The<Emphasis Type="Italic"> cnm</Emphasis> locus,a canine homologue of human autosomal forms of centronuclear myopathy,maps to chromosome 2

Myotubular/centronuclear myopathies are a nosological group of hereditary disorders characterised by severe architectural and metabolic remodelling of skeletal muscle fibres. In most myofibres, nuclei are found at an abnormal central position within a halo devoid of myofibrillar proteins. The X-linked form (myotubular myopathy) is the most prevalent and severe form in human, leading to death during early postnatal life. Maturation of fibres is not completed and fibres resemble myotubes. Linkage analysis in human has helped to identify MTM1 as the morbid gene. MTM1 encodes myotubularin, a dual protein phosphatase. In families in which myotubular myopathy segregates, detected mutations in MTM1 abolish the specific phosphatase activity targeting the second messenger phosphatidylinositol 3-phosphate. Autosomal forms (centronuclear) have a later onset and are often compatible with life. At birth, fibres are normally constituted but progressively follow remodelling with a secondary centralisation of nuclei. Their prevalence is low; hence, no linkage data can be performed and no molecular aetiology is known. In the Labrador Retriever, a spontaneous disorder strikingly mimics the clinical evolution of the human centronuclear myopathy. We have established a canine pedigree and show that the disorder segregates as an autosomal recessive trait in that pedigree. We have further mapped the dog locus to a region on chromosome 2 that is orthologous to human chromosome 10p. To date, no human MTM1 gene member has been mapped to this genetic region. This report thus describes the first spontaneous mammalian model of centronuclear myopathy and defines a new locus for this group of diseases.     

Altered Splicing of the BIN1 Muscle-Specific Exon in Humans and Dogs with Highly Progressive Centronuclear Myopathy

Amphiphysin 2, encoded by BIN1, is a key factor for membrane sensing and remodelling in different cell types. Homozygous BIN1 mutations in ubiquitously expressed exons are associated with autosomal recessive centronuclear myopathy (CNM), a mildly progressive muscle disorder typically showing abnormal nuclear centralization on biopsies. In addition, misregulation of BIN1 splicing partially accounts for the muscle defects in myotonic dystrophy (DM). However, the muscle-specific function of amphiphysin 2 and its pathogenicity in both muscle disorders are not well understood. In this study we identified and characterized the first mutation affecting the splicing of the muscle-specific BIN1 exon 11 in a consanguineous family with rapidly progressive and ultimately fatal centronuclear myopathy. In parallel, we discovered a mutation in the same BIN1 exon 11 acceptor splice site as the genetic cause of the canine Inherited Myopathy of Great Danes (IMGD). Analysis of RNA from patient muscle demonstrated complete skipping of exon 11 and BIN1 constructs without exon 11 were unable to promote membrane tubulation in differentiated myotubes. Comparative immunofluorescence and ultrastructural analyses of patient and canine biopsies revealed common structural defects, emphasizing the importance of amphiphysin 2 in membrane remodelling and maintenance of the skeletal muscle triad. Our data demonstrate that the alteration of the muscle-specific function of amphiphysin 2 is a common pathomechanism for centronuclear myopathy, myotonic dystrophy, and IMGD. The IMGD dog is the first faithful model for human BIN1-related CNM and represents a mammalian model available for preclinical trials of potential therapies.     

Defective membrane remodeling in neuromuscular diseases: insights from animal models

Proteins involved in membrane remodeling play an essential role in a plethora of cell functions including endocytosis and intracellular transport. Defects in several of them lead to human diseases. Myotubularins, amphiphysins, and dynamins are all proteins implicated in membrane trafficking and/or remodeling. Mutations in myotubularin, amphiphysin 2 (BIN1), and dynamin 2 lead to different forms of centronuclear myopathy, while mutations in myotubularin-related proteins cause Charcot-Marie-Tooth neuropathies. In addition to centronuclear myopathy, dynamin 2 is also mutated in a dominant form of Charcot-Marie-Tooth neuropathy. While several proteins from these different families are implicated in similar diseases, mutations in close homologues or in the same protein in the case of dynamin 2 lead to diseases affecting different tissues. This suggests (1) a common molecular pathway underlying these different neuromuscular diseases, and (2) tissue-specific regulation of these proteins. This review discusses the pathophysiology of the related neuromuscular diseases on the basis of animal models developed for proteins of the myotubularin, amphiphysin, and dynamin families. A better understanding of the common mechanisms between these neuromuscular disorders will lead to more specific health care and therapeutic approaches.     

Characterisation of mutations in 77 patients with X-linked myotubular myopathy,including a family with a very mild phenotype

X-linked myotubular myopathy is characterised by neonatal hypotonia, muscle weakness and respiratory distress in affected males, leading often to early death, although prolonged survival is observed in milder forms, or as a result of prolongation of ventilation support. It is caused by mutations in the MTM1 gene, which encodes a phosphatase called myotubularin, which has been highly conserved during evolution, down to yeasts ( S. cerevisiae and S. pombe). To date, 251 mutations have been identified in unrelated families, corresponding to 158 different disease-associated mutations, which are widespread throughout the gene. We have found additional mutations in 77 patients, including 35 novel ones. We identified a missense mutation N180K in a 67-year-old grandfather (the oldest known patient with an MTM1 mutation), previously suspected to have autosomal centronuclear myopathy, and in his two grandsons also mildly affected. Mild and moderate phenotypes associated with novel missense mutations and with a translation initiation defect mutation are discussed, as well as severe phenotypes associated with particular novel mutations. With the present report, 192 different mutations in the MTM1 gene have been described in 328 families. The spectrum of mutations is now enlarged from the very severe classic neonatal phenotype to very mild phenotype allowing survival to the age of 67 years.     

Myotubularin lipid phosphatase binds the hVPS15/hVPS34 lipid kinase complex on endosomes

Myotubularins constitute a ubiquitous family of phosphatidylinositol (PI) 3-phosphatases implicated in several neuromuscular disorders. Myotubularin [myotubular myopathy 1 (MTM1)] PI 3-phosphatase is shown associated with early and late endosomes. Loss of endosomal phosphatidylinositol 3-phosphate [PI(3)P] upon overexpression of wild-type MTM1, but not a phosphatase-dead MTM1C375S mutant, resulted in altered early and late endosomal PI(3)P levels and rapid depletion of early endosome antigen-1. Membrane-bound MTM1 was directly complexed to the hVPS15/hVPS34 [vacuolar protein sorting (VPS)] PI 3-kinase complex with binding mediated by the WD40 domain of the hVPS15 (p150) adapter protein and independent of a GRAM-domain point mutation that blocks PI(3,5)P(2) binding. The WD40 domain of hVPS15 also constitutes the binding site for Rab7 and, as shown previously, contributes to Rab5 binding. In vivo, the hVPS15/hVPS34 PI 3-kinase complex forms mutually exclusive complexes with the Rab GTPases (Rab5 or Rab7) or with MTM1, suggesting a competitive binding mechanism. Thus, the Rab GTPases together with MTM1 likely serve as molecular switches for controlling the sequential synthesis and degradation of endosomal PI(3)P. Normal levels of endosomal PI(3)P and PI(3,5)P(2) are crucial for both endosomal morphology and function, suggesting that disruption of endosomal sorting and trafficking in skeletal muscle when MTM1 is mutated may be a key factor in precipitating X-linked MTM.     

Mutations in BIN1 Associated with Centronuclear Myopathy Disrupt Membrane Remodeling by Affecting Protein Density and Oligomerization

The regulation of membrane shapes is central to many cellular phenomena. Bin/Amphiphysin/Rvs (BAR) domain-containing proteins are key players for membrane remodeling during endocytosis, cell migration, and endosomal sorting. BIN1, which contains an N-BAR domain, is assumed to be essential for biogenesis of plasma membrane invaginations (T-tubules) in muscle tissues. Three mutations, K35N, D151N and R154Q, have been discovered so far in the BAR domain of BIN1 in patients with centronuclear myopathy (CNM), where impaired organization of T-tubules has been reported. However, molecular mechanisms behind this malfunction have remained elusive. None of the BIN1 disease mutants displayed a significantly compromised curvature sensing ability. However, two mutants showed impaired membrane tubulation both in vivo and in vitro, and displayed characteristically different behaviors. R154Q generated smaller membrane curvature compared to WT N-BAR. Quantification of protein density on membranes revealed a lower membrane-bound density for R154Q compared to WT and the other mutants, which appeared to be the primary reason for the observation of impaired deformation capacity. The D151N mutant was unable to tubulate liposomes under certain experimental conditions. At medium protein concentrations we found ‘budding’ structures on liposomes that we hypothesized to be intermediates during the tubulation process except for the D151N mutant. Chemical crosslinking assays suggested that the D151N mutation impaired protein oligomerization upon membrane binding. Although we found an insignificant difference between WT and K35N N-BAR in in vitro assays, depolymerizing actin in live cells allowed tubulation of plasma membranes through the K35N mutant. Our results provide insights into the membrane-involved pathophysiological mechanisms leading to human disease.     

DNM2 mutations in a cohort of sporadic patients with centronuclear myopathy

Centronuclear myopathy (CNM) is a rare congenital muscle disease characterized by fibers with prominent centralized nuclei in muscle biopsies. The disease is clinically heterogeneous, ranging from severe neonatal hypotonic phenotypes to adult-onset mild muscle weakness, and can have multiple modes of inheritance in association with various genes, including MTM1, DNM2, BIN1 and RYR1. Here we analyzed 18 sporadic patients with clinical and histological diagnosis of CNM and sequenced the DNM2 gene, which codes for the dynamin 2 protein. We found DNM2 missense mutations in two patients, both in exon 8, one known (p.E368K) and one novel (p.F372C), which is found in a position of presumed pathogenicity and appeared de novo. The patients had similar phenotypes characterized by neonatal signs followed by improvement and late childhood reemergence of slowly progressive generalized muscle weakness, elongated face with ptosis and ophthalmoparesis, and histology showing fibers with radiating sarcoplasmic strands (RSS). These patients were the only ones in the series to present this histological marker, which together with previous reports in the literature suggest that, when RSS are present, direct sequencing of DNM2 mutation hot spot regions should be the first step in the molecular diagnosis of CNM, even in sporadic cases.     

抑制MTM1表达促进胚胎干细胞分化

应用随机RNAi文库,筛选了与胚胎干细胞自我更新和分化调控相关基因,发现了多个阳性候选基因,对其中的1个阳性候选基因肌管素1（myotubularin, MTM1）基因进行了深入研究．MTM1是属于蛋白酪氨酸磷酸酶（PTPase）蛋白家族的蛋白,其基因突变导致肌管性肌病．MTM1在胚胎干细胞中的功能到目前为止还不清楚．研究证实,MTM1在小鼠胚胎干细胞系CCE和R1均有表达．应用RNA干扰及集落形成实验证明,MTM1表达抑制后,处于自我更新状况胚胎干细胞集落的比例显著增加,提示MTM1在胚胎干细胞自我更新和分化的调控中起了重要的作用．     

Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy

Myotonic dystrophy is the most common muscular dystrophy in adults and the first recognized example of an RNA-mediated disease. Congenital myotonic dystrophy (CDM1) and myotonic dystrophy of type 1 (DM1) or of type 2 (DM2) are caused by the expression of mutant RNAs containing expanded CUG or CCUG repeats, respectively. These mutant RNAs sequester the splicing regulator Muscleblind-like-1 (MBNL1), resulting in specific misregulation of the alternative splicing of other pre-mRNAs. We found that alternative splicing of the bridging integrator-1 (BIN1) pre-mRNA is altered in skeletal muscle samples of people with CDM1, DM1 and DM2. BIN1 is involved in tubular invaginations of membranes and is required for the biogenesis of muscle T tubules, which are specialized skeletal muscle membrane structures essential for excitation-contraction coupling. Mutations in the BIN1 gene cause centronuclear myopathy, which shares some histopathological features with myotonic dystrophy. We found that MBNL1 binds the BIN1 pre-mRNA and regulates its alternative splicing. BIN1 missplicing results in expression of an inactive form of BIN1 lacking phosphatidylinositol 5-phosphate-binding and membrane-tubulating activities. Consistent with a defect of BIN1, muscle T tubules are altered in people with myotonic dystrophy, and membrane structures are restored upon expression of the normal splicing form of BIN1 in muscle cells of such individuals. Finally, reproducing BIN1 splicing alteration in mice is sufficient to promote T tubule alterations and muscle weakness, a predominant feature of myotonic dystrophy.     

Loss of Catalytically Inactive Lipid Phosphatase Myotubularin-related Protein 12 Impairs Myotubularin Stability and Promotes Centronuclear Myopathy in Zebrafish

X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by mutations of the myotubularin gene, MTM1. Myotubularin belongs to a large family of conserved lipid phosphatases that include both catalytically active and inactive myotubularin-related proteins (i.e., “MTMRs”). Biochemically, catalytically inactive MTMRs have been shown to form heteroligomers with active members within the myotubularin family through protein-protein interactions. However, the pathophysiological significance of catalytically inactive MTMRs remains unknown in muscle. By in vitro as well as in vivo studies, we have identified that catalytically inactive myotubularin-related protein 12 (MTMR12) binds to myotubularin in skeletal muscle. Knockdown of the mtmr12 gene in zebrafish resulted in skeletal muscle defects and impaired motor function. Analysis of mtmr12 morphant fish showed pathological changes with central nucleation, disorganized Triads, myofiber hypotrophy and whorled membrane structures similar to those seen in X-linked myotubular myopathy. Biochemical studies showed that deficiency of MTMR12 results in reduced levels of myotubularin protein in zebrafish and mammalian C2C12 cells. Loss of myotubularin also resulted in reduction of MTMR12 protein in C2C12 cells, mice and humans. Moreover, XLMTM mutations within the myotubularin interaction domain disrupted binding to MTMR12 in cell culture. Analysis of human XLMTM patient myotubes showed that mutations that disrupt the interaction between myotubularin and MTMR12 proteins result in reduction of both myotubularin and MTMR12. These studies strongly support the concept that interactions between myotubularin and MTMR12 are required for the stability of their functional protein complex in normal skeletal muscles. This work highlights an important physiological function of catalytically inactive phosphatases in the pathophysiology of myotubular myopathy and suggests a novel therapeutic approach through identification of drugs that could stabilize the myotubularin-MTMR12 complex and hence ameliorate this disorder.     

SPEG Interacts with Myotubularin,and Its Deficiency Causes Centronuclear Myopathy with Dilated Cardiomyopathy

Centronuclear myopathies (CNMs) are characterized by muscle weakness and increased numbers of central nuclei within myofibers. X-linked myotubular myopathy, the most common severe form of CNM, is caused by mutations in MTM1, encoding myotubularin (MTM1), a lipid phosphatase. To increase our understanding of MTM1 function, we conducted a yeast two-hybrid screen to identify MTM1-interacting proteins. Striated muscle preferentially expressed protein kinase (SPEG), the product of SPEG complex locus (SPEG), was identified as an MTM1-interacting protein, confirmed by immunoprecipitation and immunofluorescence studies. SPEG knockout has been previously associated with severe dilated cardiomyopathy in a mouse model. Using whole-exome sequencing, we identified three unrelated CNM-affected probands, including two with documented dilated cardiomyopathy, carrying homozygous or compound-heterozygous SPEG mutations. SPEG was markedly reduced or absent in two individuals whose muscle was available for immunofluorescence and immunoblot studies. Examination of muscle samples from Speg-knockout mice revealed an increased frequency of central nuclei, as seen in human subjects. SPEG localizes in a double line, flanking desmin over the Z lines, and is apparently in alignment with the terminal cisternae of the sarcoplasmic reticulum. Examination of human and murine MTM1-deficient muscles revealed similar abnormalities in staining patterns for both desmin and SPEG. Our results suggest that mutations in SPEG, encoding SPEG, cause a CNM phenotype as a result of its interaction with MTM1. SPEG is present in cardiac muscle, where it plays a critical role; therefore, individuals with SPEG mutations additionally present with dilated cardiomyopathy.     

Mild functional differences of dynamin 2 mutations associated to centronuclear myopathy and Charcot-Marie Tooth peripheral neuropathy

The large GTPase dynamin 2 is a key player in membrane and cytoskeletal dynamics mutated in centronuclear myopathy (CNM) and Charcot-Marie Tooth (CMT) neuropathy, two discrete dominant neuromuscular disorders affecting skeletal muscle and peripheral nerves respectively. The molecular basis for the tissue-specific phenotypes observed and the physiopathological mechanisms linked to dynamin 2 mutations are not well established. In this study, we have analyzed the impact of CNM and CMT implicated dynamin 2 mutants using ectopic expression of four CNM and two CMT mutations, and patient fibroblasts harboring two dynamin 2 CNM mutations in established cellular processes of dynamin 2 action. Wild type and CMT mutants were seen in association with microtubules whereas CNM mutants lacked microtubules association and did not disrupt interphase microtubules dynamics. Most dynamin 2 mutants partially decreased clathrin-mediated endocytosis when ectopically expressed in cultured cells; however, experiments in patient fibroblasts suggested that endocytosis is overall not defective. Furthermore, CNM mutants were seen in association with enlarged clathrin stained structures whereas the CMT mutant constructs were associated with clathrin structures that appeared clustered, similar to the structures observed in Dnm1 and Dnm2 double knock-out cells. Other roles of dynamin 2 including its interaction with BIN1 (amphiphysin 2), and its function in Golgi maintenance and centrosome cohesion were not significantly altered. Taken together, these mild functional defects are suggestive of differences between CMT and CNM disease-causing dynamin 2 mutants and suggest that a slight impairment in clathrin-mediated pathways may accumulate over time to foster the respective human diseases.     

Multiple disease-linked myotubularin mutations cause NFL assembly defects in cultured cells and disrupt myotubularin dimerization

Charcot-Marie-Tooth disease (CMT) is an inherited peripheral neuropathy that has been linked to mutations in multiple genes. Mutations in the neurofilament light ( NFL ) chain gene lead to the CMT2E form whereas mutations in the myotubularin-related protein 2 and 13 ( MTMR2 and MTMR13 ) genes lead to the CMT4B form. These two forms share characteristic pathological hallmarks on nerve biopsies including concentric sheaths ('onion bulbs') and, in at least one case, myelin loops. In addition, MTMR2 protein has been shown to interact physically with both NFL and MTMR13. Here, we present evidence that CMT-linked mutations of MTMR2 can cause NFL aggregation in a cell line devoid of endogenous intermediate filaments, SW13vim⁻. Mutations in the protein responsible for X-linked myotubular myopathy (myotubularin, MTM1) also induced NFL abnormalities in these cells. We also show that two MTMR2 mutant proteins, G103E and R283W, are unable to form dimers and undergo phosphorylation in vivo , implicating impaired complex formation in myotubularin-related pathology.     

Identification of novel interaction partners for the conserved membrane proximal region of alpha-integrin cytoplasmic domains

The alpha3Abeta1 integrin is a laminin receptor with a broad specificity for different laminin isoforms. Furthermore, it regulates the function of other integrins, like alpha2beta1, alpha5beta1 and alpha6Abeta1. In a yeast two hybrid screen of a human placenta cDNA library, we identified cDNAs coding for four different proteins that strongly interact with the conserved region of the cytoplasmic domain of the alpha3A integrin subunit. In addition to the cDNA for nucleotide exchange factor Mss4 and the putative tumour suppressor protein BIN1, two novel cDNAs were identified. Association analysis with different integrin subunits revealed them as cDNAs that encode binding proteins which react with a broad spectrum of alpha subunits. The conserved membrane proximal region of the alpha3A chain was identified as the binding site for all four proteins. They, therefore, may be involved in the regulation of general functions of integrins.     

From Dynamic Live Cell Imaging to 3D Ultrastructure: Novel Integrated Methods for High Pressure Freezing and Correlative Light-Electron Microscopy

Background

In cell biology, the study of proteins and organelles requires the combination of different imaging approaches, from live recordings with light microscopy (LM) to electron microscopy (EM).

Methodology

To correlate dynamic events in adherent cells with both ultrastructural and 3D information, we developed a method for cultured cells that combines confocal time-lapse images of GFP-tagged proteins with electron microscopy. With laser micro-patterned culture substrate, we created coordinates that were conserved at every step of the sample preparation and visualization processes. Specifically designed for cryo-fixation, this method allowed a fast freezing of dynamic events within seconds and their ultrastructural characterization. We provide examples of the dynamic oligomerization of GFP-tagged myotubularin (MTM1) phosphoinositides phosphatase induced by osmotic stress, and of the ultrastructure of membrane tubules dependent on amphiphysin 2 (BIN1) expression.

Conclusion

Accessible and versatile, we show that this approach is efficient to routinely correlate functional and dynamic LM with high resolution morphology by EM, with immuno-EM labeling, with 3D reconstruction using serial immuno-EM or tomography, and with scanning-EM.