Reduced Plasma Membrane Expression of Dysferlin Mutants Is Attributed to Accelerated Endocytosis via a Syntaxin-4-associated Pathway

Abstract

Ferlins are an ancient family of C2 domain-containing proteins, with emerging roles in vesicular trafficking and human disease. Dysferlin mutations cause inherited muscular dystrophy, and dysferlin also shows abnormal plasma membrane expression in other forms of muscular dystrophy. We establish dysferlin as a shortlived (protein half-life ϳ4 -6 h) and transitory transmembrane protein (plasma membrane half-life ϳ3 h), with a propensity for rapid endocytosis when mutated, and an association with a syntaxin-4 endocytic route. Dysferlin plasma membrane expression and endocytic rate is regulated by the C2B-FerI-C2C motif, with a critical role identified for C2C. Disruption of C2C dramatically reduces plasma membrane dysferlin (by 2.5-fold), due largely to accelerated endocytosis (by 2.5-fold). These properties of reduced efficiency of plasma membrane expression due to accelerated endocytosis are also a feature of patient missense mutant L344P (within FerI, adjacent to C2C). Importantly, dysferlin mutants that demonstrate accelerated endocytosis also display increased protein lability via endosomal proteolysis, implicating endosomal-mediated proteolytic degradation as a novel basis for dysferlin-deficiency in patients with single missense mutations. Vesicular labeling studies establish that dysferlin mutants rapidly transit from EEA1-positive early endosomes through to dextran-positive lysosomes, co-labeled by syntaxin-4 at multiple stages of endosomal transit. In summary, our studies define a transient biology for dysferlin, relevant to emerging patient therapeutics targeting dysferlin replacement. We introduce accelerated endosomal-directed degradation as a basis for lability of dysferlin missense mutants in dysferlinopathy, and show that dysferlin and syntaxin-4 similarly transit a common endosomal pathway in skeletal muscle cells.

Dysferlin Binds SNAREs (Soluble N-Ethylmaleimide-sensitive Factor (NSF) Attachment Protein Receptors) and Stimulates Membrane Fusion in a Calcium-sensitive Manner

“…However, several studies have reported that deficiencies in dysferlin result in attenuated exocytosis and the accumulation of unfused vesicles at membrane lesions, suggesting a function in membrane fusion events that occur during repair of cell membrane wounds (9,12). In agreement with this, dysferlin has been reported to bind calcium and the cell membrane lipids phosphatidylserine and phosphatidylinositol bisphosphate and to co-localize with the SNARE protein syntaxin 4 (21)(22)(23)(24). Knockdown of dysferlin also results in a reduction in lysosome exocytosis and delayed release of acid sphingomyelinase, in agreement with dysferlin's proposed role in membrane fusion (11,13).…”

“…reported to co-localize with syntaxin 4, a ubiquitously expressed SNARE protein that contributes to lysosomal exocytosis (21). To determine whether dysferlin interacts with syntaxin 4 as well as the cognate SNARE SNAP-23, dysferlin-immunoprecipitated samples from serum-starved differentiated C2C12 cells were probed by Western blot.…”

Growing muscle has different sarcolemmal properties from adult muscle: A proposal with scientific and clinical implications

“…Defects in many of these molecules are now also known to cause muscular dystrophies [52,[54][55][56]. Vesicle-mediated membrane resealing is clearly a dynamic process that involves both dysferlin and a complexity of molecules in order to patch the lesion.…”

“…Membrane resealing is a function conserved by most cells. Resealing via membrane vesicles is mediated by a mechanism resembling Ca 2þ -dependent exocytosis, which involves membrane fusion and key membrane proteins such as synaptotagmins and members of the ferlin family, with dysferlin being predominantly expressed in skeletal muscle [52][53][54].…”

Translational Research and Therapeutic Perspectives in Dysferlinopathies

“…Recent work has shown that dysferlin's plasma membrane localization is transitory; membrane anchorage never exceeds 3 hours, and the protein's half-life is only 5 hours. The syntaxin-4-associated endocytosis pathway plays a critical role in regulating this process (63).…”