Angela Lek

Ferlins: Regulators of Vesicle Fusion for Auditory Neurotransmission, Receptor Trafficking and Membrane Repair

Ferlins are a family of multiple C2 domain proteins with emerging roles in vesicle fusion and membrane trafficking. Ferlin mutations are associated with muscular dystrophy (dysferlin) and deafness (otoferlin) in humans, and infertility in Caenorhabditis elegans (Fer‐1) and Drosophila (misfire), demonstrating their importance for normal cellular functioning. Ferlins show ancient origins in eukaryotic evolution and are detected in all eukaryotic kingdoms, including unicellular eukaryotes and apicomplexian protists, suggesting origins in a common ancestor predating eukaryotic evolutionary branching. The characteristic feature of the ferlin family is their multiple tandem cytosolic C2 domains (five to seven C2 domains), the most of any protein family, and an extremely rare feature amongst eukaryotic proteins. Ferlins also bear a unique nested DysF domain and small conserved 60–70 residue ferlin‐specific sequences (Fer domains). Ferlins segregate into two subtypes based on the presence (type I ferlin) or absence (type II ferlin) of the DysF and FerA domains. Ferlins have diverse tissue‐specific and developmental expression patterns, with ferlin animal models united by pathologies arising from defects in vesicle fusion. Consistent with their proposed role in vesicle trafficking, ferlin interaction partners include cytoskeletal motors, other vesicle‐associated trafficking proteins and transmembrane receptors or channels. Herein we summarize the research history of the ferlins, an intriguing family of structurally conserved proteins with a preserved ancestral function as regulators of vesicle fusion and receptor trafficking.

Calpains, Cleaved Mini-DysferlinC72, and L-Type Channels Underpin Calcium-Dependent Muscle Membrane Repair

Dysferlin is proposed as a key mediator of calcium-dependent muscle membrane repair, although its precise role has remained elusive. Dysferlin interacts with a new membrane repair protein, mitsugumin 53 (MG53), an E3 ubiquitin ligase that shows rapid recruitment to injury sites. Using a novel ballistics assay in primary human myotubes, we show it is not full-length dysferlin recruited to sites of membrane injury but an injury-specific calpain-cleavage product, mini-dysferlinC72. Mini-dysferlinC72-rich vesicles are rapidly recruited to injury sites and fuse with plasma membrane compartments decorated by MG53 in a process coordinated by L-type calcium channels. Collective interplay between activated calpains, dysferlin, and L-type channels explains how muscle cells sense a membrane injury and mount a specialized response in the unique local environment of a membrane injury. Mini-dysferlinC72 and MG53 form an intricate lattice that intensely labels exposed phospholipids of injury sites, then infiltrates and stabilizes the membrane lesion during repair. Our results extend functional parallels between ferlins and synaptotagmins. Whereas otoferlin exists as long and short splice isoforms, dysferlin is subject to enzymatic cleavage releasing a synaptotagmin-like fragment with a specialized protein- or phospholipid-binding role for muscle membrane repair.

Dysferlin, annexin A1, and mitsugumin 53 are upregulated in muscular dystrophy and localize to longitudinal tubules of the T-system with stretch.

Mutations in dysferlin cause an inherited muscular dystrophy because of defective membrane repair. Three interacting partners of dysferlin are also implicated in membrane resealing: caveolin-3 (in limb girdle muscular dystrophy type 1C), annexin A1, and the newly identified protein mitsugumin 53 (MG53). Mitsugumin 53 accumulates at sites of membrane damage, and MG53-knockout mice display a progressive muscular dystrophy. This study explored the expression and localization of MG53 in human skeletal muscle, how membrane repair proteins are modulated in various forms of muscular dystrophy, and whether MG53 is a primary cause of human muscle disease. Mitsugumin 53 showed variable sarcolemmal and/or cytoplasmic immunolabeling in control human muscle and elevated levels in dystrophic patients. No pathogenic MG53 mutations were identified in 50 muscular dystrophy patients, suggesting that MG53 is unlikely to be a common cause of muscular dystrophy in Australia. Western blot analysis confirmed upregulation of MG53, as well as of dysferlin, annexin A1, and caveolin-3 to different degrees, in different muscular dystrophies. Importantly, MG53, annexin A1, and dysferlin localize to the t-tubule network and show enriched labeling at longitudinal tubules of the t-system in overstretch. Our results suggest that longitudinal tubules of the t-system may represent sites of physiological membrane damage targeted by this membrane repair complex.

MicroRNA-486–dependent modulation of DOCK3/PTEN/AKT signaling pathways improves muscular dystrophy–associated symptoms

Duchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding dystrophin, which results in dysfunctional signaling pathways within muscle. Previously, we identified microRNA-486 (miR-486) as a muscle-enriched microRNA that is markedly reduced in the muscles of dystrophin-deficient mice (Dmdmdx-5Cv mice) and in DMD patient muscles. Here, we determined that muscle-specific transgenic overexpression of miR-486 in muscle of Dmdmdx-5Cv mice results in reduced serum creatine kinase levels, improved sarcolemmal integrity, fewer centralized myonuclei, increased myofiber size, and improved muscle physiology and performance. Additionally, we identified dedicator of cytokinesis 3 (DOCK3) as a miR-486 target in skeletal muscle and determined that DOCK3 expression is induced in dystrophic muscles. DOCK3 overexpression in human myotubes modulated PTEN/AKT signaling, which regulates muscle hypertrophy and growth, and induced apoptosis. Furthermore, several components of the PTEN/AKT pathway were markedly modulated by miR-486 in dystrophin-deficient muscle. Skeletal muscle-specific miR-486 overexpression in Dmdmdx-5Cv animals decreased levels of DOCK3, reduced PTEN expression, and subsequently increased levels of phosphorylated AKT, which resulted in an overall beneficial effect. Together, these studies demonstrate that stable overexpression of miR-486 ameliorates the disease progression of dystrophin-deficient skeletal muscle.

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.

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.

Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair

The muscular dystrophy protein dysferlin plays a key role in the calcium-activated vesicle fusion of membrane repair. This study establishes calpains as upstream regulators of dysferlin in the membrane repair cascade and further demonstrates that similar C-terminal modules are enzymatically released from other ferlin family members.

Are protein complexes made of cores, modules and attachments?

It has recently been proposed by Gavin et al. (Nature 2006, 440, 631–636) that protein complexes in the cell exist in different forms. The proteins within each complex were proposed to exist as three different classes, being core, module or attachment proteins. This study investigates whether the core–module–attachment classification of proteins within each complex is supported by other high‐throughput protein data. Core proteins were found to have lower abundance, and shorter half‐life as compared to attachment proteins, whilst the abundance and half‐life of core and module proteins were similar. When the cell was perturbed, core proteins had smaller changes in abundance as compared to module and attachment proteins. Comparisons between six different pairwise interaction types of core, module and attachment proteins within a complex showed interaction types involving core or module proteins were more likely to be mediated by domain–domain interactions (DDIs) than interaction types involving attachment proteins. Interaction types that involve attachment proteins had a relatively higher ratio of abundance and ratio of half‐life. So we conclude that, the core, module and attachment model of protein complexes is supported by data from these proteomic scale datasets, and describe a model for a typical protein complex that considers the above results.

SMCHD1 mutations associated with a rare muscular dystrophy can also cause isolated arhinia and Bosma arhinia microphthalmia syndrome

Arhinia, or absence of the nose, is a rare malformation of unknown etiology that is often accompanied by ocular and reproductive defects. Sequencing of 40 people with arhinia revealed that 84% of probands harbor a missense mutation localized to a constrained region of SMCHD1 encompassing the ATPase domain. SMCHD1 mutations cause facioscapulohumeral muscular dystrophy type 2 (FSHD2) via a trans-acting loss-of-function epigenetic mechanism. We discovered shared mutations and comparable DNA hypomethylation patterning between these distinct disorders. CRISPR/Cas9-mediated alteration of smchd1 in zebrafish yielded arhinia-relevant phenotypes. Transcriptome and protein analyses in arhinia probands and controls showed no differences in SMCHD1 mRNA or protein abundance but revealed regulatory changes in genes and pathways associated with craniofacial patterning. Mutations in SMCHD1 thus contribute to distinct phenotypic spectra, from craniofacial malformation and reproductive disorders to muscular dystrophy, which we speculate to be consistent with oligogenic mechanisms resulting in pleiotropic outcomes.

Emerging preclinical animal models for FSHD

Facioscapulohumeral dystrophy (FSHD) is a unique and complex genetic disease that is not entirely solved. Recent advances in the field have led to a consensus genetic premise for the disorder, enabling researchers to now pursue the design of preclinical models. In this review we explore all available FSHD models (DUX4-dependent and -independent) for their utility in therapeutic discovery and potential to yield novel disease insights. Owing to the complex nature of FSHD, there is currently no single model that accurately recapitulates the genetic and pathophysiological spectrum of the disorder. Existing models emphasize only specific aspects of the disease, highlighting the need for more collaborative research and novel paradigms to advance the translational research space of FSHD.