Alix is a substrate of the Ozz-E3 ligase and modulates actin remodeling and plasma membrane stability in skeletal muscle

Alix/AIP1 is a multifunctional adaptor protein that participates in basic cellular processes, such as membrane trafficking and actin cytoskeleton assembly, by binding selectively to a variety of partner proteins. However, the mechanisms regulating Alix turnover, subcellular distribution and mode of action in muscle cells are unknown. We now report that Alix is expressed in skeletal muscle throughout myogenic differentiation. In the differentiated myotubes, specific pool of Alix colocalizes in discrete subcellular regions with Ozz, the substrate-binding component of the muscle-specific ubiquitin ligase complex, Ozz-E3. We have shown previously that Ozz-E3 plays an active role in skeletal muscle differentiation and regeneration, by ubiquitinating the sarcolemmal-associated pool of ?-catenin and the sarcomeric embryonic myosin heavy chain (MyHCemb) (Nastasi et al., 2004; Campos et al., 2010). Here, we demonstrate that Ozz interacts with and promotes the ubiquitination of Alix. This modification appears to regulate the levels of Alix in specific sub-compartments, such as the multivesicular bodies (MVBs) and the compartment containing the actin polymerisation factor cortactin. These findings suggest that the Ozz-E3 ligase regulates Alix at sites in which the endocytic machinery connects to the actin cytoskeleton. In line with this observation, we show that knockdown of Alix' expression affects the level and distribution of filamentous (F)-actin and leads to plasma membrane instability, in myotubes. These alterations result in aberrant cell morphology and exosome secretion, impaired formation of sarcolemmal protrusions, and defective cell motility. Our results point to a crucial role of Alix in the process of muscle cell migration and exosome biogenesis, by regulating the dynamics of the plasma membrane and the actin cytoskeleton.