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New approaches to the study of mitochondrial Ca2+ dynamics

AutorFuente, Sergio de la
DirectorMontero, Mayte ; Álvarez, Javier; Fonteriz, Rosalba I.
Fecha de publicación2014
EditorCSIC-UVA - Instituto de Biología y Genética Molecular (IBGM)
ResumenSeveral cellular processes are controlled by intracellular Ca2+ signals, from development and differentiation to cell death. Mitochondria play a key role in the modulation of all of these Ca2+ signals. The most important methods available to monitor [Ca2+]M are the fluorescent dyes such as rhod-2 and specifically targeted proteins such as aequorin and pericam. However, significant discrepancies, both quantitative and qualitative, exist in the literature between the results obtained with these different methods. In this study we have made a systematic comparison among the dynamic Ca2+ measurements obtained using either the fluorescent dyes rhod-2 and rhod-FF, or the Ca2+-sensitive proteins aequorin and pericam. Moreover, we have tuned up new methods in order to measure more suitably the dynamics of the [Ca2+]M. We have used the very low Ca2+-affinity fluorescent dye rhod-5N and we have developed a new double-mutated aequorin form also with very low affinity for Ca2+. Thanks to this variety of methods, we have been able to study in detail the dynamics of [Ca2+]M in HeLa cells, both intact and permeabilized. These studies have been focused in the mitochondrial Ca2+ entry and release, the effect of phosphate in [Ca2+]M dynamics and the HeLa cells response to different agonists, both in cellular population, at the single cell level and at the subcellular level. Regarding to the comparative study between different [Ca2+]M measuring methods, our results show that the measurements obtained using aequorin or pericam are consistent each other in terms of [Ca2+] dynamics. However the fluorescent dyes were not able to respond correctly to the [Ca2+]M changes, particularly during repetitive stimulations. The reason for this behavior is not clear. The loading with these dyescauses mitochondrial morphology changes and mitochondrial membrane depolarization. These changes were small and reversible at low loading concentrations (1-2μM), but they produced large and prolonged damage at higher concentrations. Our results suggest that [Ca2+]M data obtained with these dyes should be taken with care.
DescripciónTesis Doctoral presentada por Sergio de la Fuente Pérez para optar al grado de Doctor por la Universidad de Valladolid, Facultad de Medicina (Departamento de Bioquímica y Biología y Fisiología); Instituto de Biología y Genética Molecular (IBGM).-- Sujeta a Licencia Creative Commons.
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