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.

Annexin-A6 presents two modes of association with phospholipid membranes. A combined QCM-D, AFM and cryo-TEM study.

Annexins are soluble proteins that bind to biological membranes in a Ca(2+)-dependent manner. Annexin-A6 (AnxA6) is unique in the annexin family as it consists of the repeat of two annexin core modules, while all other annexins consist of a single module. AnxA6 has been proposed to participate in various membrane-related processes, including endocytosis and exocytosis, yet the molecular mechanism of association of AnxA6 with biological membranes, especially its ability to aggregate membranes, is still unclear. To address this question, we studied the association of AnxA6 with model phospholipid membranes by combining the techniques of quartz crystal microbalance with dissipation monitoring (QCM-D), (cryo-) transmission electron microscopy (TEM) and atomic force microscopy (AFM). The properties of membrane binding and membrane aggregation of AnxA6 were compared to two reference systems, annexin A5 (AnxA5), which is the annexin prototype, and a chimerical AnxA5-dimer molecule, which is able to aggregate two membranes in a symmetrical manner. We show that AnxA6 presents two modes of association with lipid membranes depending on Ca(2+)-concentration. At low Ca(2+)-concentration ( approximately 60-150microM), AnxA6 binds to membranes via its two coplanar annexin modules and is not able to associate two separate membranes. At high Ca(2+)-concentration ( approximately 2mM), AnxA6 molecules are able to bind two adjacent phospholipid membranes and present a conformation similar to the AnxA6 3D crystallographic structure. Possible biological implications of these novel membrane-binding properties of AnxA6 are discussed.

Membrane-bound 3D structures reveal the intrinsic flexibility of annexin VI.

Several quasi-ordered arrays and three two-dimensional crystal forms of annexin VI were obtained on artificial lipid monolayers. Three-dimensional reconstructions of the crystal forms exhibit marked differences in the orientations of the two lobes, revealing flexibility of the linker between the two lobes of annexin VI. Evidence is presented that the lobes may bind the monolayer in a parallel orientation, or an antiparallel orientation, in which the second lobe is turned away from the monolayer. It is hypothesized that annexin VI may also adopt several conformations in vivo, underlying different functional roles.

The structure of recombinant human annexin VI in crystals and membrane-bound.

The crystal structure of calcium-free recombinant human annexin VI was solved at a resolution of 3.2 A by using the annexin I model for Patterson search and refined to an R-factor of 19.0%. The molecule consists of two similar halves closely resembling annexin I connected by an alpha-helical segment and arranged perpendicular to each other. The calcium and membrane binding sites assigned by structural homology are therefore not located in the same plane. Analysis of the membrane-bound form of annexin VI by electron microscopy shows the two halves of the molecule coplanar with the membrane, but oriented differently to the crystal structure and suggesting a flexible arrangement. Ion channel activity has been found for annexin VI and the half molecules by electrophysiological experiments.