Limb Girdle Muscular Dystrophy 2B Research Articles

Rapid Screening for Japanese Dysferlinopathy by Fluorescent Primer Extension

Mutations in the dysferlin gene cause limb-girdle muscular dystrophy (LGMD) 2B and Miyoshi myopathy (MM), which are collectively named dysferlinopathy. Dysferlinopathy is the most frequent type of LGMD in the Japanese population. Molecular genetic analysis is essential for the diagnosis of dysferlinopathy because of its variable immunohistochemical patterns of biopsied muscles, including patterns similar to normal controls. The analysis of the entire dysferlin gene however, is time-consuming and laborious; therefore a simple and rapid screening method to detect hot spot mutations in the dysferlin gene is essential for the diagnosis of dysferlinopathy. We previously showed that 4 mutations, c.937+1G>A, c.1566C>G, c.2997G>T and c.3373delG account for 50% of all the mutations identified in Japanese dysferlinopathy patients. We performed a one-tube multiplex PCR, followed by extension of primers for each mutation with a fluorescence-labeled dideoxynucleotide to screen the 4 hot spot mutations. The multiplex primer-extension reaction was developed on samples of known mutations. The extension products were represented as 4 different peaks that corresponded to a mutated nucleotide on electropherogram. Using the developed screening method, we were able to detect mutations in these hot spots in 3 samples out of 8 clinically suspected LGMD2B/MM patients in only approximately 8 hours. These 3 cases were definitely diagnosed as LGMD2B/MM by exonic sequencing. We have developed a simple and rapid screening method which could facilitate the definitive diagnosis of dysferlinopathy, contributing to an understanding of the genotype-phenotype correlations for dysferlinopathy.

Dysferlin Deficiency and the Development of Cardiomyopathy in a Mouse Model of Limb-Girdle Muscular Dystrophy 2B

Limb-girdle muscular dystrophy 2B, Miyoshi myopathy, and distal myopathy of anterior tibialis are severely debilitating muscular dystrophies caused by genetically determined dysferlin deficiency. In these muscular dystrophies, it is the repair, not the structure, of the plasma membrane that is impaired. Though much is known about the effects of dysferlin deficiency in skeletal muscle, little is known about the role of dysferlin in maintenance of cardiomyocytes. Recent evidence suggests that dysferlin deficiency affects cardiac muscle, leading to cardiomyopathy when stressed. However, neither the morphological location of dysferlin in the cardiomyocyte nor the progression of the disease with age are known. In this study, we examined a mouse model of dysferlinopathy using light and electron microscopy as well as echocardiography and conscious electrocardiography. We determined that dysferlin is normally localized to the intercalated disk and sarcoplasm of the cardiomyocytes. In the absence of dysferlin, cardiomyocyte membrane damage occurs and is localized to the intercalated disk and sarcoplasm. This damage results in transient functional deficits at 10 months of age, but, unlike in skeletal muscle, the cell injury is sublethal and causes only mild cardiomyopathy even at advanced ages.

Genetic Manipulation of Dysferlin Expression in Skeletal Muscle: Novel Insights into Muscular Dystrophy

Mutations in the gene DYSF, which codes for the protein dysferlin, underlie Miyoshi myopathy and limb-girdle muscular dystrophy 2B in humans and produce a slowly progressing skeletal muscle degenerative disease in mice. Dysferlin is a Ca(2+)-sensing, regulatory protein that is involved in membrane repair after injury. To assess the function of dysferlin in healthy and dystrophic skeletal muscle, we generated skeletal muscle-specific transgenic mice with threefold overexpression of this protein. These mice were phenotypically indistinguishable from wild-type, and more importantly, the transgene completely rescued the muscular dystrophy (MD) disease in Dysf-null A/J mice. The dysferlin transgene rescued all histopathology and macrophage infiltration in skeletal muscle of Dysf(-/-) A/J mice, as well as promoted the rapid recovery of muscle function after forced lengthening contractions. These results indicate that MD in A/J mice is autonomous to skeletal muscle and not initiated by any other cell type. However, overexpression of dysferlin did not improve dystrophic symptoms or membrane instability in the dystrophin-glycoprotein complex-lacking Scgd (delta-sarcoglycan) null mouse, indicating that dysferlin functionality is not a limiting factor underlying membrane repair in other models of MD. In summary, the restoration of dysferlin in skeletal muscle fibers is sufficient to rescue the MD in Dysf-deficient mice, although its mild overexpression does not appear to functionally enhance membrane repair in other models of MD.

NET37, a Nuclear Envelope Transmembrane Protein with Glycosidase Homology, Is Involved in Myoblast Differentiation

The nuclear lamina and its associated proteins are important for nuclear structure and chromatin organization and also have been implicated in the regulation of cell signaling and gene expression. In this study we demonstrate that the lamina-associated nuclear envelope transmembrane protein NET37 is required for myogenic differentiation of C2C12 cells. NET37, a member of glycosidase family 31, is highly expressed in mouse skeletal muscle and is strongly up-regulated during C2C12 differentiation. By protease mapping we show that its glycosidase homology domain is located in the lumen of the nuclear envelope/endoplasmic reticulum. When NET37 is depleted from proliferating myoblasts by RNAi, myogenic differentiation is significantly impaired, and there is a concomitant delay in up-regulation of the late myogenic transcription factor myogenin. We expressed silencing-resistant NET37 mutated at a conserved residue in the glycosidase domain and found that this predicted catalytically inactive protein is unable to support myogenesis in cells depleted of wild type NET37. Therefore, the enzymatic function of NET37 appears to be important for myogenic differentiation. C2C12 cells depleted of NET37 have reduced activation of Akt after shifting to differentiation medium and are defective in insulin like growth factor-II (IGF-II) secretion, an autocrine/paracrine factor involved in Akt activation. We also observed that pro-IGF-II co-immunoprecipitates with NET37. Based on our results, we propose that NET37 has a role in IGF-II maturation in the secretory pathway during myoblast differentiation. The localization of NET37 at the nuclear envelope raises the possibility that it may coordinate myogenic events between the nuclear interior and the endoplasmic reticulum lumen via transmembrane communication.

3rd Annual Dysferlin Conference 2–5 June 2009, Boston, Massachusetts, USA

The 3rd Annual Dysferlin Conference, held from June 2–5, 2009 in Boston, MA, USA, brought together leading scientists and clinicians to discuss recent progress towards understanding and developing a therapy for the dysferlinopathies, Limb Girdle Muscular Dystrophy 2B (LGMD2B) and Miyoshi myopathy. These diseases involve progressive muscle wasting, typically beginning in the late teenage years, and are caused by mutations in the gene encoding dysferlin [1,2]. Dysferlin is required for repair of muscle fiber membrane tears [3,4], but little is known about how this defect contributes to disease pathology or whether dysferlin is also involved in other cellular processes. The conference was sponsored and organized by the Jain Foundation, and participants included 37 invited speakers, 10 additional scientists and clinicians, 40 poster presenters, 5 dysferlinopathy patients, 3 representatives of other muscular dystrophy foundations, and 7 members of the Jain Foundation scientific team. Research was presented from 11 countries around the world. Prior to the start of the main conference, a satellite meeting on the design of a natural history study for dysferlinopathy came up with specific recommendations about issues specific to this disease, including the high clinical variability, small patient numbers, and slow disease progression.

C. elegans dysferlin homologfer-1is expressed in muscle, andfer-1mutations initiate altered gene expression of muscle enriched genes

Mutations in the human dysferlin gene cause Limb Girdle Muscular Dystrophy 2B (LGMD2B). The Caenorhabditis elegans dysferlin homolog, fer-1, affects sperms development but is not known to be expressed in or have a functional roles outside of the male germline. Using several approaches, we show that fer-1 mRNA is present in C. elegans muscle cells but is absent from neurons. In mammals, loss of muscle-expressed dysferlin causes transcriptional deregulation of muscle expressed genes. To determine if similar alterations in gene expression are initiated in C. elegans due to loss of muscle-expressed fer-1, we performed whole genome Affymetrix microarray analysis of two loss-of-function fer-1 mutants. Both mutants gave rise to highly similar changes in gene expression and altered the expression of 337 genes. Using multiple analysis methods, we show that this gene set is enriched for genes known to regulate the structure and function of muscle. However, these transcriptional changes do not appear to be in response to gross sarcomeric damage, since genetically sensitized fer-1 mutants exhibit normal thin filament organization. Our data suggest that processes other than sarcomere stability may be affected by loss of fer-1 in C. elegans muscle. Therefore, C. elegans may be an attractive model system in which to explore new muscle-specific functions of the dysferlin protein and gain insights into the molecular pathogenesis of LGMD2B.

G.P.6.01 Measurements of progression in dysferlin myopathies: A preliminary prospective quantitative study

Introduction: Dysferlin myopathy is an autosomal recessive disease caused by mutations in the DYSF gene and it may present with several phenotypes. Descriptions of its clinical course are based on cross-sectional and retrospective studies. However, no prospective study has yet been performed. Material and methods: We conducted a quantitative study of the natural history of 5 limb girdle muscular dystrophy 2B (LGMD2B) and 4 Miyoshi myopathy (MM)), over an 18-month follow-up period. We performed a standardized protocol that included manual muscle testing (MMT), quantitative muscle testing (QMT), functional scales, timed tests, creatine kinase and respiratory and cardiac function.

T.P.6.04 Comparison of skeletal muscle potential of mesenchymal stem-cells from different sources injected in SJL mice

One of the aims of the HGRC-USP in Brazil is to compare the potential of stem cells from different sources to originate skeletal muscle. Limb-girdle muscular dystrophies (LGMD) are a heterogeneous group of disorders characterized by progressive degeneration of skeletal muscle caused by the absence or defective muscular proteins. The murine model for Limb-Girdle Muscular Dystrophy 2B (LGMD2B), the SJL mice, carries a deletion in the dysferlin gene that causes a reduction in the protein levels to 15% of normal.

New aspects on patients affected by dysferlin deficient muscular dystrophy

Mutations in the dysferlin gene lead to limb girdle muscular dystrophy 2B, Miyoshi myopathy and distal anterior compartment myopathy. A cohort of 36 patients affected by dysferlinopathy is described, in...

The Dysferlin homolog fer‐1 is required for efficient cholinergic synaptic transmission in C. elegans

Limb Girdle Muscular Dystrophy 2B (LGMD2B) is a progressive and incurable muscle disorder. Mutations in the Dysferlin gene cause LGMD2B, but the molecular mechanisms underlying disease pathogenesis are unclear. To better understand the pathogenesis of this disease, we studied the C. elegans Dysferlin homolog fer‐1. Previous studies have characterized fer‐1 only with regards to its roles in spermatogenesis. We found that fer‐1 is also expressed in muscle, where it regulates the localization of post‐synaptic acetylcholine receptors at the neuromuscular junction, suggesting a new role for Dysferlin as a regulator of cholinergic synaptic transmission. Consistent with these data, fer‐1 mutants were resistant to drugs that increase cholinergic signaling, and electrophysiological approaches confirm that fer‐1 mutants have a defect in acetylcholine‐dependent synaptic signaling. Acute administration of a cholinesterase inhibitor improved the excitability and functional properties of fer‐1 muscle. Given the strong evolutionary conservation in synaptic physiology between C. elegans and humans, these data suggest that disruption of cholinergic signaling may contribute to LGMD2B pathology and that drugs enhancing cholinergic signaling might be an effective therapeutic strategy for treatment.Supported by NIH Training Grant in Muscle Biology (T32 AR‐053461‐02) and ITMAT (UL1RR024134) grant.

Amyloidose bei Muskeldystrophie

Mutations in the gene encoding dysferlin (DYSF) cause limb-girdle muscular dystrophy 2B (LGMD2B) and Miyoshi myopathy (MM). We were able to examine eight patients suspected of LGMD2B clinically, histochemically. The genotype was determined in every case. We found sarcolemmal and interstitial amyloid deposits in four muscle sections. All of the mutations associated with amyloid were located in the N-terminal region of dysferlin, and dysferlin clearly proved to be a component of the amyloid deposits. Dysferlin-deficient muscular dystrophy is the first muscular dystrophy in which amyloidosis is involved. This fact must be considered in the process of developing therapeutic strategies. The influence of the amyloid deposits on the pathogenesis of the disease and the possible involvement of other organs in the progressive course are as yet unclear.

Partial Epilepsy in an Adolescent Male With Limb-Girdle Muscular Dystrophy 1B

Muscular dystrophies are inherited muscle disorders associated with different gene mutations. Fukuyama congenital muscular dystrophy is associated with cobblestone lissencephaly and epilepsy frequently. Rarely, other types of muscular dystrophies are also associated with epilepsy including Duchenne and Becker muscular dystrophy, facioscapulohumeral dystrophy, congenital muscular dystrophy with partial and complete deficiency of laminin alpha2 chain, and limb-girdle muscular dystrophy 2A with calpain deficiency. We now report another rare case of partial epilepsy and limb-girdle muscular dystrophy type 1B with lamin A/C gene mutation.

Dysferlin Deficiency Shows Compensatory Induction of Rab27A/Slp2a That May Contribute to Inflammatory Onset

Mutations in the dysferlin gene cause limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi myopathy. Dysferlin-deficient cells show abnormalities in vesicular traffic and membrane repair although onset of symptoms is not commonly seen until the late teenage years and is often associated with subacute onset and marked muscle inflammation. To identify molecular networks specific to dysferlin-deficient muscle that might explain disease pathogenesis, muscle mRNA profiles from 10 mutation-positive LGMD2B/MM patients were compared with a disease control [LGMD2I; (n = 9)], and normal muscle samples (n = 11). Query of inflammatory pathways suggested LGMD2B-specific increases in co-stimulatory signaling between dendritic cells and T cells (CD86, CD28, and CTLA4), associated with localized expression of both versican and tenascin. LGMD2B muscle also showed an increase in vesicular trafficking pathway proteins not normally observed in muscle (synaptotagmin-like protein Slp2a/SYTL2 and the small GTPase Rab27A). We propose that Rab27A/Slp2a expression in LGMD2B muscle provides a compensatory vesicular trafficking pathway that is able to repair membrane damage in the absence of dysferlin. However, this same pathway may release endocytotic vesicle contents, resulting in an inflammatory microenvironment. As dysferlin deficiency has been shown to enhance phagocytosis by macrophages, together with our findings of abnormal myofiber endocytosis pathways and dendritic-T cell activation markers, these results suggest a model of immune and inflammatory network over-stimulation that may explain the subacute inflammatory presentation.

G.O.6 Sjl dystrophic mice express large amount of human muscle proteins following systemic delivery of human adipose-derived stem cells

Limb-girdle muscular dystrophies (LGMD) are a heterogeneous group of disorders characterized by progressive degeneration of skeletal muscle caused by the absence or defective muscular proteins. The murine model for limb-girdle muscular dystrophy 2B (LGMD2B), the sjl mice, carry a deletion in the dysferlin gene that causes a reduction in the protein levels to 15% of normal. The mice show muscle weakness that begins at 4–6 weeks and is nearly complete by 8 months of age. The possibility to restore the defective muscle protein and improve muscular performance by cell therapy is a promising approach for the treatment of LGMD or other forms of progressive muscular dystrophies (PMD).

SJL Dystrophic Mice Express a Significant Amount of Human Muscle Proteins Following Systemic Delivery of Human Adipose-Derived Stromal Cells Without Immunosuppression

Limb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of disorders characterized by progressive degeneration of skeletal muscle caused by the absence of or defective muscular proteins. The murine model for limb-girdle muscular dystrophy 2B (LGMD2B), the SJL mice, carries a deletion in the dysferlin gene that causes a reduction in the protein levels to 15% of normal. The mice show muscle weakness that begins at 4-6 weeks and is nearly complete by 8 months of age. The possibility of restoring the defective muscle protein and improving muscular performance by cell therapy is a promising approach for the treatment of LGMDs or other forms of progressive muscular dystrophies. Here we have injected human adipose stromal cells (hASCs) into the SJL mice, without immunosuppression, aiming to assess their ability to engraft into recipient dystrophic muscle after systemic delivery; form chimeric human/mouse muscle fibers; express human muscle proteins in the dystrophic host and improve muscular performance. We show for the first time that hASCs are not rejected after systemic injection even without immunosuppression, are able to fuse with the host muscle, express a significant amount of human muscle proteins, and improve motor ability of injected animals. These results may have important applications for future therapy in patients with different forms of muscular dystrophies.

Dysferlinopathy: Clinical aspects in India

Mutations in the dysferlin gene on Chromosome 2p cause dysferlinopathies which include two distinct clinical entities, Miyoshi myopathy (MM) (OMIM no. 254130) and limb girdle muscular dystrophy 2B (LGMD2B) (OMIM no. 253601). They have been diagnosed as different diseases clinically depending on the affected muscles at the onset. In this issue of Neurology India, Nalini and Gayathri reported the differences in their clinical findings in India,[1] while the histopathological characteristics of their muscle specimens were not any different. The authors have presented the largest study of dysferlinopathy in India which focused on clinical aspects, according to our knowledge . MM and LGMD2B are autosomal recessive, slow progressive muscular dystrophies recognized by primary deficiency of dysferlin expression on their patients’ muscles. The patients who have dysferlin deficiency are affected in the teens or early adulthood, and show dramatic elevation of serum creatine kinase (CK).[2] Liu and colleagues discovered dysferlin, a skeletal muscle gene, which is found to be mutated in both MM and LGMD2B.[3] The study employing dysferlin-null mice showed that the mice developed progressive muscular dystrophy and exhibited elevated serum CK like human dysferlinopathy. [4] Dysferlin-deficient muscle fibers were defective in Ca2+-dependent sarcolemma resealing and it suggested the membrane repair system did not work during contraction-induced injuries. We reported that dantrolene, a well-known Ca2+ release inhibitor during muscle contraction, was effective for some Japanese MM siblings.[5] Altogether these facts might support that both dysferlinopathies had the same pathological background. Although both diseases have the same genetic and pathological background, they are distinguishable clinically. Basically, the calf muscles are affected at the initial stage in MM, but LGMD2B begins with proximal muscle weakness. The investigators focused on the clinical differences between MM and LGMD2B employing 28 Indian patients who had been diagnosed as dysferlinopathy by immunohistochemical analysis since 6.4±4.2 years. They found that the occurrence of distal muscle weakness in upper limbs was seen specifically in MM patients. It was pretty interesting, because originally Miyoshi and colleagues focused on the lower limb weakness.[2] Another Japanese group focused on the clinical differences between patients with and without G3370T or G3510A mutation in dysferlin gene, but in only MM. They found that the clinical onset was delayed in the patients with G3370T mutation and higher serum CK was seen in the patients with G3510A mutation.[6] The same group has recently reported that a 53-year-old man with LGMD2B (homozygous G3370T) mutation had chorea in addition to the myopathy. This is perhaps explained by the expression of smaller brain isoform of dysferlin in putamen.[7] These findings imply that the future genetic analysis might be able to provide new correlation between dysferlin mutation and clinical phenotype and much longer follow-up could uncover other interesting central nervous system-derived symptoms. As the authors noticed, they had to still exclude calpainopathy (LGMD2A) (OMIM no.253600) and caveolinopathy (LGMD1C) (OMIM no.607801). Three cases of LGMD2A showed a double defect of both calpain-3 and dysferlin in dystrophy muscles[8] and dysferlin expression was patchy in LGMD1C muscles.[9] Further genetic analysis or caveolin-3 immunostaining for LGMD1C might be helpful for differential diagnosis in the future. Nevertheless the present paper has outlined clinical details about both forms of dysfelinopathy in India preciously, and it will be further informative to uncover the geographical and ethnical differences of dysferlinopathy in the world.

Distinctive patterns of microRNA expression in primary muscular disorders

The primary muscle disorders are a diverse group of diseases caused by various defective structural proteins, abnormal signaling molecules, enzymes and proteins involved in posttranslational modifications, and other mechanisms. Although there is increasing clarification of the primary aberrant cellular processes responsible for these conditions, the decisive factors involved in the secondary pathogenic cascades are still mainly obscure. Given the emerging roles of microRNAs (miRNAs) in modulation of cellular phenotypes, we searched for miRNAs regulated during the degenerative process of muscle to gain insight into the specific regulation of genes that are disrupted in pathological muscle conditions. We describe 185 miRNAs that are up- or down-regulated in 10 major muscular disorders in humans [Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophies types 2A and 2B, Miyoshi myopathy, nemaline myopathy, polymyositis, dermatomyositis, and inclusion body myositis]. Although five miRNAs were found to be consistently regulated in almost all samples analyzed, pointing to possible involvement of a common regulatory mechanism, others were dysregulated only in one disease and not at all in the other disorders. Functional correlation between the predicted targets of these miRNAs and mRNA expression demonstrated tight posttranscriptional regulation at the mRNA level in DMD and Miyoshi myopathy. Together with direct mRNA-miRNA predicted interactions demonstrated in DMD, some of which are involved in known secondary response functions and others that are involved in muscle regeneration, these findings suggest an important role of miRNAs in specific physiological pathways underlying the disease pathology.

Dysfunction of dysferlin-deficient hearts

Mutations in the gene encoding dysferlin cause limb-girdle muscular dystrophy 2B (LGMD2B), a disorder that is believed to spare the heart. We observed dilated cardiomyopathy in two out of seven LGMD2B patients and cardiac abnormalities in three others. Cardiac biopsies showed that dysferlin was completely absent from the sarcolemma and appeared to be trapped within the cardiomyocytes. SJL/J mice (33-week-old) had diminished end-systolic pressure and reduced dP/dt; however, the hearts were histologically normal. Gene expression profiles of cardiac tissue were obtained and later confirmed by quantitative RT-PCR. Dysferlin-deficient and control mice had different gene expression patterns in terms of cardiomyocyte Z-disc and signal transduction proteins. CapZ, LIM-domain-binding protein 3 (LDB3, MLP), cypher (ZASP), desmin, and the cardiac ankyrin-repeated protein (CARP) were differentially expressed, compared to controls. Mechanical stress induced by the nonselective beta-adrenergic agonist isoproterenol (5 mg/kg body weight) given daily for 10 days resulted in reduced fractional shortening and increased cardiac fibrosis in SJL/J mice as compared to controls. Isoproterenol also caused metalloproteinase-2 upregulation in SJL/J mice. In A/J mice, the effect of isoproterenol injection was even more dramatic and lead to premature death as well as marked sarcolemmal injury as demonstrated by Evans blue dye penetration. Our data suggest that disturbances in dysferlin as well as Z-line proteins and transcription factors particularly under mechanical stress cause cardiomyopathy.

G.P.5.01 Inflammatory or ‘congenital myopathy’ type findings in muscle biopsies of patients with LMNA mutations

Muscular dystrophy associated to lamin A/C gene (LMNA) mutations is characterized by muscle weakness, joint contractures, dilated cardiomyopathy and arrhythmias. It includes autosomal dominant and rare recessive forms of Emery–Dreifuss as well as limb-girdle muscular dystrophy 1B and dilated cardiomyopathy with conduction system disease. We here report clinical onset, follow up and pathological findings of four cases. Muscle biopsies were studied at 5years of age for case 1; 6 and 71/2years for case 2, 14years for case 3, and 31years for case 4, by histochemical, immunohistochemical and electron microscopy techniques.

AHNAK a novel component of the dysferlin protein complex, redistributes to the cytoplasm with dysferlin during skeletal muscle regeneration

Mutations in dysferlin cause limb girdle muscular dystrophy 2B, Miyoshi myopathy and distal anterior compartment myopathy. Dysferlin is proposed to play a role in muscle membrane repair. To gain functional insight into the molecular mechanisms of dysferlin, we have searched for dysferlin-interacting proteins in skeletal muscle. By coimmunoprecipitation coupled with mass spectrometry, we demonstrate that AHNAK interacts with dysferlin. We defined the binding sites in dysferlin and AHNAK as the C2A domain in dysferlin and the carboxyterminal domain of AHNAK by glutathione S-transferase (GST)-pull down assays. As expected, the N-terminal domain of myoferlin also interacts with the carboxyterminal domain of AHNAK. In normal skeletal muscle, dysferlin and AHNAK colocalize at the sarcolemmal membrane and T-tubules. In dysferlinopathies, reduction or absence of dysferlin correlates with a secondary muscle-specific loss of AHNAK. Moreover, in regenerating rat muscle, dysferlin and AHNAK showed a marked increase and cytoplasmic localization, consistent with the direct interaction between them. Our data suggest that dysferlin participates in the recruitment and stabilization of AHNAK to the sarcolemma and that AHNAK plays a role in dysferlin membrane repair process. It may also have significant implications for understanding the biology of AHNAK-containing exocytotic vesicles, "enlargosomes," in plasma membrane remodeling and repair.