) and the other present only in cardiac and skeletal muscle (COX VIa-M). We have now isolated a full-length cDNA specifying human COX VIa-M. The deduced mature COX VIa-M polypeptide is 62% identical to the human COX VIa-L isoform, but is approximately 80% identical to the bovine and rat COX VIa-M isoforms, suggesting that the two COX VIa isoform-encoding genes arose prior to the mammalian radiation. Transcriptional analysis showed a tissue-specific pattern: whereas COXVIa-L is transcribed ubiquitously, COXVIa-M is transcribed only in heart and skeletal muscle. The cDNA specifying COX VIa-M is a prime candidate for use in investigations of Mendelian-inherited COX deficiencies with primary involvement of muscle.

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

Mitochondrial DNA is not fragmented during apoptosis.

We have exposed mouse thymocytes and P-815 mastocytoma cells to four different conditions reported to cause apoptosis: 1) incubation in the absence of mitogenic factors; 2) incubation in the presence of dexamethasone; 3) stimulation with external ATP; 4) treatment with high concentrations of the K+ ionophore valinomycin. These treatments caused DNA fragmentation to a varying extent in the two cell types. High stringency hybridization with a cDNA probe specific to a mitochondrial DNA sequence revealed that during apoptosis induced by lack of mitogenic factors, dexamethasone, or extracellular ATP, mitochondrial DNA was not fragmented. On the contrary, valinomycin caused extensive degradation of mitochondrial DNA. These results support the notion that DNA fragmentation during apoptosis is a specific nuclear event and suggest that other agents, such as valinomycin, may act less selectively.

Adenosine Triphosphate↗
PubMed1991

The most conserved nuclear-encoded polypeptide of cytochrome c oxidase is the putative zinc-binding subunit: primary structure of subunit V from the slime mold Dictyostelium discoideum.

A full-length 515 base pairs cDNA for cytochrome c oxidase subunit V of D. discoideum was isolated from a lambda gt11 expression library. The encoded polypeptide, whose identity was confirmed by partial protein sequencing, is 119 amino acids long (Mr = 13,352) and does not contain a cleavable presequence. The protein, which is homologous to human subunit Vb and yeast subunit IV, exhibits the highest degree of sequence conservation found among nuclear-encoded subunits of cytochrome c oxidase from distantly related organisms. All the invariant residues are clustered in two regions of the C-terminus which include the putative amino acids involved in the coordination of the Zn ion tightly associated to eukaryotic oxidase.

Amino Acid Sequence↗
PubMed1991

Nucleotide sequence of a cDNA coding for the mitochondrial precursor protein of cytochrome c oxidase subunit IV from the slime mold Dictyostelium discoideum.

Subunit-specific polyclonal antibodies were used to isolate cDNA clones encoding subunit IV of Dictyostelium discoideum cytochrome c oxidase. DNA sequence analysis reveals an open reading frame of 149 amino acids. As shown by sequencing of the protein N-terminus, the subunit is synthesized with a 24 residue cleavable presequence which leads to a mature polypeptide of 14305 Da. The slime mold subunit exhibits a low but significant degree of similarity with subunit Va of human and subunit VI of yeast cytochrome c oxidase.

Amino Acid Sequence↗
PubMed1991

Characterization of a cDNA encoding subunit VI of cytochrome c oxidase from the slime mold Dictyostelium discoideum.

The primary structure of subunit VI of cytochrome c oxidase from the slime mold Dictyostelium discoideum has been determined by sequencing cDNA and N-terminus of the protein. The 92 amino acid residues long polypeptide (Mr = 10,535) shows homology with subunit IV of mammalian and subunit V of yeast cytochrome c oxidase. Though smaller and synthesized without a cleavable presequence, the slime mold oxidase subunit maintains the presence of a putative membrane spanning region.

Amino Acid Sequence↗