Abstract
Dysferlin is a transmembrane C-2 domain-containing protein involved in vesicle trafficking and membrane remodeling in skeletal muscle cells. However, the mechanism by which dysferlin regulates these cellular processes remains unclear. Since actin dynamics is critical for vesicle trafficking and membrane remodeling, we studied the role of dysferlin in Ca2+-induced G-actin incorporation into filaments in four different immortalized myoblast cell lines (DYSF2, DYSF3, AB320, and ER) derived from patients harboring mutations in the dysferlin gene. As compared with immortalized myoblasts obtained from a control subject, dysferlin expression and G-actin incorporation were significantly decreased in myoblasts from dysferlinopathy patients. Stable knockdown of dysferlin with specific shRNA in control myoblasts also significantly reduced G-actin incorporation. The impaired G-actin incorporation was restored by the expression of full-length dysferlin as well as dysferlin N-terminal or C-terminal regions, both of which contain three C2 domains. DYSF3 myoblasts also exhibited altered distribution of annexin A2, a dysferlin partner involved in actin remodeling. However, dysferlin N-terminal and C-terminal regions appeared to not fully restore such annexin A2 mislocation. Then, our results suggest that dysferlin regulates actin remodeling by a mechanism that does to not involve annexin A2.
Highlights
Dysferlin is a transmembrane protein containing seven cytosolic C2 domains, which bind Ca2+ and acidic phospholipids with different affinities [1,2]
Dysferlin and cytoskeletal actin play critical roles at various differentiation stages of skeletal muscle physiology; they are required for vesicle trafficking and exocytosis in myoblasts [6,7,27,46,47] and plasmalemma repair in myofibers [3,4,5]
Annexin A2, and actin remodeling has been observed during plasmalemmal repair
Summary
Dysferlin is a transmembrane protein containing seven cytosolic C2 domains, which bind Ca2+ and acidic phospholipids with different affinities [1,2]. Dysferlin plays a role in other cellular processes in the skeletal muscle tissue including cytokine secretion and membrane receptor recycling in myoblasts [6,7] as well as biogenesis, remodeling, and maintenance of the T-tubule system [8,9]. Skeletal muscle cells derived from dysferlinopathy patients [19] and dysferlin-deficient mice [3] display a defective Ca2+-dependent plasmalemma repair. Skeletal muscle cells express two cytoskeletal actin isoforms, β-actin and γ-actin, that localize in sub-plasmalemmal regions [28,29]. Skeletal muscle-specific ablation of β-actin or γ-actin causes a progressive myopathy, characterized by myofiber degeneration/regeneration and muscle weakness [29,30], emphasizing the critical role of the cytoskeletal actin network in the function of skeletal muscle cells
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