1,720,964 research outputs found
Identification and characterization of a novel MICU1 splice variant
Full text linkThe ability of mitochondria to take up Ca2+ plays a fundamental role in the regulation of several biological processes [1]. In the last five years, the molecular and functional characterization of the MCU machinery pictures this Ca2+ channel as one of the most sophisticated ion channels described so far [2]. These groundbreaking discoveries have opened a new era for the study of mitochondrial Ca2+ in cell physiology and will allow to deepen the knowledge on the tissues-specific properties of MCU [3] that are still poorly understood.
In this regard, we identified an alternative splice isoform (hereafter named MICU1.1) of the positive MCU modulator MICU1, characterized by the addition of a micro-exon coding for 4 amino acids (EFWQ), conserved in vertebrates. Interestingly, while MICU1 is ubiquitously expressed, MICU1.1 shows a peculiar tissues distribution, being highly expressed in tissues that display the greatest level of mitochondrial Ca2+ uptake, skeletal muscle and lower levels are found in brain.
Immunoprecipitation experiments performed in HeLa cells assessed that MICU1.1 efficiently interacts with MCU and MICU2. Furthermore, MICU1.1 is able to form homo- and heterodimers with MICU2, as well as MICU1. Nonetheless, the overexpression of MICU1.1 in HeLa cells causes a major increase of mitochondrial Ca2+ uptake upon histamine stimulation compared to conventional MICU1, without affecting neither cytosolic Ca2+ values nor the mitochondrial membrane potential. Strikingly, MICU2 overexpression in cells expressing MICU1.1 is unable to block the increase of mitochondrial Ca2+ uptake induced by MICU1.1. On the contrary, the co-expression of MICU1.1 together with MICU2 further increases mitochondrial Ca2+ uptake speed compared to cells overexpressing MICU1.1 alone. On the other hand, MICU1.1, when bound to MICU2, is able to act as gatekeeper of the channel at resting Ca2+ levels as well as MICU1-MICU2 heterodimer. Importantly, we found that MICU1.1-MICU2 overexpression induces the shift of the threshold of MCU opening towards lower Ca2+ concentrations.
Consistently with previous results on MICU1, MICU1.1 function is dependent on its ability to bind Ca2+. Indeed, a MICU1.1 mutant, insensitive to Ca2+, displays a dominant-negative effect on mitochondrial Ca2+ uptake. On the contrary, MICU1.1 is less affected by the dominant-negative effect of mutated MICU2, insensitive to Ca2+ binding.
We also analysed the contribution of the extra-exon to the particular behaviour of MICU1.1. We observed that single mutations or deletions of these residues do not influence the effect of MICU1.1 on mitochondrial Ca2+ uptake. On the contrary, the substitution of all the four amino acids of the extra-exon with four alanine residues is sufficient to recapitulate MICU1 behaviour.
In conclusion, we characterized a transcript variant of MICU1, which is specifically expressed in excitable tissues, prevalently in skeletal muscle, and that shows a higher ability to activate MCU compared to conventional MICU1. Interestingly, MICU1.1 exerts a peculiar function when bound to MICU2. Overall, our data demonstrate a skeletal muscle-specific mitochondrial Ca2+ uptake machinery with a presumably unique function. Thus, in this tissue, mitochondrial Ca2+ can exert new, unexplored roles. Future experiments have to be performed to clarify its physiological and pathological relevance
High-Throughput Screening Using Photoluminescence Probe to Measure Intracellular Calcium Levels
No full textAequorin, a 22 kDa protein produced by the jellyfish Aequorea victoria, was the first probe used to measure Ca2+ concentrations ([Ca2+]) of specific intracellular organelles in intact cells. After the binding of Ca2+ to three high-affinity binding sites, an irreversible reaction occurs leading to the emission of photons that is proportional to [Ca2+]. While native aequorin is suitable for measuring cytosolic [Ca2+] after cell stimulation in a range from 0.5 to 10 mu M, it cannot be used in organelles where [Ca2+] is much higher, such as in the lumen of endoplasmic/sarcoplasmic reticulum (ER/SR) and mitochondria. However, some modifications made on aequorin itself or on coelenterazine, its lipophilic prosthetic luminophore, and the addition of targeting sequences or the fusion with resident proteins allowed the specific organelle localization and the measurements of intra-organelle Ca2+ levels. In the last years, the development of multiwell plate readers has opened the possibility to perform aequorin-based high-throughput screenings and has overcome some limitation of the standard method. Here we present the procedure for expressing, targeting, and reconstituting aequorin in intact cells and for measuring Ca2+ in the bulk cytosol, mitochondria, and ER by a high-throughput screening system
Neither too much nor too little: mitochondrial calcium concentration as a balance between physiological and pathological conditions
Full text linkCa2+ ions serve as pleiotropic second messengers in the cell, regulating several cellular processes. Mitochondria play a fundamental role in Ca2+ homeostasis since mitochondrial Ca2+ (mitCa2+) is a key regulator of oxidative metabolism and cell death. MitCa2+ uptake is mediated by the mitochondrial Ca2+ uniporter complex (MCUc) localized in the inner mitochondrial membrane (IMM). MitCa2+ uptake stimulates the activity of three key enzymes of the Krebs cycle, thereby modulating ATP production and promoting oxidative metabolism. As Paracelsus stated, "Dosis sola facit venenum,"in pathological conditions, mitCa2+ overload triggers the opening of the mitochondrial permeability transition pore (mPTP), enabling the release of apoptotic factors and ultimately leading to cell death. Excessive mitCa2+ accumulation is also associated with a pathological increase of reactive oxygen species (ROS). In this article, we review the precise regulation and the effectors of mitCa2+ in physiopathological processes
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Molecular structure and pathophysiological roles of the Mitochondrial Calcium Uniporter
Full text linkAbstractMitochondrial Ca2+ uptake regulates a wide array of cell functions, from stimulation of aerobic metabolism and ATP production in physiological settings, to induction of cell death in pathological conditions. The molecular identity of the Mitochondrial Calcium Uniporter (MCU), the highly selective channel responsible for Ca2+ entry through the IMM, has been described less than five years ago. Since then, research has been conducted to clarify the modulation of its activity, which relies on the dynamic interaction with regulatory proteins, and its contribution to the pathophysiology of organs and tissues. Particular attention has been placed on characterizing the role of MCU in cardiac and skeletal muscles.In this review we summarize the molecular structure and regulation of the MCU complex in addition to its pathophysiological role, with particular attention to striated muscle tissues. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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