Marisa Brini

Transient and Long-Lasting Openings of the Mitochondrial Permeability Transition Pore Can Be Monitored Directly in Intact Cells by Changes in Mitochondrial Calcein Fluorescence

The occurrence and the mode of opening of the mitochondrial permeability transition pore (MTP) were investigated directly in intact cells by monitoring the fluorescence of mitochondrial entrapped calcein. When MH1C1 cells and hepatocytes were loaded with calcein AM, calcein was also present within mitochondria, because (i) its mitochondrial signal was quenched by the addition of tetramethylrhodamine methyl ester and (ii) calcein-loaded mitochondria could be visualized after digitonin permeabilization. Under the latter condition, the addition of Ca2+ induced a prompt and massive release of the accumulated calcein, which was prevented by CsA, indicating that calcein release could, in principle, probe MTP opening in intact cells as well. To study this process, we developed a procedure by which the cytosolic calcein signal was quenched by Co2+. In hepatocytes and MH1C1 cells coloaded with Co2+ and calcein AM, treatment with MTP inducers caused a rapid, though limited, decrease in mitochondrial calcein fluorescence, which was significantly reduced by CsA. We also observed a constant and spontaneous decrease in mitochondrial calcein fluorescence, which was completely prevented by CsA. Thus MTP likely fluctuates rapidly between open and closed states in intact cells.

Generation, control, and processing of cellular calcium signals.

Referee: Guiseppe Inesi, M.D., Ph.D., Professor and Chairman, School of Medicine, Dept. of Biochemistry and Molecular Biology, Univeristy of Maryland, Baltimore.

Calcium Pumps in Health and Disease

Ca2+-ATPases (pumps) are key actors in the regulation of Ca2+ in eukaryotic cells and are thus essential to the correct functioning of the cell machinery. They have high affinity for Ca2+ and can efficiently regulate it down to very low concentration levels. Two of the pumps have been known for decades (the SERCA and PMCA pumps); one (the SPCA pump) has only become known recently. Each pump is the product of a multigene family, the number of isoforms being further increased by alternative splicing of the primary transcripts. The three pumps share the basic features of the catalytic mechanism but differ in a number of properties related to tissue distribution, regulation, and role in the cellular homeostasis of Ca2+. The molecular understanding of the function of the pumps has received great impetus from the solution of the three-dimensional structure of one of them, the SERCA pump. These spectacular advances in the structure and molecular mechanism of the pumps have been accompanied by the emergence and rapid expansion of the topic of pump malfunction, which has paralleled the rapid expansion of knowledge in the topic of Ca2+-signaling dysfunction. Most of the pump defects described so far are genetic: when they are very severe, they produce gross and global disturbances of Ca2+ homeostasis that are incompatible with cell life. However, pump defects may also be of a type that produce subtler, often tissue-specific disturbances that affect individual components of the Ca2+-controlling and/or processing machinery. They do not bring cells to immediate death but seriously compromise their normal functioning.

Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells

BACKGROUND It has recently been demonstrated that the green fluorescent protein (GFP) of the jellyfish Aequorea victoria retains its fluorescent properties when recombinantly expressed in both prokaryotic (Escherichia coli) and eukaryotic (Caenorhabditis elegans and Drosophila melanogaster) living cells; it can therefore be used as a powerful marker of gene expression in vivo. The specific targeting of recombinant GFP within cells would allow it to be used for even more applications, but no information is yet available on the possibility of targeting GFP to intracellular organelles. RESULTS In this study, we show that the GFP cDNA can be expressed at high levels in cultured mammalian cells; the recombinant polypeptide is highly fluorescent and is exclusively localized in the cytosol. Furthermore, we have modified the GFP cDNA to include a mitochondrial targeting sequence (and a strong immunological epitope at the amino terminus of the encoded polypeptide). When transiently transfected into mammalian cells, this construct drives the expression of a strongly fluorescent GFP chimera which selectively localizes to the mitochondria. We also describe two of the many possible applications of this recombinant GFP in physiological studies. The targeted chimera allows the visualization of mitochondrial movement in living cells. Also, unlike dyes such as rhodamine, it reveals morphological changes induced in mitochondria by drugs that collapse the organelle membrane potential. Moreover, when GFP is cotransfected with a membrane receptor, such as the alpha 1-adrenergic receptor, the fluorescence of the GFP in intact cells can be used in recognizing the transfected cells. Thus, specific changes in intracellular Ca2+ concentration that occur in cells expressing the recombinant receptor can be identified using a classical fluorescent Ca2+ indicator. CONCLUSION GFP is an invaluable new tool for studies of molecular biology and cell physiology. As a marker of transfection in vivo, it provides a simple means of identifying genetically modified cells to be used in physiological studies. More importantly, chimeric GFP, which in principle can be targeted to any subcellular location, can be used to monitor complex phenomena in intact living cells, such as changes in shape and distribution of organelles, and it has the potential to be used as a probe of physiological parameters.

Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells

Direct monitoring of the free Ca2+ concentration in the lumen of the endoplasmic reticulum (ER) is an important but still unsolved experimental problem. We have shown that a Ca(2+)‐sensitive photoprotein, aequorin, can be addressed to defined subcellular compartments by adding the appropriate targeting sequences. By engineering a new aequorin chimera with reduced Ca2+ affinity, retained in the ER lumen via interaction of its N‐terminus with the endogenous resident protein BiP, we show here that, after emptying the ER, Ca2+ is rapidly re‐accumulated up to concentrations of > 100 microM, thus consuming most of the reporter photoprotein. An estimate of the steady‐state Ca2+ concentration was obtained using Sr2+, a well‐known Ca2+ surrogate which elicits a significantly slower rate of aequorin consumption. Under conditions in which the rate and extent of Sr2+ accumulation in the ER closely mimick those of Ca2+, the steady‐state mean lumenal Sr2+ concentration ([Sr2+]er) was approximately 2 mM. Receptor stimulation causes, in a few seconds, a 3‐fold decrease of the [Sr2+]er, whereas specific inhibition of the ER Ca2+ ATPase leads to an approximately 10‐fold drop in a few minutes.

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.

Mitochondria, calcium and cell death: A deadly triad in neurodegeneration

Mitochondrial Ca(2+) accumulation is a tightly controlled process, in turn regulating functions as diverse as aerobic metabolism and induction of cell death. The link between Ca(2+) (dys)regulation, mitochondria and cellular derangement is particularly evident in neurodegenerative disorders, in which genetic models and environmental factors allowed to identify common traits in the pathogenic routes. We will here summarize: i) the current view of mechanisms and functions of mitochondrial Ca(2+) homeostasis, ii) the basic principles of organelle Ca(2+) transport, iii) the role of Ca(2+) in neuronal cell death, and iv) the new information on the pathogenesis of Alzheimers, Huntingtons and Parkinsons diseases, highlighting the role of Ca(2+) and mitochondria.

Calcium pumps: structural basis for and mechanism of calcium transmembrane transport.

Eukaryotic cells remove calcium from the cytosol using P-type pumps in the plasma membrane and in the sarco(endo)plasmic reticulum. These pumps share membrane topography and general mechanism of action, but differ in regulatory properties. Recent advances in the field include the three-dimensional structure of the sarco(endo)plasmic reticulum and further understanding of the transcriptional regulation of the plasma membrane P-type pump by calcium.

A calcium signaling defect in the pathogenesis of a mitochondrial DNA inherited oxidative phosphorylation deficiency

In recent years, genetic defects of the mitochondrial genome (mtDNA) were shown to be associated with a heterogeneous group of disorders, known as mitochondrial diseases, but the cellular events deriving from the molecular lesions and the mechanistic basis of the specificity of the syndromes are still incompletely understood. Mitochondrial calcium (Ca2+) homeostasis depends on close contacts with the endoplasmic reticulum and is essential in modulating organelle function. Given the strong dependence of mitochondrial Ca2+ uptake on the membrane potential and the intracellular distribution of the organelle, both of which may be altered in mitochondrial diseases, we investigated the occurrence of defects in mitochondrial Ca2+ handling in living cells with either the tRNALys mutation of MERRF (myoclonic epilepsy with ragged-red fibers) or the ATPase mutation of NARP (neurogenic muscle weakness, ataxia and retinitis pigmentosa). There was a derangement of mitochondrial Ca2+ homeostasis in MERRF, but not in NARP cells, whereas cytosolic Ca2+ responses were normal in both cell types. Treatment of MERRF cells with drugs affecting organellar Ca2+ transport mostly restored both the agonist-dependent mitochondrial Ca2+ uptake and the ensuing stimulation of ATP production. These results emphasize the differences in the cellular pathogenesis of the various mtDNA defects and indicate specific pharmacological approaches to the treatment of some mitochondrial diseases.

Calcium signalling: a historical account, recent developments and future perspectives

Abstract. Ca2+ is a uniquely important messenger that penetrates into cells through gated channels to transmit signals to a large number of enzymes. The evolutionary choice of Ca2+ was dictated by its unusual chemical properties, which permit its reversible complexation by specific proteins in the presence of much larger amounts of other potentially competing cations. The decoding of the Ca2+ signal consists in two conformational changes of the complexing proteins, of which calmodulin is the most important. The first occurs when Ca2+ is bound, the second (a collapse of the elongated protein) when interaction with the targeted enzymes occurs. Soluble proteins such as calmodulin contribute to the buffering of cell Ca2+, but membrane intrinsic transporting proteins are more important. Ca2+ is transported across the plasma membrane (channel, a pump, a Na+/Ca2+ exchanger) and across the membrane of the organelles. The endoplasmic reticulum is the most dynamic store: it accumulates Ca2+ by a pump, and releases it via channels gated by either inositol 1,4,5-trisphosphate (IP3) and cyclic adenosine diphosphate ribose (cADPr). The mitochondrion is more sluggish, but it is closed-connected with the reticulum, and senses microdomains of high Ca2+ close to IP3 or cADPr release channels. The regulation of Ca2+ in the nucleus, where important Ca2+-sensitive processes reside, is a debated issue. Finally, if the control of cellular Ca2+ homeostasis somehow fails (excess penetration), mitochondria ‘buy time’ by precipitating inside Ca2+ and phosphate. If injury persists, Ca2+-death eventually ensues.