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.

Actin and an unconventional myosin motor, TgMyoF, control the organization and dynamics of the endomembrane network in Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that relies on three distinct secretory organelles, the micronemes, rhoptries, and dense granules, for parasite survival and disease pathogenesis. Secretory proteins destined for these organelles are synthesized in the endoplasmic reticulum (ER) and sequentially trafficked through a highly polarized endomembrane network that consists of the Golgi and multiple post-Golgi compartments. Currently, little is known about how the parasite cytoskeleton controls the positioning of the organelles in this pathway, or how vesicular cargo is trafficked between organelles. Here we show that F-actin and an unconventional myosin motor, TgMyoF, control the dynamics and organization of the organelles in the secretory pathway, specifically ER tubule movement, apical positioning of the Golgi and post-Golgi compartments, apical positioning of the rhoptries, and finally, the directed transport of Rab6-positive and Rop1-positive vesicles. Thus, this study identifies TgMyoF and actin as the key cytoskeletal components that organize the endomembrane system in T. gondii.

Differential requirements for cyclase-associated protein (CAP) in actin-dependent processes of Toxoplasma gondii.

Toxoplasma gondii contains a limited subset of actin binding proteins. Here we show that the putative actin regulator cyclase-associated protein (CAP) is present in two different isoforms and its deletion leads to significant defects in some but not all actin dependent processes. We observe defects in cell-cell communication, daughter cell orientation and the juxtanuclear accumulation of actin, but only modest defects in synchronicity of division and no defect in the replication of the apicoplast. 3D electron microscopy reveals that loss of CAP results in a defect in formation of a normal central residual body, but parasites remain connected within the vacuole. This dissociates synchronicity of division and parasite rosetting and reveals that establishment and maintenance of the residual body may be more complex than previously thought. These results highlight the different spatial requirements for F-actin regulation in Toxoplasma which appear to be achieved by partially overlapping functions of actin regulators.

Membrane Repair Assay for Human Skeletal Muscle Cells.

The characterization of the membrane repair machinery in human skeletal muscle has become crucial, since it has been shown that some muscular dystrophies result from a defect of this fundamental physiological process. Deciphering membrane repair mechanism requires the development of methodologies allowing studying the response of skeletal muscle cells to sarcolemma damage and identifying candidate proteins playing a role in the membrane repair machinery. Here, we describe a protocol that is based on the creation of cell membrane disruption by infrared laser irradiation in human myotubes. Membrane disruption and repair are assayed by monitoring the incorporation into myotubes of the membrane probe FM1-43. This methodology has recently enabled us to show that Annexin-A5 is required for membrane repair in human skeletal muscle cells (Carmeille et al., Biochim Biophys Acta 1863:2267-2279, 2016).

Membrane repair of human skeletal muscle cells requires Annexin-A5.

Defect in membrane repair contributes to the development of limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. In healthy skeletal muscle, unraveling membrane repair mechanisms requires to establish an exhaustive list of the components of the resealing machinery. Here we show that human myotubes rendered deficient for Annexin-A5 (AnxA5) suffer from a severe defect in membrane resealing. This defect is rescued by the addition of recombinant AnxA5 while an AnxA5 mutant, which is unable to form 2D protein arrays, has no effect. Using correlative light and electron microscopy, we show that AnxA5 binds to the edges of the torn membrane, as early as a few seconds after sarcolemma injury, where it probably self-assembles into 2D arrays. In addition, we observed that membrane resealing is associated with the presence of a cluster of lipid vesicles at the wounded site. AnxA5 is present at the surface of these vesicles and may thus participate in plugging the cell membrane disruption. Finally, we show that AnxA5 behaves similarly in myotubes from a muscle cell line established from a patient suffering from LGMD2B, a myopathy due to dysferlin mutations, which indicates that trafficking of AnxA5 during sarcolemma damage is independent of the presence of dysferlin.

Annexin-A5 promotes membrane resealing in human trophoblasts.

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 Ca(2+)-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 µm²-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.