Characterization of the sarcolemma in limb-girdle muscular dystrophy

Limb-girdle muscular dystrophies (LGMD) are a heterogeneous group of slowly progressive muscular dystrophies. Mutations in the dysferlin gene cause LGMD 2B, Miyoshi myopathy (MM) and distal anterior compartment myopathy (DACM) commonly referred to as dysferlinopathies. Dysferlin is a transmembrane protein and its subcellular localization is predominantly allocated to the plasma membrane, but also to developing T-tubules and intracellular vesicles. At the sarcolemma, dysferlin was shown to be crucial for efficient membrane repair, although it has been proposed to have a role in many other cellular functions. LGMD 1C is caused by mutations in the caveolin-3 (cav-3) gene. Cav-3 is the muscle-specific isoform of the caveolin protein family which represent, together with the cavin protein family, the major structural and functional components of caveolae. Caveolae are plasma membrane invaginations and belong to a specialized type of membrane microdomains referred to as lipid rafts. Cav-3 is known to regulate dysferlin localization and rate of endocytosis at the plasma membrane. Recently, recessive mutations in the anoctamin gene could also be related to LGMDs. As seen for dysferlinopathies, ANO5 mutations can lead to a LGMD or MM clinical phenotype. Anoctamin-5 is a member of the anoctamin protein family and is thought to function as calcium- activated chloride channel. In order to reveal the molecular mechanisms underlying LGMD and to investigate the putative interactions of dysferlin, cav-3, and ano5, experiments on primary skeletal muscle cell lines with disease-related mutations in DYSF, CAV3, and ANO5 have been analyzed in this study. Immunolabeling studies revealed that dysferlin and cav-3 are partially colocalized in vesicular structures at the plasma membrane of human primary myotubes. Biochemical purification of lipid rafts from differentiated myotubes showed that dysferlin is associated with lipid rafts linked to the actin- cytoskeleton. Transmission electron microscopy analysis of myotubes derived from skeletal muscle of LGMD patients revealed alterations of caveolae abundance at the plasma membrane correlating with the disease-causing mutations. Ultrastructural studies on the subcellular localization of dysferlin showed plasma membrane, but also intracellular localization in cytosolic vesicles. Immunopurification of intracellular dysferlin-containing vesicles revealed the presence of cav-3 and other known dysferlin interacting partners. These vesicles were found to contain approximately 500 proteins detected by proteomics. Results from this study show that caveolae play a crucial role in the context of LGMD. Especially for LGMD 2B, caveolae abundance at the plasma membrane in human primary myotubes can be correlated with LGMD-causing mutations. Furthermore, we propose that dysferlin function during membrane repair is mediated by actin-linked lipid raft formation. Our results confirm that dysferlin is localized in cytosolic vesicles, which are involved in multiple cellular processes such as vesicle transport, endo- and exocytosis, cell-adherence, and lipid raft dynamics. Taken together, the close association of dysferlin and cav-3, especially in context to caveolae, could be demonstrated in this study.

Author dc:creator

• Kunz, Séverine

• limb-girdle muscular dystrophy
• dysferlin
• caveolin-3
• caveolae
• lipid rafts
• anoctamin-5
• electron microscopy

Licence dc:rights.uri

• http://www.fu-berlin.de/sites/refubium/rechtliches/Nutzungsbedingungen

Language dc:language

eng

Identifier URI

http://dx.doi.org/10.17169/refubium-9263

OAI record oai:identifier

https://refubium.fu-berlin.de/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Arefubium.fu-berlin.de%3Afub188%2F5064