Ferlin Family Research Articles

Phylogenetic analysis of ferlin genes reveals ancient eukaryotic origins

BackgroundThe ferlin gene family possesses a rare and identifying feature consisting of multiple tandem C2 domains and a C-terminal transmembrane domain. Much currently remains unknown about the fundamental function of this gene family, however, mutations in its two most well-characterised members, dysferlin and otoferlin, have been implicated in human disease. The availability of genome sequences from a wide range of species makes it possible to explore the evolution of the ferlin family, providing contextual insight into characteristic features that define the ferlin gene family in its present form in humans.ResultsFerlin genes were detected from all species of representative phyla, with two ferlin subgroups partitioned within the ferlin phylogenetic tree based on the presence or absence of a DysF domain. Invertebrates generally possessed two ferlin genes (one with DysF and one without), with six ferlin genes in most vertebrates (three DysF, three non-DysF). Expansion of the ferlin gene family is evident between the divergence of lamprey (jawless vertebrates) and shark (cartilaginous fish). Common to almost all ferlins is an N-terminal C2-FerI-C2 sandwich, a FerB motif, and two C-terminal C2 domains (C2E and C2F) adjacent to the transmembrane domain. Preservation of these structural elements throughout eukaryotic evolution suggests a fundamental role of these motifs for ferlin function. In contrast, DysF, C2DE, and FerA are optional, giving rise to subtle differences in domain topologies of ferlin genes. Despite conservation of multiple C2 domains in all ferlins, the C-terminal C2 domains (C2E and C2F) displayed higher sequence conservation and greater conservation of putative calcium binding residues across paralogs and orthologs. Interestingly, the two most studied non-mammalian ferlins (Fer-1 and Misfire) in model organisms C. elegans and D. melanogaster, present as outgroups in the phylogenetic analysis, with results suggesting reproduction-related divergence and specialization of species-specific functions within their genus.ConclusionsOur phylogenetic studies provide evolutionary insight into the ferlin gene family. We highlight the existence of ferlin-like proteins throughout eukaryotic evolution, from unicellular phytoplankton and apicomplexan parasites, through to humans. We characterise the preservation of ferlin structural motifs, not only of C2 domains, but also the more poorly characterised ferlin-specific motifs representing the DysF, FerA and FerB domains. Our data suggest an ancient role of ferlin proteins, with lessons from vertebrate biology and human disease suggesting a role relating to vesicle fusion and plasma membrane specialization.

Myoferlin regulation by NFAT in muscle injury, regeneration and repair

Ferlin proteins mediate membrane-fusion events in response to Ca(2+). Myoferlin, a member of the ferlin family, is required for normal muscle development, during which it mediates myoblast fusion. We isolated both damaged and intact myofibers from a mouse model of muscular dystrophy using laser-capture microdissection and found that the levels of myoferlin mRNA and protein were increased in damaged myofibers. To better define the components of the muscle-injury response, we identified a discreet 1543-bp fragment of the myoferlin promoter, containing multiple NFAT-binding sites, and found that this was sufficient to drive high-level myoferlin expression in cells and in vivo. This promoter recapitulated normal myoferlin expression in that it was downregulated in healthy myofibers and was upregulated in response to myofiber damage. Transgenic mice expressing GFP under the control of the myoferlin promoter were generated and GFP expression in this model was used to track muscle damage in vivo after muscle injury and in muscle disease. Myoferlin modulates the response to muscle injury through its activity in both myoblasts and mature myofibers.

EHD1 and EHD2 interact with Fer1L5 to mediate myoblast fusion

Myoferlin and dysferlin regulate myoblast fusion and muscle membrane resealing, respectively. Correspondingly, myoferlin is most highly expressed in singly nucleated myoblasts while dysferlin expression is highest in mature, multinucleated myotubes. The mammalian ferlins are calcium‐sensing, C2 domain‐containing proteins involved in vesicle trafficking. Myoferlin mediates endocytic recycling and participates in trafficking the insulin like growth factor receptor. We 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 abundantly expressed in nascent myotubes containing only 2–4 nuclei. We also found that fer1l5 protein binds directly to the endocytic recycling proteins, EHD1 and EHD2, and that this interaction is mediated by the second C2 domain in fer1l5. Loss of EHD1 and/or EHD2 inhibits myoblast fusion, and EHD2 is required for normal translocation of fer1L5 to the plasma membrane. Abolishing the nucleotide binding ability of the EHD2 ATPase domain recapitulates the fer1L5 translocation defect. 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.

A new role for Dysferlin in endothelial cell adhesion and neo‐vascularization

Dysferlin is a member of the Ferlin family of proteins known to regulate plasma membrane integrity and are linked to muscular dystrophy. However, the peripheral roles of Dysferlin in non‐muscle cells remain virtually unknown. By performing proteomic analysis of caveolae/lipid rafts, we reported that endothelial cells (EC) express Myoferlin and that it mediates plasma membrane translocation of vascular endothelial growth factor receptor‐2 (VEGFR‐2). Herein, we show by Northern and Western blot as well as by immunohistochemistry that EC and intact blood vessels of rodent and human origin all express Dysferlin but that its presence is not required for VEGFR‐2 signaling. Instead, loss of Dysferlin in EC results in deficient adhesion, detachment and growth arrest in sub‐confluent cells whereas confluent EC monolayers are remarkably resistant to cell detachment. In Dysferlin‐null mice, delivery of VEGF, an EC‐specific angiogenic agonist, results in blunted neo‐angiogenesis compared to WT mice. Mechanistically, loss of Dysferlin causes poly‐ubiquitination and proteasomal degradation of platelet endothelial cellular adhesion molecule‐1 (PECAM‐1/CD31). Our discovery that Dysferlin is a regulator of EC adhesion and neo‐vascularization broadens the functional scope of this family of proteins and suggest that specialization exist between the different Ferlins and their respective membrane‐based cargo.

Placental Dysferlin Expression is Reduced in Severe Preeclampsia

Dysferlin (DYSF) and myoferlin (MYOF), members of the ferlin family of membrane proteins, are co-expressed in human placental syncytiotrophoblast (STB). Although the role of these ferlin proteins in the placenta has yet to be established, it has been suggested that DYSF and MYOF may contribute to the stability of the apical STB plasma membrane. The release of STB-derived cellular debris increases in the setting of preeclampsia (PE), suggesting relative destabilization of the hemochorial interface. To test whether PE was associated with alterations in placental expression of DYSF and/or MYOF, a cross-sectional study was performed using specimens of villous placenta collected form women with severe PE ( n = 10) and normotensive controls ( n = 10). DYSF and MYOF expression were examined using quantitative real–time RT-PCR, immunoblotting, and immunofluorescence labeling of tissue specimens. Placental DYSF expression was 57% lower at the mRNA level ( p = 0.03) and 38% lower at the protein level ( p = 0.026) in severe PE as compared to normotensive subjects. There were no differences in placental MYOF protein or mRNA expression between these groups. No appreciable changes in the distribution of DYSF or MYOF within placental villi was observed in PE relative to control specimens. We conclude that DYSF expression is reduced in severe PE relative to gestational age-matched controls. As DYSF has a role in membrane repair, these data suggest a role for DYSF in the stability of the apical STB plasma membrane and may account, at least in part, for the increased shedding of microparticles from this membrane in PE.

Myoferlin is critical for endocytosis in endothelial cells

Myoferlin is a member of the ferlin family of proteins that promotes endomembrane fusion with the plasma membrane in muscle cells and endothelial cells. In addition, myoferlin is necessary for the surface expression of vascular endothelial growth factor receptor 2 through the formation of a protein complex with dynamin-2 (Dyn-2). Since Dyn-2 is necessary for the fission of endocytic vesicles from the plasma membrane, we tested the hypothesis that myoferlin may regulates aspects of receptor-dependent endocytosis. Here we show that myoferlin gene silencing decreases both clathrin and caveolae/raft-dependent endocytosis, whereas ectopic myoferlin expression in COS-7 cells increases endocytosis by up to 125%. Interestingly, we have observed that inhibition of Dyn-2 activity or caveolin-1 (Cav-1) expression impairs endocytosis as well as membrane resealing after injury, indicating that Dyn-2 and Cav-1 also participate in both membrane fission and fusion processes. Mechanistically, myoferlin partially colocalizes with Dyn-2 and Cav-1 and forms a protein complex with Cav-1 solubilized from tissue extracts. Together, these data describe a new role for myoferlin in receptor-dependent endocytosis and an overlapping role for myoferlin-Dyn-2-Cav-1 protein complexes in membrane fusion and fission events.

While Dysferlin and Myoferlin Are Coexpressed in the Human Placenta, Only Dysferlin Expression Is Responsive to Trophoblast Fusion in Model Systems1

The syncytiotrophoblast is a specialized epithelium derived from mononuclear cytotrophoblasts that fuse to form this extensive syncytium. Dysferlin is expressed primarily in the apical plasma membrane of the syncytiotrophoblast in the human placenta. Here, we document the presence of another member of the ferlin family, myoferlin, in the placenta and show that it too is expressed primarily in the syncytiotrophoblast. Additionally, we examined the trophoblastic cell lines BeWo, JAR, and JEG-3 for the expression of dysferlin and myoferlin and determined the extent to which their expression was modulated by cell-cell fusion. In trophoblastic cells, there was a positive correlation between cell fusion and increased dysferlin expression but not myoferlin expression. Regarding expression, these trophoblastic cell lines recapitulate the distribution of dysferlin in mononuclear cytotrophoblasts and the syncytiotrophoblast in vivo.

Solution Structure of the Inner DysF Domain of Myoferlin and Implications for Limb Girdle Muscular Dystrophy Type 2B

Mutations in the protein dysferlin, a member of the ferlin family, lead to limb girdle muscular dystrophy type 2B and Myoshi myopathy. The ferlins are large proteins characterised by multiple C2 domains and a single C-terminal membrane-spanning helix. However, there is sequence conservation in some of the ferlin family in regions outside the C2 domains. In one annotation of the domain structure of these proteins, an unusual internal duplication event has been noted where a putative domain is inserted in between the N- and C-terminal parts of a homologous domain. This domain is known as the DysF domain. Here, we present the solution structure of the inner DysF domain of the dysferlin paralogue myoferlin, which has a unique fold held together by stacking of arginine and tryptophans, mutations that lead to clinical disease in dysferlin.

Dysferlin Homozygous Mutation G1418D Causes Limb-Girdle Type 2B in a Mexican Family

Dysferlin protein (DYSF) is a ferlin family member found in sarcolemma and is involved in membrane repair, muscle differentiation, membrane fusion, etc. The deficiency of DYSF due to mutations is associated with different pathologic phenotypes including the autosomal recessive limb-girdle type 2B phenotype (LGMD2B), a distal anterior compartment myopathy (DMAT), and the Miyoshi myopathy (MM). In this study, we determined a missense mutation c.4253G>A on the DYSF gene in a Mexican family from an endogamic population. This mutation was assumed to be the cause of dystrophy because only homozygous individuals of the family manifest a clinical phenotype. Structural implications caused by G/D substitution at amino acid position 1418 are discussed in terms of potential importance of the dysferlin neighboring sequence.

Myoferlin Regulates Vascular Endothelial Growth Factor Receptor-2 Stability and Function

Myoferlin and dysferlin are members of the ferlin family of membrane proteins. Recent studies have shown that mutation or genetic disruption of myoferlin or dysferlin promotes muscular dystrophy-related phenotypes in mice, which are the result of impaired plasma membrane integrity. However, no biological functions have been ascribed to myoferlin in non-muscle tissues. Herein, using a proteomic analysis of endothelial cell (EC) caveolae/lipid raft microdomains we identified myoferlin in these domains and show that myoferlin is highly expressed in ECs and vascular tissues. The loss of myoferlin results in lack of proliferation, migration, and nitric oxide (NO) release in response to vascular endothelial growth factor (VEGF). Western blotting and surface biotinylation experiments show that loss of myoferlin reduces the expression level and autophosphorylation of VEGF receptor-2 (VEGFR-2) in native ECs. In a reconstituted cell system, transfection of myoferlin increases VEGFR-2 membrane expression and autophosphorylation in response to VEGF. In vivo, VEGFR-2 levels and VEGF-induced permeability are impaired in myoferlin-deficient mice. Mechanistically, myoferlin forms a complex with dynamin-2 and VEGFR-2, which prevents CBL-dependent VEGFR-2 polyubiquitination and proteasomal degradation. These data are the first to report novel biological activities for myoferlin and reveal the role of membrane integrity to VEGF signaling.

Mutation impact on dysferlin inferred from database analysis and computer-based structural predictions

Dysferlin is a large sarcolemmal protein implicated in the repair of surface membrane tears in muscle cells. Mutations in dysferlin result in limb girdle muscular dystrophy type 2B and Miyoshi myopathy. Using a cDNA based approach we identified eight new pathogenic dysferlin alleles. To better understand how missense mutations could lead to reduced or absent dysferlin expression levels, we mapped missense mutations from our own and from published databases ( n = 55) to the secondary protein structure of dysferlin, deduced by computerized structural prediction tools. We found the protein to be very sensitive to the alteration of residues that were predicted to be buried inside the protein structure. We identified seven putative C2 domains, one more than commonly reported, of both type I and type II topology in dysferlin. Missense mutations often affected those structures as well as residues that were highly conserved between members of the ferlin family. Thus, alteration of structurally important residues in dysferlin could lead to improper folding and degradation of the mutant protein.

FER-1 regulates Ca2+-mediated membrane fusion during C. elegans spermatogenesis

FER-1 is required for fusion of specialized vesicles, called membranous organelles, with the sperm plasma membrane during Caenorhabditis elegans spermiogenesis. To investigate its role in membranous organelle fusion, we examined ten fer-1 mutations and found that they all cause the same defect in membrane fusion. FER-1 and the ferlin protein family are membrane proteins with four to seven C2 domains. These domains commonly mediate Ca2+ -dependent lipid-processing events. Most of the fer-1 mutations fall within these C2 domains, showing that they have distinct, non-redundant functions. We found that membranous organelle fusion requires intracellular Ca2+ and that C2 domain mutations alter Ca2+ sensitivity. This suggests that the C2 domains are involved in Ca2+ sensing and further supports their independent function. Using two immunological approaches we found three FER-1 isoforms, two of which might arise from FER-1 by proteolysis. By both light and electron microscopy, these FER-1 proteins were found to be localized to membranous organelle membranes. Dysferlin, a human homologue of FER-1 involved in muscular dystrophy, is required for vesicle fusion during Ca2+ -induced muscle membrane repair. Our results suggest that the ferlin family members share a conserved mechanism to regulate cell-type-specific membrane fusion.

S1-04-04 Mutations in tau gene lead to neurodegeneration by making tau a more favorable substrate for abnormal hyperphosphorylation

Myoferlin and dysferlin are closely related members of the ferlin family of Ca2 +-regulated vesicle fusion proteins. Dysferlin is proposed to play a role in Ca2 +-triggered vesicle fusion during membrane repair. Myoferlin regulates endocytosis, recycling of growth factor receptors and adhesion proteins, and is linked to the metastatic potential of cancer cells. Our previous studies establish that dysferlin is cleaved by calpains during membrane injury, with the cleavage motif encoded by alternately-spliced exon 40a. Herein we describe the cleavage of myoferlin, yielding a membrane-associated dual C2 domain ‘mini-myoferlin’. Myoferlin bears two enzymatic cleavage sites: a canonical cleavage site encoded by exon 38 within the C2DE domain; and a second cleavage site in the linker adjacent to C2DE, encoded by alternately-spliced exon 38a, homologous to dysferlin exon 40a. Both myoferlin cleavage sites, when introduced into dysferlin, can functionally substitute for exon 40a to confer Ca2 +-triggered calpain cleavage in response to membrane injury. However, enzymatic cleavage of myoferlin is complex, showing both constitutive or Ca2 +-enhanced cleavage in different cell lines, that is not solely dependent on calpains-1 or -2. The functional impact of myoferlin cleavage was explored through signalling protein phospho-protein arrays revealing specific activation of ERK1/2 by ectopic expression of cleavable myoferlin, but not an uncleavable isoform. In summary, we molecularly define two enzymatic cleavage sites within myoferlin and demonstrate ‘mini-myoferlin’ can be detected in human breast cancer tumour samples and cell lines. These data further illustrate that enzymatic cleavage of ferlins is an evolutionarily preserved mechanism to release functionally specialized mini-modules.

Myoferlin, a candidate gene and potential modifier of muscular dystrophy.

Dysferlin, the gene product of the limb girdle muscular dystrophy (LGMD) 2B locus, encodes a membrane-associated protein with homology to Caenorhabditis elegans fer-1. Humans with mutations in dysferlin ( DYSF ) develop muscle weakness that affects both proximal and distal muscles. Strikingly, the phenotype in LGMD 2B patients is highly variable, but the type of mutation in DYSF cannot explain this phenotypic variability. Through electronic database searching, we identified a protein highly homologous to dysferlin that we have named myoferlin. Myoferlin mRNA was highly expressed in cardiac muscle and to a lesser degree in skeletal muscle. However, antibodies raised to myoferlin showed abundant expression of myoferlin in both cardiac and skeletal muscle. Within the cell, myoferlin was associated with the plasma membrane but, unlike dysferlin, myoferlin was also associated with the nuclear membrane. Ferlin family members contain C2 domains, and these domains play a role in calcium-mediated membrane fusion events. To investigate this, we studied the expression of myoferlin in the mdx mouse, which lacks dystrophin and whose muscles undergo repeated rounds of degeneration and regeneration. We found upregulation of myoferlin at the membrane in mdx skeletal muscle. Thus, myoferlin ( MYOF ) is a candidate gene for muscular dystrophy and cardiomyopathy, or possibly a modifier of the muscular dystrophy phenotype.