216 research outputs found
Differential recruitment of PKC isoforms in HeLa cells during redox stress.
No full textThe protein kinase C (PKC) family is a major transducer of several intracellular pathways. In confirmation of this important role, PKCs exhibit high molecular heterogeneity, because they occur in at least 10 different isoforms differing in biochemical properties and sensitivity to activators. In this report we focused on the ability of different redox agents to induce modification of intracellular distribution of specific PKC isoforms in HeLa cells. To this end we utilized a panel of green fluorescent protein (GFP) chimeras and a high-speed digital imaging system. We observed a remarkable complexity of PKC signalling patterns occurring during redox stress with marked differences among PKC isoforms also belonging to the same subgroup. Moreover our results suggest that modifications of the intracellular redox state can modulate the responsiveness of specific PKC isoforms and, in turn, change the sensitivity of the different isoforms to cell stimulation
The versatility of mitochondrial calcium signals: From stimulation of cell metabolism to induction of cell death
No full textBoth the contribution of mitochondria to intracellular calcium (Ca2+) signalling and the role of mitochondrial Ca2+ uptake in shaping the cytoplasmic response and controlling mitochondrial function are areas of intense investigation. These studies rely on the appropriate use of emerging techniques coupled with judicious data interpretation to a large extent. The development of targeted probes based on the molecular engineering of luminescent proteins has allowed the specific measurement of Ca2+ concentration ([Ca2+]) and adenosine trisphosphate concentration ([ATP]) in intracellular organelles or cytoplasmic subdomains. This approach has given novel information on different aspects of mitochondrial homeostasis. (c) 2008 Elsevier B.V. All rights reserved
H-Ras-driven tumoral maintenance is sustained through caveolin-1-dependent alterations in calcium signaling
No full textA growing body of research has highlighted the complex range of tumoral traits acquired during H-Ras-driven transformation and maintenance, which include proliferative signaling, growth suppressor evasion and resistance to cell death. Clear molecular information about these processes is not yet available, but recent evidence has provided solid support for the importance of mitochondria. Here, we show that the induction of oncogenic H-Ras leads to changes in intracellular calcium (Ca2+), evaluate the temporal relationship between oncogene expression and mitochondrial physiology, and demonstrate that Ca2+ homeostasis is altered by caveolin-1, a protein that has a key role in tumor maintenance. Our results indicate that tumor-suppressor caveolin-1 is a core component of the Ca2+-dependent apoptotic pathway and participates in the regulation of critical mitochondrial functions during tumor development. The compromised caveolin-1/Ca2+ axis contributes to failure in both mitochondrial metabolism and apoptosis, thereby sustaining the neoplastic phenotype. These results illustrate a direct link between Ca2+ regulation and mitochondrial biology in cance
Biosensors for the Detection of Calcium and pH
No full textThe wide diffusion of molecular biology techniques, with the possibility of modifying and expressing in virtually all cell types exogenous cDNAs, has been responsible for the large expansion in the use of protein probes in cell biology.
Two groups of reporter proteins are currently employed: the chemiluminescent proteins (e.g. aequorin of Aequorea victoria), and the fluorescent proteins (e.g. green fluorescent protein GFP of Aequorea victoria).
The study of isolated mitochondria, dating back to the 60ties, has provided a wealth of information on the biochemical routes allowing these organelles, deriving from the adaptation of primordial symbionts, to couple oxidation of substrates to the production of ATP. Moreover, recent work highlighted the role of signals reaching the mitochondria in the activation of apoptosis.
In this context, it is an exciting task to study mitochondrial function in living cells. For this purpose, new tools are needed, that combine a specific mitochondrial distribution to the sensitivity to the parameter of interest. Recombinant reporter proteins are emerging as the tools of choice, as targeting sequences can be appended that direct them to the compartment of interest.
In particular, we will describe the development, and use, of protein chimeras (deriving from proteins naturally present in the medusa Aequorea victoria) specifically targeted to the mitochondria, either to the matrix or to the intermembrane space. Aequorin, the pioneer of the targeted recombinant probes, is a Ca2+-sensitive photoprotein, that emits light upon binding of Ca2+ to three high affinity binding sites. Then, we will describe the use of mutants of GFP as Ca2+ and pH probes
homeostasis
No full textBackground: Hailey-Hailey disease (HHD), also known as familial benign chronic pemphigus, is a rare autosomal dominant inherited intraepidermal blistering genodermatosis. Mutations in the ATP2C1 gene encoding for the Golgi secretory pathway Ca2+/Mn2+-ATPasi protein 1 (SPCA1) affect the processing of desmosomal components and the epidermal suprabasal cell-cell adhesion by deregulating the keratinocyte cytosolic Ca2+ concentration. We report the unexpected, dramatic, and persistent clinical improvement of the skin lesions of a patient affected with longstanding HHD with daily intake of a solution containing magnesium chloride hexahydrate (MgCl2). Materials and methods: We investigated the effect of MgCl2 on the intracellular Ca2+ homeostasis and on the activity of particular Ca2+-effectors in HeLa cells transfected with chimeric aequorins (cytAEQ, mtAEQ, erAEQ and GoAEQ) targeted to different subcellular compartments (cytosol, mitochondria, endoplasmic reticulum, and Golgi, respectively)...Background: Hailey-Hailey disease (HHD), also known as familial benign chronic pemphigus, is a rare autosomal dominant inherited intraepidermal blistering genodermatosis. Mutations in the ATP2C1 gene encoding for the Golgi secretory pathway Ca2+/Mn2+-ATPasi protein 1 (SPCA1) affect the processing of desmosomal components and the epidermal suprabasal cell-cell adhesion by deregulating the keratinocyte cytosolic Ca2+ concentration. We report the unexpected, dramatic and persistent clinical improvement of the skin lesions of a patient affected with long standing HHD with daily intake of a solution containing magnesium chloride hexahydrate (MgCl2).
Methods: We investigated the effect of MgCl2 on the intracellular Ca2+ homeostasis and on the activity of particular Ca2+-effectors in HeLa cells transfected with chimeric aequorins (cytAEQ, mtAEQ, erAEQ and GoAEQ) targeted to different subcellular compartments (cytosol, mitochondria, endoplasmic reticulum and Golgi, respectively).
Results: Experimental investigations on HeLa cells showed the effect of MgCl2 on the function of Ca2+-extrusor systems, resulting in increased cytosolic and mitochondrial Ca2+ levels, without altering the mechanisms of intraluminal Ca2+-filling and Ca2+-release of stores.
Conclusions: Based on our clinical observation and experimental results, it can be hypothesized that MgCl2 could act as an inhibitor of the Ca2+-extruding activity in keratinocytes favoring intracellular Ca2+-disponibility and Ca2+-dependent mechanisms in desmosome assembly. This may represent the molecular basis of the good response of the HHD clinical features with MgCl2 solution in the patient described
Implication of copper and iron sources and availability for plant growth and development
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Mitochondrial Stress Responses and "Mito-Inflammation" in Cystic Fibrosis
No full textCystic fibrosis (CF) is a genetic disease associated to mutations in the cystic fibrosis transmembrane conductance regulator gene, which results in the alteration of biological fluid and electrolyte homeostasis. The characteristic pathological manifestation is represented by exaggerated proinflammatory response in lung of CF patients, driven by recurrent infections and worsen by hypersecretion of proinflammatory mediators and progressive tissue destruction. Treating inflammation remains a priority in CF. However, current anti-inflammatory treatments, including non-steroidal agents, are poorly effective and present dramatic side effects in CF patients. Different studies suggest an intimate relationship between mitochondria and CF lung disease, supporting the hypothesis that a decline in mitochondrial function endorses the development of the hyperinflammatory phenotype observed in CF lung. This allowed the implementation of a new concept: the "mito-inflammation," a compartmentalization of inflammatory process, related to the role of mitochondria in engage and sustain the inflammatory responses, resulting a druggable target to counteract the amplification of inflammatory signals in CF. Here, we will offer an overview of the contribution of mitochondria in the pathogenesis of CF lung disease, delving into mitochondrial quality control responses, which concur significantly to exacerbation of CF lung inflammatory responses. Finally, we will discuss the new therapeutic avenues that aim to target the mito-inflammation, an alternative therapeutic advantage for mitochondrial quality control that improves CF patient's inflammatory state
Efficacy of magnesium chloride in the treatment of Hailey–Hailey disease: some further considerations
No full textBased on our clinical observation and experimental results, it can be hypothesized that MgCl2 could act as an inhibitor of the Ca2+-extruding activity in keratinocytes favoring intracellular Ca2+-disponibility and Ca2+-dependent mechanisms in desmosome assembly. This may represent the molecular basis of the treatment of Hailey-Hailey disease with MgCl2 solution
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