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

CHC22 and CHC17 clathrins have distinct biochemical properties and display differential regulation and function

Clathrins are cytoplasmic proteins that play essential roles in endocytosis and other membrane traffic pathways. Upon recruitment to intracellular membranes, the canonical clathrin triskelion assembles into a polyhedral protein coat that facilitates vesicle formation and captures cargo molecules for transport. The triskelion is formed by trimerization of three clathrin heavy-chain subunits. Most vertebrates have two isoforms of clathrin heavy chains, CHC17 and CHC22, generating two clathrins with distinct cellular functions. CHC17 forms vesicles at the plasma membrane for receptor-mediated endocytosis and at the trans-Golgi network for organelle biogenesis. CHC22 plays a key role in intracellular targeting of the insulin-regulated glucose transporter 4 (GLUT4), accumulates at the site of GLUT4 sequestration during insulin resistance, and has also been implicated in neuronal development. Here, we demonstrate that CHC22 and CHC17 share morphological features, in that CHC22 forms a triskelion and latticed vesicle coats. However, cellular CHC22-coated vesicles were distinct from those formed by CHC17. The CHC22 coat was more stable to pH change and was not removed by the enzyme complex that disassembles the CHC17 coat. Moreover, the two clathrins were differentially recruited to membranes by adaptors, and CHC22 did not support vesicle formation or transferrin endocytosis at the plasma membrane in the presence or absence of CHC17. Our findings provide biochemical evidence for separate regulation and distinct functional niches for CHC17 and CHC22 in human cells. Furthermore, the greater stability of the CHC22 coat relative to the CHC17 coat may be relevant to its excessive accumulation with GLUT4 during insulin resistance. [Abstract copyright: Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

The CHC22 Clathrin-GLUT4 Transport Pathway Contributes to Skeletal Muscle Regeneration

Mobilization of the GLUT4 glucose transporter from intracellular storage vesicles provides a mechanism for insulin-responsive glucose import into skeletal muscle. In humans, clathrin isoform CHC22 participates in formation of the GLUT4 storage compartment in skeletal muscle and fat. CHC22 function is limited to retrograde endosomal sorting and is restricted in its tissue expression and species distribution compared to the conserved CHC17 isoform that mediates endocytosis and several other membrane traffic pathways. Previously, we noted that CHC22 was expressed at elevated levels in regenerating rat muscle. Here we investigate whether the GLUT4 pathway in which CHC22 participates could play a role in muscle regeneration in humans and we test this possibility using CHC22-transgenic mice, which do not normally express CHC22. We observed that GLUT4 expression is elevated in parallel with that of CHC22 in regenerating skeletal muscle fibers from patients with inflammatory and other myopathies. Regenerating human myofibers displayed concurrent increases in expression of VAMP2, another regulator of GLUT4 transport. Regenerating fibers from wild-type mouse skeletal muscle injected with cardiotoxin also showed increased levels of GLUT4 and VAMP2. We previously demonstrated that transgenic mice expressing CHC22 in their muscle over-sequester GLUT4 and VAMP2 and have defective GLUT4 trafficking leading to diabetic symptoms. In this study, we find that muscle regeneration rates in CHC22 mice were delayed compared to wild-type mice, and myoblasts isolated from these mice did not proliferate in response to glucose. Additionally, CHC22-expressing mouse muscle displayed a fiber type switch from oxidative to glycolytic, similar to that observed in type 2 diabetic patients. These observations implicate the pathway for GLUT4 transport in regeneration of both human and mouse skeletal muscle, and demonstrate a role for this pathway in maintenance of muscle fiber type. Extrapolating these findings, CHC22 and GLUT4 can be considered markers of muscle regeneration in humans

Increased Muscle Stress-Sensitivity Induced by Selenoprotein N Inactivation in Mouse: A Mammalian Model for SEPN1-Related Myopathy

Selenium is an essential trace element and selenoprotein N (SelN) was the first selenium-containing protein shown to be directly involved in human inherited diseases. Mutations in the SEPN1 gene, encoding SelN, cause a group of muscular disorders characterized by predominant affection of axial muscles. SelN has been shown to participate in calcium and redox homeostasis, but its pathophysiological role in skeletal muscle remains largely unknown. To address SelN function in vivo, we generated a Sepn1-null mouse model by gene targeting. The Sepn1−/− mice had normal growth and lifespan, and were macroscopically indistinguishable from wild-type littermates. Only minor defects were observed in muscle morphology and contractile properties in SelN-deficient mice in basal conditions. However, when subjected to challenging physical exercise and stress conditions (forced swimming test), Sepn1−/− mice developed an obvious phenotype, characterized by limited motility and body rigidity during the swimming session, as well as a progressive curvature of the spine and predominant alteration of paravertebral muscles. This induced phenotype recapitulates the distribution of muscle involvement in patients with SEPN1-Related Myopathy, hence positioning this new animal model as a valuable tool to dissect the role of SelN in muscle function and to characterize the pathophysiological process

EHD2 is a mechanotransducer connecting caveolae dynamics with gene transcription

Caveolae are small invaginated pits that function as dynamic mechanosensors to buffer tension variations at the plasma membrane. Here we show that under mechanical stress, the EHD2 ATPase is rapidly released from caveolae, SUMOylated, and translocated to the nucleus, where it regulates the transcription of several genes including those coding for caveolae constituents. We also found that EHD2 is required to maintain the caveolae reservoir at the plasma membrane during the variations of membrane tension induced by mechanical stress. Metal-replica electron microscopy of breast cancer cells lacking EHD2 revealed a complete absence of caveolae and a lack of gene regulation under mechanical stress. Expressing EHD2 was sufficient to restore both functions in these cells. Our findings therefore define EHD2 as a central player in mechanotransduction connecting the disassembly of the caveolae reservoir with the regulation of gene transcription under mechanical stress

Cargo regulates clathrin coated pit invagination via clathrin light chain phosphorylation

Clathrin light chains (CLCs) control selective uptake of a range of G protein–coupled receptors (GPCRs), although the mechanism by which this occurs has remained elusive thus far. In particular, site-specific phosphorylation of CLCb controls the uptake of the purinergic GPCR P2Y12, but it is dispensable for the constitutive uptake of the transferrin receptor (TfR). We demonstrate that phosphorylation of CLCb is required for the maturation of clathrin-coated pits (CCPs) through the transition of flat lattices into invaginated buds. This transition is dependent on efficient clathrin exchange regulated by CLCb phosphorylation and mediated through auxilin. Strikingly, this rearrangement is required for the uptake of P2Y12 but not TfR. These findings link auxilin-mediated clathrin exchange to early stages of CCP invagination in a cargo-specific manner. This supports a model in which CCPs invaginate with variable modes of curvature depending on the cargo they incorporate

Caractérisation et rôle des isoformes de la triadine dans la physiologie des cellules musculaires

Au cours de cette étude, j'ai recherché quel était le rôle des différentes isoformes de la triadine, Trisk 95, Trisk 51, Trisk 49 et Trisk 32 dans les cellules musculaires. Dans un premier temps, j'ai étudié le rôle des triadines Trisk 95 et Trisk 51. Pour se faire, j'ai induit leur surexpression grâce à des vecteurs viraux, soit dans des cultures primaires, soit chez la souris nouveau-née. Trisk 95 et Trisk 51 sont associées au RyR et la surexpression de Trisk 95 peut bloquer la libération de calcium induite par dépolarisation, alors que dans les mêmes conditions, la surexpression de Trisk 51 n'a pas d'effet. J'ai aussi étudié l'effet de la surexpression de Trisk 95 sur l'entrée capacitive, qui est le mécanisme par lequel une entrée de Ca2+ extracellulaire sera induite pour remplir les stocks intracellulaires vidés. La surexpression de Trisk 95 diminue l'entrée capacitive dans des cultures primaires. La surexpression à long terme de Trisk 95 et Trisk 51 chez l'animal n'a que peu d'effet sur les autres protéines du complexe de mobilisation du Ca2+. Par contre, elle perturbe la localisation de la cavéoline-3, une protéine de structure impliquée dans la formation des triades. Dans un second temps, j'ai caractérisé la localisation et les partenaires de Trisk 49 et de Trisk 32 par des marquages immunofluorescents et de la microscopie confocale. Ces deux isoformes ne sont pas localisées à la triade. Les expériences d'immunoprécipitation montrent que Trisk 32 pourrait à la fois être associée au RyR et à un autre type de canal intracellulaire de libération de Ca2+, le récepteur de l'inositol 1,4,5-tris-phosphate (IP3R). Ainsi, les triadines pourraient constituer une famille de protéines, impliquées dans la régulation de différents types de canaux intracellulaires, et pourraient jouer un rôle dans la formation et le maintien de certains compartiments du RS.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

The Value of Flexibility in Power Markets

The concept of flexibility is not one you find in standard microeconomics textbooks , yet it already plays a major role in the remuneration of the resources that generate and consume electricity every day and is likely to play an even larger role with the penetration of large intermittent renewable capacities. In this paper we attempt to quantify the net revenues that can be captured by a flexible resource able to react to the short term price variations on the day-ahead and intraday markets in Germany. We find that the difference between day-ahead and intraday revenues for a flexible resource has been increasing (although the profitability has been decreasing on both markets). This difference is more pronounced once 15mn price variations can be captured by a flexible resource. The net revenues from the local 15mn auction (which is held 3 hours after the hourly "coupled" day-ahead auction) are more than eight times higher than the day-ahead hourly auction bu

Anneaux ou tresses ?: La microscopie corrélative dévoile la structure de l’actine axonale

International audienceNo abstract availabl