Abstract
Dysferlin is a protein of the ferlin family predominantly expressed in heart and skeletal muscle. The 230kDa type II transmembrane protein contains seven C2 domains and localizes to the T-tubule system and the plasma membrane in skeletal muscle. Mutations in the dysferlin gene lead to LGMD2B and Miyoshi Myopathy. Dysferlin is involved in skeletal muscle membrane repair, regeneration and biogenesis of the T-tubule system but the precise molecular function of dysferlin is so far unknown. Here we show that full-length dysferlin is able to bind phospholipids, especially PI (4,5) P<sub>2</sub> (PIP2), an important phospholipid within the T-tubule membrane. In C2C12 myotubes dysferlin colocalized with PIP2 at the T-tubule system. This T-tubule localization of dysferlin was altered after depletion of PIP2 indicating importance of PIP2 for T-tubule integrity. In non-muscle cells co-expression of dysferlin with a sensor for PIP2 lead to recruitment of PIP2 from the plasma membrane to an intracellular localization and colocalization with dysferlin. Furthermore, co-expression of a constitutively active mutant of Arf6 or the PIP2 kinase with dysferlin induced dysferlin recruitment to PIP2-containing vacuoles. These results indicate PIP2-binding of dysferlin which was corroborated by PIP strip analysis and liposome flotation experiments <i>in vitro</i>. Truncated dysferlins and pathogenic mutations of dysferlin failed to be recruited to PIP2-containing structures and did not bind membranes in liposome flotation experiments indicating that the full-length protein is necessary for PIP2 binding. Taken together, these results suggest that full-length dysferlin is able to bind lipid membranes with preference of the negatively charged phospholipid PIP2. Binding to PIP2 was abolished by truncations and pathogenic mutations of the dysferlin gene which indicates that PIP2-binding may be important in dysferlin-deficient muscle pathology.
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