Endocytic recycling proteins EHD1 and EHD2 interact with fer-1-like-5 (Fer1L5) and mediate myoblast fusion

The mammalian ferlins are calcium-sensing, C2 domain-containing proteins involved in vesicle trafficking. Myoferlin and dysferlin regulate myoblast fusion and muscle membrane resealing, respectively. Correspondingly, myoferlin is most highly expressed in singly nucleated myoblasts, whereas dysferlin expression is increased in mature, multinucleated myotubes. Myoferlin also mediates endocytic recycling and participates in trafficking the insulin-like growth factor receptor. We have now characterized a novel member of the ferlin family, Fer1L5, because of its high homology to dysferlin and myoferlin. We found that Fer1L5 protein is expressed in small myotubes that contain only two to four nuclei. We also found that Fer1L5 protein binds directly to the endocytic recycling proteins EHD1 and EHD2 and that the second C2 domain in Fer1L5 mediates this interaction. Reduction of EHD1 and/or EHD2 inhibits myoblast fusion, and EHD2 is required for normal translocation of Fer1L5 to the plasma membrane. The characterization of Fer1L5 and its interaction with EHD1 and EHD2 underscores the complex requirement of ferlin proteins and mediators of endocytic recycling for membrane trafficking events during myotube formation.     

Modular Dispensability of Dysferlin C2 Domains Reveals Rational Design for Mini-dysferlin Molecules

Dysferlin is a large transmembrane protein composed of a C-terminal transmembrane domain, two DysF domains, and seven C2 domains that mediate lipid- and protein-binding interactions. Recessive loss-of-function mutations in dysferlin lead to muscular dystrophies, for which no treatment is currently available. The large size of dysferlin precludes its encapsulation into an adeno-associated virus (AAV), the vector of choice for gene delivery to muscle. To design mini-dysferlin molecules suitable for AAV-mediated gene transfer, we tested internally truncated dysferlin constructs, each lacking one of the seven C2 domains, for their ability to localize to the plasma membrane and to repair laser-induced plasmalemmal wounds in dysferlin-deficient human myoblasts. We demonstrate that the dysferlin C2B, C2C, C2D, and C2E domains are dispensable for correct plasmalemmal localization. Furthermore, we show that the C2B, C2C, and C2E domains and, to a lesser extent, the C2D domain are dispensable for dysferlin membrane repair function. On the basis of these results, we designed small dysferlin molecules that can localize to the plasma membrane and reseal laser-induced plasmalemmal injuries and that are small enough to be incorporated into AAV. These results lay the groundwork for AAV-mediated gene therapy experiments in dysferlin-deficient mouse models.     

Proteasomal inhibition restores biological function of mis-sense mutated dysferlin in patient-derived muscle cells

Dysferlin is a transmembrane protein implicated in surface membrane repair of muscle cells. Mutations in dysferlin cause the progressive muscular dystrophies Miyoshi myopathy, limb girdle muscular dystrophy 2B, and distal anterior compartment myopathy. Dysferlinopathies are inherited in an autosomal recessive manner, and many patients with this disease harbor mis-sense mutations in at least one of their two pathogenic DYSF alleles. These patients have significantly reduced or absent dysferlin levels in skeletal muscle, suggesting that dysferlin encoded by mis-sense alleles is rapidly degraded by the cellular quality control system. We reasoned that mis-sense mutated dysferlin, if salvaged from degradation, might be biologically functional. We used a dysferlin-deficient human myoblast culture harboring the common R555W mis-sense allele and a DYSF-null allele, as well as control human myoblast cultures harboring either two wild-type or two null alleles. We measured dysferlin protein and mRNA levels, resealing kinetics of laser-induced plasmalemmal wounds, myotube formation, and cellular viability after treatment of the human myoblast cultures with the proteasome inhibitors lactacystin or bortezomib (Velcade). We show that endogenous R555W mis-sense mutated dysferlin is degraded by the proteasomal system. Inhibition of the proteasome by lactacystin or Velcade increases the levels of R555W mis-sense mutated dysferlin. This salvaged protein is functional as it restores plasma membrane resealing in patient-derived myoblasts and reverses their deficit in myotube formation. Bortezomib and lactacystin did not cause cellular toxicity at the regimen used. Our results raise the possibility that inhibition of the degradation pathway of mis-sense mutated dysferlin could be used as a therapeutic strategy for patients harboring certain dysferlin mis-sense mutations.     

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

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 short-lived (protein half-life ∼4–6 h) and transitory transmembrane protein (plasma membrane half-life ∼3 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.     

Proteomic identification of dysferlin-interacting protein complexes in human vascular endothelium

Dysferlin is a membrane-anchored protein known to facilitate membrane repair in skeletal muscles following mechanical injury. Mutations of dysferlin gene impair sarcolemma integrity, a hallmark of certain forms of muscular dystrophy in patients. Dysferlin contains seven calcium-dependent C2 binding domains, which are required to promote fusion of intracellular membrane vesicles.Emerging evidence reveal the unexpected expression of dysferlin in non-muscle, non-mechanically active tissues, such as endothelial cells, which cast doubts over the belief that ferlin proteins act exclusively as membrane repair proteins. We and others have shown that deficient trafficking of membrane bound proteins in dysferlin-deficient cells, suggesting that dysferlin might mediate trafficking of client proteins. Herein, we describe the intracellular trafficking and movement of GFP-dysferlin positive vesicles in unfixed reconstituted cells using live microscopy. By performing GST pull-down assays followed by mass spectrometry, we identified dysferlin binding protein complexes in human vascular endothelial cells. Together, our data further support the claims that dysferlin not only mediates membrane repair but also trafficking of client proteins, ultimately, help bridging dysferlinopathies to aberrant membrane signaling.     

AhR deficiency impairs expression of LPS-induced inflammatory genes in mice

Mutations in the gene encoding dysferlin cause two distinct muscular dystrophy phenotypes: limb-girdle muscular dystrophy type 2B (LGMD-2B) and Miyoshi myopathy (MM). Dysferlin is a large transmembrane protein involved in myoblast fusion and membrane resealing. Zebrafish represent an ideal animal model to use for studying muscle disease including abnormalities of dysferlin. cDNAs of zebrafish dysferlin were cloned (6.3 kb) and the predicted amino acid sequences, showed 68% similarity to predicted amino acid sequences of mammalian dysferlin. The expression of dysferlin was mainly in skeletal muscle, heart and eye, and the expression could be detected as early as 11 h post fertilization (hpf). Three different antisense oligonucleotide morpholinos were targeted to inhibit translation of this dysferlin mRNA and the morpholino-injected fish showed marked muscle disorganization which could be detected by birefringence assay. Western blot analysis using dysferlin antibodies showed that the expression of dysferlin was reduced in each of the three morphants. Dysferlin expression was shown to be reduced at the myosepta of zebrafish muscle using immunohistochemistry, although the expression of other muscle membrane components, dystrophin, laminin, β-dystroglycan were detected normally. Our data suggest that zebrafish dysferlin expression is involved in stabilizing muscle structures and its downregulation causes muscle disorganization.     

Mutation at a new allele of the dysferlin gene causes Miyoshi myopathy: A case report

Miyoshi myopathy (MM) is a rare autosomal recessive disorder caused by dysferlin (DYSF) gene mutation. Miyoshi myopathy-inducing mutation sites in the DYSF gene have been discovered worldwide. In the present study, a patient with progressive lower extremity weakness is reported, for which MM was diagnosed according to clinical manifestations, muscle biopsy, and immunohistochemistry. In addition, the DYSF gene of the patient and his parents was sequenced and analyzed and two heterozygous mutations of the DYSF gene (c.4756C> T and c.5316dupC) were discovered. The first mutation correlated with MM while the second was a new mutation. The patient was diagnosed with a compound heterozygous mutation. The mutation site is a new member of pathogenic MM gene mutations.     

Proteomic investigation of the molecular pathophysiology of dysferlinopathy

Mutations in dysferlin gene cause several types of muscular dystrophy in humans, including the limb-girdle muscular dystrophy type 2B and the distal muscular dystrophy of Miyoshi. The dysferlin gene product is a membrane-associated protein belonging to the ferlins family of proteins. The function of the dysferlin protein and the cause of deterioration and regression of muscle fibres in its absence, are incompletely known. A functional clue may be the presence of six hydrophilic domains, C2, that bind calcium and mediate the interaction of proteins with cellular membranes. Dysferlin seems to be involved in the membrane fusion or repair. Molecular diagnosis of dysferlinopathies is now possible and the types of gene alterations that have been characterized so far include missense mutations, deletions and insertions.     

Dysferlin is essential for endocytosis in the sea star oocyte

Dysferlin is a calcium-binding transmembrane protein involved in membrane fusion and membrane repair. In humans, mutations in the dysferlin gene are associated with muscular dystrophy. In this study, we isolated plasma membrane-enriched fractions from full-grown immature oocytes of the sea star, and identified dysferlin by mass spectrometry analysis. The full-length dysferlin sequence is highly conserved between human and the sea star. We learned that in the sea star Patiria miniata, dysferlin RNA and protein are expressed from oogenesis to gastrulation. Interestingly, the protein is highly enriched in the plasma membrane of oocytes. Injection of a morpholino against dysferlin leads to a decrease of endocytosis in oocytes, and to a developmental arrest during gastrulation. These results suggest that dysferlin is critical for normal endocytosis during oogenesis and for embryogenesis in the sea star and that this animal may be a useful model for studying the relationship of dysferlin structure as it relates to its function.