Caveolin 3 Is Associated with the Calcium Release Complex and Is Modified via in Vivo Triadin Modification†

International audienceThe triadin isoforms Trisk 95 and Trisk 51 are both components of the skeletal muscle calcium release complex. To investigate the specific role of Trisk 95 and Trisk 51 isoforms in muscle physiology, we overexpressed Trisk 95 or Trisk 51 using adenovirus-mediated gene transfer in skeletal muscle of newborn mice. Overexpression of either Trisk 95 or Trisk 51 alters the muscle fiber morphology, while leaving unchanged the expression of the ryanodine receptor, the dihydropyridine receptor, and calsequestrin. We also observe an aberrant expression of caveolin 3 in both Trisk 95- and Trisk 51-overexpressing skeletal muscles. Using a biochemical approach, we demonstrate that caveolin 3 is associated with the calcium release complex in skeletal muscle. Taking advantage of muscle and non-muscle cell culture models and triadin null mouse skeletal muscle, we further dissect the molecular organization of the caveolin 3-containing calcium release complex. Our data demonstrate that the association of caveolin 3 with the calcium release complex occurs via a direct interaction with the transmembrane domain of the ryanodine receptor. Taken together, these data suggest that caveolin 3-containing membrane domains and the calcium release complex are functionally linked and that Trisk 95 and Trisk 51 are instrumental to the regulation of this interaction, the integrity of which may be crucial for muscle physiology

Role of triadin in the organization of reticulum membrane at the muscle triad.

International audienceThe terminal cisternae represent one of the functional domains of the skeletal muscle sarcoplasmic reticulum (SR). They are closely apposed to plasma membrane invaginations, the T-tubules, with which they form structures called triads. In triads, the physical interaction between the T-tubule-anchored voltage-sensing channel DHPR and the SR calcium channel RyR1 is essential because it allows the depolarization-induced calcium release that triggers muscle contraction. This interaction between DHPR and RyR1 is based on the peculiar membrane structures of both T-tubules and SR terminal cisternae. However, little is known about the molecular mechanisms governing the formation of SR terminal cisternae. We have previously shown that ablation of triadins, a family of SR transmembrane proteins that interact with RyR1, induced skeletal muscle weakness in knockout mice as well as a modification of the shape of triads. Here we explore the intrinsic molecular properties of the longest triadin isoform Trisk 95. We show that when ectopically expressed, Trisk 95 can modulate reticulum membrane morphology. The membrane deformations induced by Trisk 95 are accompanied by modifications of the microtubule network organization. We show that multimerization of Trisk 95 by disulfide bridges, together with interaction with microtubules, are responsible for the ability of Trisk 95 to structure reticulum membrane. When domains responsible for these molecular properties are deleted, anchoring of Trisk 95 to the triads in muscle cells is strongly decreased, suggesting that oligomers of Trisk 95 and microtubules contribute to the organization of the SR terminal cisternae in a triad

Fonctions des triadines dans le muscle squelettique. Caractérisation de l'isoforme Trisk 32.

Triadin is a protein of the skeletal muscle. Four isoforms have been cloned: Trisk 95, Trisk 51, Trisk 49, and Trisk 32. They are transmembrane proteins of the sarcoplasmic reticulum (SR). Trisk 95 and Trisk 51 are in the triad junction where they are associated with a calcium release channel, the ryanodine receptor (RyR). Trisk 49 and Trisk 32 are localised in the longitudinal SR. It has been shown that Trisk 95 is able to regulate RyR Ca2+ releases. The aim of this work was to study triadins functions in the skeletal muscle with different and complementary approches. In the first part of this work, Trisk 95 and Trisk 51 were overexpressed in vivo in mouse muscles. These muscles where then characterised and the results showed an association between RyR and caveolin-3, a protein of the plasma membrane. The second part of this work is related to the study of Trisk 32. I studied more precisely the localisation of Trisk 32 and showed that it is localised in the triad, in the longitudinal SR, and close to mitochondria. Co-immunoprecipiation experiments highlighted new partners for Trisk 32 such as RyR and the IP3 receptor, another calcium channel of the SR. Thanks to itspartners, it can be assumed that Trisk 32 is involved in the regulation of many Ca2+ dependent processes. At least, triadin gene has been knocked out in mouse. This mouse model clearly presents an obvious muscle weakness. The muscle presented ultrastructural disorders. These results suggest that in addition to it function as a Ca2+ release channel regulator, triadin could have a scaffolding function.La triadine est une famille de protéines du muscle squelettique. Quatre isoformes de la triadine ont été clonées: Trisk 95, Trisk 51, Trisk 49 et Trisk 32. Ce sont des protéines transmembranaires du reticulum sarcoplasmique (RS). Trisk 95 et Trisk 51 sont localisées dans la triade où elles sont associées au récepteur de la ryanodine (RyR), un canal calcique. Trisk 49 et Trisk 32 sont localisées dans le RS longitudinal. Il a été montré que Trisk 95 régule les relâchements de Ca2+ du RyR. L'objectif de ce travail de thèse a été d'étudier les fonctions des triadines dans le muscle squelettique grâce à différentes approches et techniques complémentaires. Dans un premier temps, Trisk 95 et de Trisk 51 ont été étudiées par surexpression in vivo dans les muscles de souris. La caractérisation de ces muscles a permis de mettre en évidence l'association du RyR avec la cavéoline, une protéine de la membrane plasmique. Dans un second temps, la fonction de Trisk 32 a été étudiée dans le muscle squelettique. L'étude précise de sa localisation a permis de montrer qu'elle est localisée dans la triade, dans le RS longitudinal, et à proximité des mitochondries. Des expériences de co-immunoprécipitation ont révélé qu'elle est associée avec le RyR et avec le récepteur de l'IP3. De par ses partenaires, Trisk 32 semble jouer un rôle dans la régulation de nombreux mécanismes impliquant le Ca2+. Enfin, le gène de la triadine a été invalidé chez la souris. Cette souris KO triadine présente une faiblesse musculaire et des défauts dans l'ultrastructure de la triade. Ces résultats indiquent qu'en plus de sa fonction de régulation des relâchements de Ca2+ la triadine pourrait avoir un rôle structural

Fonctions des traidines dans le muscle squelettique (caractérisation de l'isoforme Trisk 32)

La triadine est une famille de protéines du muscle squelettique. Quatre isoformes de la triadine 0 été clonées: Trisk 95, Trisk 51, Trisk 49 et Trisk 32. Ce sont des protéines transmembranaires d reticulum sarcoplasmique (RS). Trisk 95 et Trisk 51 sont localisées dans la triade où elles so associées au récepteur de la ryanodine (RyR), un canal calcique. Trisk 49 et Trisk 32 sont localisée dans le RS longitudinal. Il a été montré que Trisk 95 régule les relâchements de Ca2+ du Ry L'objectif de ce travail de thèse a été d'étudier les fonctions des triadines dans le muscle squelettiqu grâce à différentes approches et techniques complémentaires. Dans un premier temps, Trisk 95 et Trisk 51 ont été étudiées par surexpression in vivo dans les muscles de souris. La caractérisation d~ ces muscles a permis de mettre en évidence l'association du RyR avec la cavéoline, une protéine d~ la membrane plasmique. Dans un second temps, la fonction de Trisk 32 a été étudiée dans le musd squelettique. L'étude précise de sa localisation a permis de montrer qu'elle est localisée dans 1: triade, dans le RS longitudinal, et à proximité des mitochondries. Des expériences de co immunoprécipitation ont révélé qu'elle est associée avec le RyR et avec le récepteur de l'IP3. De par ses partenaires, Trisk 32 semble jouer un rôle dans la régulation de nombreux mécanisme impliquant le Ca2+. Enfin, le gène de la triadine a été invalidé chez la souris. Cette souris KO triadinl présente une faiblesse musculaire et des défauts dans l'ultrastructure de la triade. Ces résultat indiquent qu'en plus de sa fonction de régulation des relâchements de Ca2+ la triadine pourrait avoi un rôle structural.Triadin is a protein of the skeletal muscle. Four isoforms have been cloned: Trisk 95, Trisk 51, Tris 49, and Trisk 32. They are transmembrane proteins of the sarcoplasmic reticulum (SR). Trisk 95 an Trisk 51 are in the triad junction where they are associated with a calcium release channel, th ryanodine receptor (RyR). Trisk 49 and Trisk 32 are localised in the longitudinal SR. It has bee shown that Trisk 95 is able to regulate RyR Ca2+ releases. The aim of this work was to study triadin functions in the skeletal muscle with different and complementary approches. ln the first part of this work, Trisk 95 and Trisk 51 were overexpressed in vivo in mouse muscles. These muscles where the] characterised and the results showed a functionnal association of triadin and caveolin-3, a protein 0 the plasma membrane, through a direct association of RyR with caveolin-3. The second part of thi work is related to the study of Trisk 32. l studied more precisely the localisation of Trisk 32 an< showed that it is localised in the triad, in the longitudinal SR, and close to mitochondria Coimmunoprecipiation experiments highlighted new partners for Trisk 32 such as RyR and the IP receptor, another calcium channel of the SR. Thanks to its partners, it can be assumed that Trisk 32 i involved in the regulation of many Ca2+ dependant processes. At least, triadin gene has been knockec out in mouse. This mouse model clearly presents an objective muscle weakness. The musd presented ultrastructural disorders. These results suggest that in addition to it function as a Ca2 release channel regulator, triadin could have a scaffolding function.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

The calcium release complex in skeletal muscle and its associated diseases

International audienceIntroduction The coupling between the stimulation of a muscle fibre and the massive intracellular calcium release, that is the molecular basis of muscle contraction, is fulfilled by a macromolecular complex, the calcium release complex (CRC). Exegesis A very precise molecular architecture is required for CRC correct function, as well as a specific membrane organisation in muscle cells. Conclusion Several muscle pathologies are associated with mutations in the two calcium channels of the CRC. However, elucidation of mechanisms involved in CRC function are still required to characterise muscle pathologies of unknown origin

Low concentrations of vorinostat decrease EB1 expression in GBM cells and affect microtubule dynamics, cell survival and migration

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Nutrient Requirements during Pregnancy and Lactation

A woman’s nutritional status during pregnancy and breastfeeding is not only critical for her health, but also for that of future generations. Nutritional requirements during pregnancy differ considerably from those of non-pregnant women. Thus, a personalized approach to nutritional advice is recommended. Currently, some countries recommend routine supplementation for all pregnant women, while others recommend supplements only when necessary. Maternal physiological adaptations, as well as nutritional requirements during pregnancy and lactation, will be reviewed in the literature examining the impacts of dietary changes. All of these data have been studied deeply to facilitate a discussion on dietary supplement use and the recommended doses of nutrients during pregnancy and lactation. The aim of this review is to evaluate the knowledge in the scientific literature on the current recommendations for the intake of the most common micronutrients and omega-3 fatty acids during pregnancy and lactation in the United States, Canada, and Europe. Taking into account these considerations, we examine minerals, vitamins, and omega-3 fatty acid requirements. Finally, we conclude by discussing the potential benefits of each form of supplementation