1,721,237 research outputs found
The renaissance of mitochondrial calcium transport
No full textAlthough the capacity of mitochondria for accumulating Ca2+ down the electrical gradient generated by the respiratory chain has been known for over three decades, the physiological significance of this phenomenon has been re-evaluated only recently. Indeed, it was long believed that the low affinity of the mitochondrial Ca2+ transporters would allow significant uptake only in conditions of cellular Ca2+ overload. Conversely, the direct measurement of [Ca2+] in the mitochondrial matrix revealed major [Ca2+] increases upon agonist stimulation. In this review, we will summarize: (a) the mechanisms that allow this large response, reconciling the biochemical properties of the transporters and the large amplitude of the mitochondrial [Ca2+] rises, and (b) the biological role of mitochondrial Ca2+ signalling, that encompasses the regulation of mitochondrial function and the modulation of the spatio-temporal pattern of cytosolic [Ca2+] increases
When calcium goes wrong: Genetic alterations of a ubiquitous signaling route
No full textIn all eukaryotic cells, the cytosolic concentration of calcium ions ([Ca2+]c) is tightly controlled by complex interactions among transporters, pumps, channels and binding proteins. Finely tuned changes in [Ca2+]c modulate a variety of intracellular functions, and disruption of Ca2+ handling leads to cell death. Here we review the human genetic diseases associated with perturbations in the Ca2+ signaling machinery. Despite the importance of Ca2+ in physiology and pathology, the number of known genetic diseases that can be attributed to defects in proteins directly involved in Ca2+ homeostasis is limited to few examples, which will be discussed. This paucity in contrast with the wide molecular repertoire may depend on the extreme severity of the phenotype (leading to death in utero) or, conversely, on functional compensation due to redundancy. In the latter case, it stands to reason that other genetic defects in calcium signaling have yet to be identified owing to their subtle phenotype
High tide of calcium in mitochondria
No full textMitochondria in intact cells can transiently accumulate calcium during cell stimulation. The heterogeneity of the response, the extremely high calcium concentrations reached in the mitochondrial matrix, and the ensuing modulation of secretion add further complexity to the spatiotemporal aspects of signalling through calcium ions
Microdomains of intracellular Ca2+: Molecular determinants and functional consequences
No full textCalcium ions are ubiquitous and versatile signaling molecules, capable of decoding a variety of extracellular stimuli (hormones, neurotransmitters, growth factors, etc.) into markedly different intracellular actions, ranging from contraction to secretion, from proliferation to cell death. The key to this pleiotropic role is the complex spatiotemporal organization of the [Ca(2+)] rise evoked by extracellular agonists, which allows selected effectors to be recruited and specific actions to be initiated. In this review, we discuss the structural and functional bases that generate the subcellular heterogeneity in cellular Ca(2+) levels at rest and under stimulation. This complex choreography requires the concerted action of many different players; the central role is, of course, that of the calcium ion, with the main supporting characters being all the entities responsible for moving Ca(2+) between different compartments, while the cellular architecture provides a determining framework within which all the players have their exits and their entrances. In particular, we concentrate on the molecular mechanisms that lead to the generation of cytoplasmic Ca(2+) microdomains, focusing on their different subcellular location, mechanism of generation, and functional role
Transfected aequorin in the measurement of cytosolic Ca2+ concentration ([Ca2+]c). A critical evaluation.
No full textTargeted 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
CD95 Stimulation and ceramide have a different impact on the morphology and physiology of mitochondria and endoplasmic reticulum.
No full textStimulation with C2 ceramide (1) or with antibodies to the death receptor CD95/Fas/APO-1 (2) induces apoptosis in a wide variety of cell types. Both stimuli cause cytochrome c release, caspase activation and DNA fragmentation. Less known are the effects of these apoptotic agents on organelles such as mitochondria and endoplasmic reticulum (ER). We studied the modifications occurring to mitochondria and ER in HeLa cells during the onset of apoptosis, showing that ceramide and CD95 stimulation have a different impact on morphology and physiology of mitochondria and ER. In C2 ceramide-stimulated cells, mitochondria were fragmented, spherical and smaller than in control cells; these changes occurred as early as 30 min from the stimulation and were not detected in anti-CD95 treated cells where the mitochondrial network did not apparently change its morphology even after several hours. This could be due to a direct damaging effect of ceramide on mitochondrial structures but also to the fact that ceramide itself induces a rapid Ca2+ release from the ER (3). In addition, ceramide induced a dramatic reduction in the mitochondrial Ca2+ response upon stimulation of the ER with histamine, while on the contrary, anti-CD95-stimulated HeLa cells showed a histamine-induced Ca2+ response similar to that of unstimulated control cells. The reduction in mitochondrial Ca2+ response in cells treated with C2 ceramide did not depend on a diminished capacity of mitochondria to take-up Ca2+ released by the ER but primarily on the fact that [Ca2+]ER was lower in ceramide-treated cells. Morphological changes occurring to the ER during ceramide treatment are under investigation. These findings suggest that Ca2+ is involved in the apoptotic pathway of C2 ceramide and show that different apoptotic stimuli have a different impact on the morphology and physiology of mitochondria and ER.
Bibliography:
1) Okazaki T, Kondo T, Kitano T, Tashima M. Diversity and complexity of ceramide signalling in apoptosis. Cell Signal 1998, 10:685-92.
2) Sharma K, Wang RX, Zhang LY, Yin DL, Luo XY, Solomon JC, Jiang RF, Markos K, Davidson W, Scott DW, Shi YF. Death the Fas way: regulation and pathophysiology of CD95 and its ligand. Pharmacol Ther 2000, 88:333-47.
3) Pinton P, Ferrari D, Rapizzi E, Di Virgilio F, Pozzan T, Rizzuto R. The Ca2+ concentration of the endoplasmic reticulum is a key determinant of ceramide-induced apoptosis: significance for the molecular mechanism of Bcl-2 action. EMBO 2001, 20:2690 2701
Identification of calreticulin isoforms in the central nervous system
No full textIn 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 compartment
The basics of mitochondrial cAMP signalling: Where, when, why
Full text linkCytosolic cAMP signalling in live cells has been extensively investigated in the past, while only in the last decade the existence of an intramitochondrial autonomous cAMP homeostatic system began to emerge. Thanks to the development of novel tools to investigate cAMP dynamics and cAMP/PKA-dependent phosphorylation within the matrix and in other mitochondrial compartments, it is now possible to address directly and in intact living cells a series of questions that until now could be addressed only by indirect approaches, in isolated organelles or through subcellular fractionation studies. In this contribution we discuss the mechanisms that regulate cAMP dynamics at the surface and inside mitochondria, and its crosstalk with organelle Ca2+ handling. We then address a series of still unsolved questions, such as the intramitochondrial localization of key elements of the cAMP signaling toolkit, e.g., adenylate cyclases, phosphodiesterases, protein kinase A (PKA) and Epac. Finally, we discuss the evidence for and against the existence of an intramitochondrial PKA pool and the functional role of cAMP increases within the organelle matrix
Cap formation by various ligands on lymphocytes shows the same dependence on high cellular ATP levels
No full text- …
