Annexins and Membrane Repair Dysfunctions in Muscular Dystrophies

Muscular dystrophies constitute a group of genetic disorders that cause weakness and progressive loss of skeletal muscle mass. Among them, Miyoshi muscular dystrophy 1 (MMD1), limb girdle muscular dystrophy type R2 (LGMDR2/2B), and LGMDR12 (2L) are characterized by mutation in gene encoding key membrane-repair protein, which leads to severe dysfunctions in sarcolemma repair. Cell membrane disruption is a physiological event induced by mechanical stress, such as muscle contraction and stretching. Like many eukaryotic cells, muscle fibers possess a protein machinery ensuring fast resealing of damaged plasma membrane. Members of the annexins A (ANXA) family belong to this protein machinery. ANXA are small soluble proteins, twelve in number in humans, which share the property of binding to membranes exposing negatively-charged phospholipids in the presence of calcium (Ca2+). Many ANXA have been reported to participate in membrane repair of varied cell types and species, including human skeletal muscle cells in which they may play a collective role in protection and repair of the sarcolemma. Here, we discuss the participation of ANXA in membrane repair of healthy skeletal muscle cells and how dysregulation of ANXA expression may impact the clinical severity of muscular dystrophies

Review conclusions by Ernst and Canter regarding spinal manipulation refuted

In the April 2006 issue of the Journal of Royal Society of Medicine, Ernst and Canter authored a review of the most recent systematic reviews on the effectiveness of spinal manipulation for any condition. The authors concluded that, except for back pain, spinal manipulation is not an effective intervention for any condition and, because of potential side effects, cannot be recommended for use at all in clinical practice. Based on a critical appraisal of their review, the authors of this commentary seriously challenge the conclusions by Ernst and Canter, who did not adhere to standard systematic review methodology, thus threatening the validity of their conclusions. There was no systematic assessment of the literature pertaining to the hazards of manipulation, including comparison to other therapies. Hence, their claim that the risks of manipulation outweigh the benefits, and thus spinal manipulation cannot be recommended as treatment for any condition, was not supported by the data analyzed. Their conclusions are misleading and not based on evidence that allow discrediting of a large body of professionals using spinal manipulation

Imaging Membrane Repair in Single Cells Using Correlative Light and Electron Microscopy

Many cells possess the ability to repair plasma membrane disruption in physiological conditions. Growing evidence indicates a correlation between membrane repair and many human diseases. For example, a negative correlation is observed in muscle where failure to reseal sarcolemma may contribute to the development of muscular dystrophies. Instead, a positive correlation is observed in cancer cells where membrane repair may be exacerbated during metastasis. Here we describe a protocol that combines laser technology for membrane damage, immunostaining with gold nanoparticles and imaging by fluorescence microscopy and transmission electron microscopy (TEM), which allows the characterization of the molecular machinery involved in membrane repair. Fluorescence microscopy enables to determine the subcellular localization of candidate proteins in damaged cells while TEM offers high-resolution ultrastructural analysis of the m-disruption site, which enables to decipher the membrane repair mechanism. Here we focus on the study of human skeletal muscle cells, for obvious clinical interest, but this protocol is also suitable for other cell types

Trafficking of Annexins during Membrane Repair in Human Skeletal Muscle Cells

Defects in membrane repair contribute to the development of muscular dystrophies, such as Miyoshi muscular dystrophy 1, limb girdle muscular dystrophy (LGMD), type R2 or R12. Deciphering membrane repair dysfunctions in the development of muscular dystrophies requires precise and detailed knowledge of the membrane repair machinery in healthy human skeletal muscle cells. Using correlative light and electron microscopy (CLEM), we studied the trafficking of four members of the annexin (ANX) family, in myotubes damaged by laser ablation. Our data support a model in which ANXA4 and ANXA6 are recruited to the disruption site by propagating as a wave-like motion along the sarcolemma. They may act in membrane resealing by proceeding to sarcolemma remodeling. On the other hand, ANXA1 and A2 exhibit a progressive cytoplasmic recruitment, likely by interacting with intracellular vesicles, in order to form the lipid patch required for membrane resealing. Once the sarcolemma has been resealed, ANXA1 is released from the site of the membrane injury and returns to the cytosol, while ANXA2 remains accumulated close to the wounding site on the cytoplasmic side. On the other side of the repaired sarcolemma are ANXA4 and ANXA6 that face the extracellular milieu, where they are concentrated in a dense structure, the cap subdomain. The proposed model provides a basis for the identification of cellular dysregulations in the membrane repair of dystrophic human muscle cells

Annexin-A6 in Membrane Repair of Human Skeletal Muscle Cell: A Role in the Cap Subdomain

Defects in membrane repair contribute to the development of some muscular dystrophies, highlighting the importance to decipher the membrane repair mechanisms in human skeletal muscle. In murine myofibers, the formation of a cap subdomain composed notably by annexins (Anx) is critical for membrane repair. We applied membrane damage by laser ablation to human skeletal muscle cells and assessed the behavior of annexin-A6 (AnxA6) tagged with GFP by correlative light and electron microscopy (CLEM). We show that AnxA6 was recruited to the site of membrane injury within a few seconds after membrane injury. In addition, we show that the deficiency in AnxA6 compromises human sarcolemma repair, demonstrating the crucial role played by AnxA6 in this process. An AnxA6-containing cap-subdomain was formed in damaged human myotubes in about one minute. Through transmission electron microscopy (TEM), we observed that extension of the sarcolemma occurred during membrane resealing, which participated in forming a dense lipid structure in order to plug the hole. By properties of membrane folding and curvature, AnxA6 helped in the formation of this tight structure. The compaction of intracellular membranes—which are used for membrane resealing and engulfed in extensions of the sarcolemma—may also facilitate elimination of the excess of lipid and protein material once cell membrane has been repaired. These data reinforce the role played by AnxA6 and the cap subdomain in membrane repair of skeletal muscle cells

Régulation du gène vitellogénine chez la truite arc-en-ciel (études moléculaire et fonctionnelle de l'interaction entre le récepteur aux oestrogènes et des élèments de réponse aux oestrogènes)

Le récepteur aux oestrogènes (ER) contrôle l'expression d'un ensemble de gènes impliqués dans la croissance et la différenciation cellulaire ou les fonctions reproductrices. La régulation des gènes cibles est reélisée par la fixation du récepteur a une séquence sur l'ADN, appelée Element de Réponse aux Oestrogènes (ERE). L'ER présente un maximum d'affinité pour la séquence ERE consensus : AGGTCAnnnTGACCT. Malgré le caractère présumé optimal de cette séquence, elle n'est pas la plus fréquemment observée dans les gènes oestrogènes-dépendants. La forte affinité de l'interaction ER/ERE constitue-t-elle donc un gage d'activiation maximale et de régulation optimale ? Le gène vitellogénine de la truite arc-en-ciel a été étudié comme modèle, son promoteur ne présentant aucune ERE pour consensus. Par reconstitution du mécanisme transcriptionnel chez la levure, nous avons mis en évidence une seule séquence ERE pour ce gène (ERErt:GGGGCAnnnTAACCT). Nous avons observé que, in vivo, le récepteur aux oestrogènes de la truite arc-en-ciel (rtER) présente une dépendance partielle à l'oestradiol pour un gène rapporteur contenant dans son promoteur l'ERE consensus, alors que la dépendance est totale en présence de l'ERErt. Parallèlement, les paramètres thermodynamiques, cinétiques et dynamiques de l'interaction spécifique ER/EREcs et ER/ERErt, mesures in vitro, par spectroscopie de fluoresecnece, ont démontré la contribution des interactions électrostatiques dans ces deux complexes (plus marquées pour le complexe ER/ERErt) mais aussi l'effet de complexation sur la dynamique de la protéine (fluctuations moindres pour le complexe ER/ERErt).LORIENT-BU (561212106) / SudocSudocFranceF

Efficient strategy to increase the surface functionalization of core-shell superparamagnetic nanoparticles using dendron grafting

Core-shell c-Fe2O3/polymer 300 nm superparamagnetic nanoparticles, grafted by fluorescent dendrons using a convergent approach, showed an increase in their surface functionalization compared to grafting using a linear analogue

Review: Annexin-A5 and cell membrane repair

Annexins are soluble proteins that bind to biological membranes containing negatively charged phospholipids, principally phosphatidylserine, in a Ca2+-dependent manner. Annexin-A5 (AnxA5), the smallest member of the annexin family, presents unique properties of membrane binding and self-assembly into ordered two-dimensional (2D) arrays on membrane surfaces. We have previously reported that AnxA5 plays a central role in the machinery of membrane repair by enabling rapid resealing of plasma membrane disruption in murine perivascular cells. AnxA5 promotes membrane repair via the formation of a protective 2D bandage at membrane damaged site. Here, we review current knowledge on cell membrane repair and present recent findings on the role of AnxA5 in membrane resealing of human trophoblasts. (C) 2015 Published by IFPA and Elsevier Ltd

The unprecedented membrane deformation of the human nuclear envelope, in a magnetic field, indicates formation of nuclear membrane invaginations

International audienceHuman nuclear membrane (hNM) invaginations are thought to be crucial in fusion, fission and remodeling of cells and present in many human diseases. There is however little knowledge, if any, about their lipid composition and dynamics. We therefore isolated nuclear envelope lipids from human kidney cells, analyzed their composition and determined the membrane dynamics after resuspension in buffer. The hNM lipid extract was composed of a complex mixture of phospholipids, with high amounts of phosphatidylcholines, phosphatidylinositols (PI) and cholesterol. hNM dynamics was determined by solid-state NMR and revealed that the lamellar gel-to-fluid phase transition occurs below 0 °C, reflecting the presence of elevated amounts of unsaturated fatty acid chains. Fluidity was higher than the plasma membrane, illustrating the dual action of Cholesterol (ordering) and PI lipids (disordering). The most striking result was the large magnetic field-induced membrane deformation allowing to determine the membrane bending elasticity, a property related to hydrodynamics of cells and organelles. Human Nuclear Lipid Membranes were at least two orders of magnitude more elastic than the classical plasma membrane suggesting a physical explanation for the formation of nuclear membrane invaginations

Biochimica et biophysica acta

Annexin-A5 (AnxA5) is the smallest member of the annexins, a group of soluble proteins that bind to membranes containing negatively-charged phospholipids, principally phosphatidylserine, in a Ca2+-dependent manner. AnxA5 presents unique properties of binding and self-assembling on membrane surfaces, forming highly ordered two-dimensional (2D) arrays. We showed previously that AnxA5 plays a central role in the machinery of cell membrane repair of murine perivascular cells, promoting the resealing of membrane damages via the formation of 2D protein arrays at membrane disrupted sites and preventing the extension of membrane ruptures. As the placenta is one of the richest source of AnxA5 in humans, we investigated whether AnxA5 was involved in membrane repair in this organ. We addressed this question at the level of human trophoblasts, either mononucleated cytotrophoblasts or multinucleated syncytiotrophoblasts, in choriocarcinoma cells and primary trophoblasts. Using established procedure of laser irradiation and fluorescence microscopy, we observed that both human cytotrophoblasts and syncytiotrophoblasts repair efficiently a mum2-size disruption. Compared to wild-type cells, AnxA5-deficient trophoblasts exhibit severe defect of membrane repair. Through specifically binding to the disrupted site as early as a few seconds after membrane wounding, AnxA5 promotes membrane resealing of injured human trophoblasts. In addition, we observed that a large membrane area containing the disrupted site was released in the extracellular milieu. We propose mechanisms ensuring membrane resealing and subsequent lesion removal in human trophoblasts. This article is part of a Special Issue entitled: 13th European Symposium on Calcium