Paola Pizzo

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.

Mitochondrial Ca2+ as a key regulator of cell life and death.

Mitochondrial Ca2+ homeostasis is today at the center of wide interest in the scientific community because of its role both in the modulation of numerous physiological responses and because of its involvement in cell death. In this review, we briefly summarize a few basic features of mitochondrial Ca2+ handling in vitro and within living cells, and its involvement in the modulation of Ca2+-dependent signaling. We then discuss the role of mitochondrial Ca2+ in the control of apoptotic death, focusing in particular on the effects of pro- and anti-apoptotic proteins of the Bcl-2 family. Finally, the potential involvement of Ca2+ and mitochondria in the development of two diseases, Ullrich muscular dystrophy and familial Alzheimers disease, is briefly discussed.

Mitofusin 2 ablation increases endoplasmic reticulum–mitochondria coupling

Significance The privileged interrelationship between mitochondria and the endoplasmic reticulum (ER) plays a key role in a variety of physiological functions, from lipid metabolism to Ca2+ signalling, and its modulation influences apoptotic susceptibility, mitophagy, and cellular bioenergetics. Among the several proteins known to influence ER–mitochondria interactions, mitofusin 2 (Mfn2) has been proposed to form a physical tether. In this study, we demonstrate that Mfn2 instead works as an ER–mitochondria tethering antagonist preventing an excessive, potentially toxic, proximity between the two organelles. Cells in which Mfn2 is ablated or reduced have an increased number of ER–mitochondria close contacts, potentiated Ca2+ transfer between the two organelles, and greater sensitivity to cell-death stimuli that implies mitochondria Ca2+ overload toxicity. The organization and mutual interactions between endoplasmic reticulum (ER) and mitochondria modulate key aspects of cell pathophysiology. Several proteins have been suggested to be involved in keeping ER and mitochondria at a correct distance. Among them, in mammalian cells, mitofusin 2 (Mfn2), located on both the outer mitochondrial membrane and the ER surface, has been proposed to be a physical tether between the two organelles, forming homotypic interactions and heterocomplexes with its homolog Mfn1. Recently, this widely accepted model has been challenged using quantitative EM analysis. Using a multiplicity of morphological, biochemical, functional, and genetic approaches, we demonstrate that Mfn2 ablation increases the structural and functional ER–mitochondria coupling. In particular, we show that in different cell types Mfn2 ablation or silencing increases the close contacts between the two organelles and strengthens the efficacy of inositol trisphosphate (IP3)-induced Ca2+ transfer from the ER to mitochondria, sensitizing cells to a mitochondrial Ca2+ overload-dependent death. We also show that the previously reported discrepancy between electron and fluorescence microscopy data on ER–mitochondria proximity in Mfn2-ablated cells is only apparent. By using a different type of morphological analysis of fluorescent images that takes into account (and corrects for) the gross modifications in mitochondrial shape resulting from Mfn2 ablation, we demonstrate that an increased proximity between the organelles is also observed by confocal microscopy when Mfn2 levels are reduced. Based on these results, we propose a new model for ER–mitochondria juxtaposition in which Mfn2 works as a tethering antagonist preventing an excessive, potentially toxic, proximity between the two organelles.

Modulation of the endoplasmic reticulum–mitochondria interface in Alzheimer’s disease and related models

It is well-established that subcompartments of endoplasmic reticulum (ER) are in physical contact with the mitochondria. These lipid raft-like regions of ER are referred to as mitochondria-associated ER membranes (MAMs), and they play an important role in, for example, lipid synthesis, calcium homeostasis, and apoptotic signaling. Perturbation of MAM function has previously been suggested in Alzheimer’s disease (AD) as shown in fibroblasts from AD patients and a neuroblastoma cell line containing familial presenilin-2 AD mutation. The effect of AD pathogenesis on the ER–mitochondria interplay in the brain has so far remained unknown. Here, we studied ER–mitochondria contacts in human AD brain and related AD mouse and neuronal cell models. We found uniform distribution of MAM in neurons. Phosphofurin acidic cluster sorting protein-2 and σ1 receptor, two MAM-associated proteins, were shown to be essential for neuronal survival, because siRNA knockdown resulted in degeneration. Up-regulated MAM-associated proteins were found in the AD brain and amyloid precursor protein (APP)Swe/Lon mouse model, in which up-regulation was observed before the appearance of plaques. By studying an ER–mitochondria bridging complex, inositol-1,4,5-triphosphate receptor–voltage-dependent anion channel, we revealed that nanomolar concentrations of amyloid β-peptide increased inositol-1,4,5-triphosphate receptor and voltage-dependent anion channel protein expression and elevated the number of ER–mitochondria contact points and mitochondrial calcium concentrations. Our data suggest an important role of ER–mitochondria contacts and cross-talk in AD pathology.

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.

Presenilin 2 modulates endoplasmic reticulum (ER)–mitochondria interactions and Ca2+ cross-talk

Presenilin mutations are the main cause of familial Alzheimers disease (FAD). Presenilins also play a key role in Ca2+ homeostasis, and their FAD-linked mutants affect cellular Ca2+ handling in several ways. We previously have demonstrated that FAD-linked presenilin 2 (PS2) mutants decrease the Ca2+ content of the endoplasmic reticulum (ER) by inhibiting sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) activity and increasing ER Ca2+ leak. Here we focus on the effect of presenilins on mitochondrial Ca2+ dynamics. By using genetically encoded Ca2+ indicators specifically targeted to mitochondria (aequorin- and GFP-based probes) in SH-SY5Y cells and primary neuronal cultures, we show that overexpression or down-regulation of PS2, but not of presenilin 1 (PS1), modulates the Ca2+ shuttling between ER and mitochondria, with its FAD mutants strongly favoring Ca2+ transfer between the two organelles. This effect is not caused by a direct PS2 action on mitochondrial Ca2+-uptake machinery but rather by an increased physical interaction between ER and mitochondria that augments the frequency of Ca2+ hot spots generated at the cytoplasmic surface of the outer mitochondrial membrane upon stimulation. This PS2 function adds further complexity to the multifaceted nature of presenilins and to their physiological role within the cell. We also discuss the importance of this additional effect of FAD-linked PS2 mutants for the understanding of FAD pathogenesis.

H2O2 in plant peroxisomes: an in vivo analysis uncovers a Ca2+-dependent scavenging system

Oxidative stress is a major challenge for all cells living in an oxygen-based world. Among reactive oxygen species, H2O2, is a well known toxic molecule and, nowadays, considered a specific component of several signalling pathways. In order to gain insight into the roles played by H2O2 in plant cells, it is necessary to have a reliable, specific and non-invasive methodology for its in vivo detection. Hence, the genetically encoded H2O2 sensor HyPer was expressed in plant cells in different subcellular compartments such as cytoplasm and peroxisomes. Moreover, with the use of the new green fluorescent protein (GFP)-based Cameleon Ca2+ indicator, D3cpv-KVK-SKL, targeted to peroxisomes, we demonstrated that the induction of cytoplasmic Ca2+ increase is followed by Ca2+ rise in the peroxisomal lumen. The analyses of HyPer fluorescence ratios were performed in leaf peroxisomes of tobacco and pre- and post-bolting Arabidopsis plants. These analyses allowed us to demonstrate that an intraperoxisomal Ca2+ rise in vivo stimulates catalase activity, increasing peroxisomal H2O2 scavenging efficiency.

After half a century mitochondrial calcium in‐ and efflux machineries reveal themselves

Mitochondrial Ca2+ uptake and release play a fundamental role in the control of different physiological processes, such as cytoplasmic Ca2+ signalling, ATP production and hormone metabolism, while dysregulation of mitochondrial Ca2+ handling triggers the cascade of events that lead to cell death. The basic mechanisms of mitochondrial Ca2+ homeostasis have been firmly established for decades, but the molecular identities of the channels and transporters responsible for Ca2+ uptake and release have remained mysterious until very recently. Here, we briefly review the main findings that have led to our present understanding of mitochondrial Ca2+ homeostasis and its integration in cell physiology. We will then discuss the recent work that has unravelled the biochemical identity of three key molecules: NCLX, the mitochondrial Na+/Ca2+ antiporter, MCU, the pore‐forming subunit of the mitochondrial Ca2+ uptake channel, and MICU1, one of its regulatory subunits.

Lipid rafts and T cell receptor signaling: a critical re-evaluation.

The current model suggesting that raft integrity is required for T cell activation is mostly (but not exclusively) based on the use of drugs, such as methyl‐β ‐cyclodextrin (Mβ CD), that disorganize rafts and inhibit T cell receptor (TCR)‐induced Ca2 + influx. Here we show that conditions that disrupt lipid raft integrity do not inhibit TCR triggering in Jurkat cells andnormal T lymphocytes. Indeed, we found that the reported inhibition of TCR‐induced Ca2 + influx by Mβ CD treatment is mainly due to (a) nonspecific depletion of intracellular Ca2 + stores and (b) plasma membrane depolarization of T cells. When these side‐effects are taken into account, raft disorganization does not alter TCR‐dependent Ca2 + signaling. In line with these results, also TCR‐induced tyrosine phosphorylation is not inhibited by dispersion of lipid rafts. By contrast, in the same conditions, Ca2 + signaling via the glycosylphosphatidylinositol (GPI)‐anchored protein CD59 is totally abolished. These results indicate that, while signaling through GPI‐anchored proteins requires lipid raft integrity, CD3‐dependent TCR activation occurs independently of cholesterol extraction.

Mitochondrial Ca2+ homeostasis: mechanism, role, and tissue specificities

Mitochondria from every tissue are quite similar in their capability to accumulate Ca2+ in a process that depends on the electrical potential across the inner membrane; it is catalyzed by a gated channel (named mitochondrial Ca2+ uniporter), the molecular identity of which has only recently been unraveled. The release of accumulated Ca2+ in mitochondria from different tissues is, on the contrary, quite variable, both in terms of speed and mechanism: a Na+-dependent efflux in excitable cells (catalyzed by NCLX) and a H+/Ca2+ exchanger in other cells. The efficacy of mitochondrial Ca2+ uptake in living cells is strictly dependent on the topological arrangement of the organelles with respect to the source of Ca2+ flowing into the cytoplasm, i.e., plasma membrane or intracellular channels. In turn, the structural and functional relationships between mitochondria and other cellular membranes are dictated by the specific architecture of different cells. Mitochondria not only modulate the amplitude and the kinetics of local and bulk cytoplasmic Ca2+ changes but also depend on the Ca2+ signal for their own functionality, in particular for their capacity to produce ATP. In this review, we summarize the processes involved in mitochondrial Ca2+ handling and its integration in cell physiology, highlighting the main common characteristics as well as key differences, in different tissues.