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International audience; For long-lived contractile cells, such as striated muscle cells, maintaining proteome integrity is a challenging task. These cells require hundreds of components that must be properly synthesized, folded, and incorporated into the basic contractile unit, the sarcomere. Muscle protein quality control in cells is mainly guaranteed by the ubiquitin-proteasome system (UPS), the lysosome-autophagy system, and various molecular chaperones. Recent studies establish the concept of dedicated UPS in the regulation of sarcomere assembly during development and in adult life to maintain the intricate and interwoven organization of protein complexes in muscle. Failure of sarcomere protein quality control often represents the basis of severe myopathies and cardiomyopathies in human, further highlighting its importance in producing and maintaining the contractile machinery of muscle cells in shape.

Endothelium protection is critical, because of the impact of vascular leakage and edema on pathological conditions such as brain ischemia. Whereas deficiency of class II phosphoinositide 3‐kinase alpha (PI3KC2α) results in an increase in vascular permeability, we uncover a crucial role of the beta isoform (PI3KC2β) in the loss of endothelial barrier integrity following injury. Here, we studied the role of PI3KC2β in endothelial permeability and endosomal trafficking in vitro and in vivo in ischemic stroke. Mice with inactive PI3KC2β showed protection against vascular permeability, edema, cerebral infarction, and deleterious inflammatory response. Loss of PI3KC2β in human cerebral microvascular endothelial cells stabilized homotypic cell–cell junctions by increasing Rab11‐dependent VE‐cadherin recycling. These results identify PI3KC2β as a potential new therapeutic target to prevent aggravating lesions following ischemic stroke.

Following their generation by lipid kinases and phosphatases, phosphoinositides regulate important biological processes such as cytoskeleton rearrangement, membrane remodeling/trafficking, and gene expression through the interaction of their phosphorylated inositol head group with a variety of protein domains such as PH, PX, and FYVE. Therefore, it is important to determine the specificity of phosphoinositides toward effector proteins to understand their impact on cellular physiology. Several methods have been developed to identify and characterize phosphoinositide effectors, and liposomes-based methods are preferred because the phosphoinositides are incorporated in a membrane, the composition of which can mimic cellular membranes. In this report, we describe the experimental setup for liposome flotation assay and a recently developed method called protein-lipid interaction by fluorescence (PLIF) for the characterization of phosphoinositide-binding specificities of proteins.

Our knowledge of the role and biology of the different phosphoinositides has greatly expanded over recent years. Reversible phosphorylation by specific kinases and phosphatases of positions 3, 4, and 5 on the inositol ring is a highly dynamic process playing a critical role in the regulation of the spatiotemporal recruitment and binding of effector proteins. The specific phosphoinositide kinases and phosphatases are key players in the control of many cellular functions, including proliferation, survival, intracellular trafficking, or cytoskeleton reorganization. Several of these enzymes are mutated in human diseases. The impact of the fatty acid composition of phosphoinositides in their function is much less understood. There is an important molecular diversity in the fatty acid side chains of PI. While stearic and arachidonic fatty acids are the major acyl species in PIP, PIP

Our knowledge of the role and biology of the different phosphoinositides has greatly expanded over recent years. Reversible phosphorylation by specific kinases and phosphatases of positions 3, 4, and 5 on the inositol ring is a highly dynamic process playing a critical role in the regulation of the spatiotemporal recruitment and binding of effector proteins. The specific phosphoinositide kinases and phosphatases are key players in the control of many cellular functions, including proliferation, survival, intracellular trafficking, or cytoskeleton reorganization. Several of these enzymes are mutated in human diseases. The impact of the fatty acid composition of phosphoinositides in their function is much less understood. There is an important molecular diversity in the fatty acid side chains of PI. While stearic and arachidonic fatty acids are the major acyl species in PIP, PIP2, and PIP3, other fatty acid combinations are also found. The role of these different molecular species is still unknown, but it is important to quantify these different molecules and their potential changes during cell stimulation to better characterize this emerging field. Here, we describe a sensitive high-performance liquid chromatography-mass spectrometry method that we used for the first time to profile the changes in phosphoinositide molecular species (summed fatty acyl chain profiles) in human and mouse platelets under resting conditions and following stimulation. This method can be applied to other hematopoietic primary cells isolated from human or experimental animal models.

Following their generation by lipid kinases and phosphatases, phosphoinositides regulate important biological processes such as cytoskeleton rearrangement, membrane remodeling/trafficking, and gene expression through the interaction of their phosphorylated inositol head group with a variety of protein domains such as PH, PX, and FYVE. Therefore, it is important to determine the specificity of phosphoinositides toward effector proteins to understand their impact on cellular physiology. Several methods have been developed to identify and characterize phosphoinositide effectors, and liposomes-based methods are preferred because the phosphoinositides are incorporated in a membrane, the composition of which can mimic cellular membranes. In this report, we describe the experimental setup for liposome flotation assay and a recently developed method called protein-lipid interaction by fluorescence (PLIF) for the characterization of phosphoinositide-binding specificities of proteins.

Our knowledge on the expression, regulation and roles of the different phosphoinositide 3-kinases (PI3Ks) in platelet signaling and functions has greatly expanded these last twenty years. Much progress has been made in understanding the roles and regulations of class I PI3Ks which produce the lipid second messenger phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P3). Selective pharmacological inhibitors and genetic approaches have allowed researchers to generate an impressive amount of data on the role of class I PI3Kα, β, δ and γ in platelet activation and in thrombosis. Furthermore, platelets do also express two class II PI3Ks (PI3KC2α and PI3KC2β), thought to generate PtdIns(3,4)P2 and PtdIns3P, and the sole class III PI3K (Vps34), known to synthesize PtdIns3P. Recent studies have started to reveal the importance of PI3KC2α and Vps34 in megakaryocytes and platelets, opening new perspective in our comprehension of platelet biology and thrombosis. In this review, we will summarize previous and recent advances on platelet PI3Ks isoforms. The implication of these kinases and their lipid products in fundamental platelet biological processes and thrombosis will be discussed. Finally, the relevance of developing potential antithrombotic strategies by targeting PI3Ks will be examined.

The ubiquitin proteasome system and autophagy are major protein turnover mechanisms in muscle cells, which ensure stemness and muscle fibre maintenance. Muscle cells contain a high proportion of cytoskeletal proteins, which are prone to misfolding and aggregation; pathological processes that are observed in several neuromuscular diseases called proteinopathies. Despite advances in deciphering the mechanisms underlying misfolding and aggregation, little is known about how muscle cells manage cytoskeletal degradation. Here, we describe a process by which muscle cells degrade the misfolded intermediate filament proteins desmin and vimentin by the proteasome. This relies on the MTM1–UBQLN2 complex to recognize and guide these misfolded proteins to the proteasome and occurs prior to aggregate formation. Thus, our data highlight a safeguarding function of the MTM1–UBQLN2 complex that ensures cytoskeletal integrity to avoid proteotoxic aggregate formation. Gavriilidis et al. show that MTM1, which is mutated in X-linked centronuclear myopathy, and UBQLN2 recognize misfolded desmin and vimentin and trigger their degradation to clear misfolded intermediated filaments prior to aggregate formation.

Desmin is a muscle-specific type III intermediate filament essential for proper muscular structure and function. In human, mutations affecting desmin expression or promoting its aggregation lead to skeletal (desmin-related myopathies), or cardiac (desmin-related cardiomyopathy) phenotypes, or both. Patient muscles display intracellular accumulations of misfolded proteins and desmin-positive insoluble granulofilamentous aggregates, leading to a large spectrum of molecular alterations. Increasing evidence shows that desmin function is not limited to the structural and mechanical integrity of cells. This novel perception is strongly supported by the finding that diseases featuring desmin aggregates cannot be easily associated with mechanical defects, but rather involve desmin filaments in a broader spectrum of functions, such as in organelle positioning and integrity and in signaling. Here, we review desmin functions and related diseases affecting striated muscles. We detail emergent cellular functions of desmin based on reported phenotypes in patients and animal models. We discuss known desmin protein partners and propose an overview of the way that this molecular network could serve as a signal transduction platform necessary for proper muscle function.

Scientific Reports 6: Article number: 2514010.1038/srep25140; published online: April292016; updated: May312016 In the PDF version of this Article, there is a typographical error in the Discussion section, “Human type 2 diabetes patients exhibited increased plasma levels of TIMP1 and TIMP237.” should read: “Human type 2 diabetes patients exhibited increased plasma levels of TIMP1 and TIMP235.”

MMP11 overexpression is a bad prognostic factor in various human carcinomas. Interestingly, this proteinase is not expressed in malignant cells themselves but is secreted by adjacent non-malignant mesenchymal/stromal cells, such as cancer associated fibroblasts (CAFs) and adipocytes (CAAs), which favors cancer cell survival and progression. As MMP11 negatively regulates adipogenesis in vitro, we hypothesized that it may play a role in whole body metabolism and energy homeostasis. We used an in vivo gain- (Mmp11-Tg mice) and loss- (Mmp11−/− mice) of-function approach to address the systemic function of MMP11. Strikingly, MMP11 overexpression protects against type 2 diabetes while Mmp11−/− mice exhibit hallmarks of metabolic syndrome. Moreover, Mmp11-Tg mice were protected from diet-induced obesity and display mitochondrial dysfunction, due to oxidative stress, and metabolic switch from oxidative phosphorylation to aerobic glycolysis. This Warburg-like effect observed in adipose tissues might provide a rationale for the deleterious impact of CAA-secreted MMP11, favouring tumor progression. MMP11 overexpression also leads to increased circulating IGF1 levels and the activation of the IGF1/AKT/FOXO1 cascade, an important metabolic signalling pathway. Our data reveal a major role for MMP11 in controlling energy metabolism, and provide new clues for understanding the relationship between metabolism, cancer progression and patient outcome.

International audience; 1 MMP11 overexpression is a bad prognostic factor in various human carcinomas. Interestingly, this proteinase is not expressed in malignant cells themselves but is secreted by adjacent non-malignant mesenchymal/stromal cells, such as cancer associated fibroblasts (CAFs) and adipocytes (CAAs), which favors cancer cell survival and progression. As MMP11 negatively regulates adipogenesis in vitro, we hypothesized that it may play a role in whole body metabolism and energy homeostasis. We used an in vivo gain-(Mmp11-Tg mice) and loss-(Mmp11−/− mice) of-function approach to address the systemic function of MMP11. Strikingly, MMP11 overexpression protects against type 2 diabetes while Mmp11−/− mice exhibit hallmarks of metabolic syndrome. Moreover, Mmp11-Tg mice were protected from diet-induced obesity and display mitochondrial dysfunction, due to oxidative stress, and metabolic switch from oxidative phosphorylation to aerobic glycolysis. This Warburg-like effect observed in adipose tissues might provide a rationale for the deleterious impact of CAA-secreted MMP11, favouring tumor progression. MMP11 overexpression also leads to increased circulating IGF1 levels and the activation of the IGF1/AKT/FOXO1 cascade, an important metabolic signalling pathway. Our data reveal a major role for MMP11 in controlling energy metabolism, and provide new clues for understanding the relationship between metabolism, cancer progression and patient outcome. More and more epidemiological data attribute a pernicious effect to metabolic disorders, notably obesity and diabetes , on cancer progression. Moreover, metformin, a drug commonly administered for type 2 diabetes, displays antitumor properties 1. To date, most studies have emphasized tumor aggressiveness in a context of already constituted metabolic disorders. Less attention has been given to the ability of cancer cells to impact on metabolism via molecules abnormally expressed by primary tumors and/or metastases. This effect may be of interest and should therefore be evaluated to unravel the link between metabolism and cancer. Matrix metalloproteinase 11 (MMP11; previously named stromelysin-3) 2

Autophagy is a cellular process whereby cytoplasmic substrates are targeted for degradation in the lysosome via the membrane structures autophagosomes. This process is initiated by specific phosphoinositides, PtdIns3P and PtdIns5P, which play a key role in autophagy by recruiting effectors such as Atg18/WIPI2. Therefore, quantifying those lipids is important to better understand the assembly of the complex autophagic machinery. Herein, we describe in detail methods to quantify PtdIns3P and PtdIns5P by specific mass assays feasible in most laboratories.

The myotubularin family of phosphoinositide phosphatases includes several members mutated in neuromuscular diseases or associated with metabolic syndrome, obesity, and cancer. Catalytically dead phosphatases regulate their active homologs by heterodimerization and potentially represent key players in the phosphatase-kinase balance. Although the enzymatic specificity for phosphoinositides indicates a role for myotubularins in endocytosis and membrane trafficking, recent findings in cellular and animal models suggest that myotubularins regulate additional processes including cell proliferation and differentiation, autophagy, cytokinesis, and cytoskeletal and cell junction dynamics. In this review, we discuss how myotubularins regulate such diverse processes, emphasizing newly identified functions in a physiological and pathological context. A better understanding of myotubularin pathophysiology will pave the way towards therapeutic strategies.

Muscle contraction relies on a highly organized intracellular network of membrane organelles and cytoskeleton proteins. Among the latter are the intermediate filaments (IFs), a large family of proteins mutated in more than 30 human diseases. For example, mutations in the DES gene, which encodes the IF desmin, lead to desmin-related myopathy and cardiomyopathy. Here, we demonstrate that myotubularin (MTM1), which is mutated in individuals with X-linked centronuclear myopathy (XLCNM; also known as myotubular myopathy), is a desmin-binding protein and provide evidence for direct regulation of desmin by MTM1 in vitro and in vivo. XLCNM-causing mutations in MTM1 disrupted the MTM1-desmin complex, resulting in abnormal IF assembly and architecture in muscle cells and both mouse and human skeletal muscles. Adeno-associated virus-mediated ectopic expression of WT MTM1 in Mtm1-KO muscle reestablished normal desmin expression and localization. In addition, decreased MTM1 expression and XLCNM-causing mutations induced abnormal mitochondrial positioning, shape, dynamics, and function. We therefore conclude that MTM1 is a major regulator of both the desmin cytoskeleton and mitochondria homeostasis, specifically in skeletal muscle. Defects in IF stabilization and mitochondrial dynamics appear as common physiopathological features of centronuclear myopathies and desmin-related myopathies.

Myotubular myopathy and centronuclear myopathies (CNM) are congenital myopathies characterized by generalized muscle weakness and mislocalization of muscle fiber nuclei. Genetically distinct forms exist, and mutations in BIN1 were recently identified in autosomal recessive cases (ARCNM). Amphiphysins have been implicated in membrane remodeling in brain and skeletal muscle. Our objective was to decipher the pathogenetic mechanisms underlying different forms of CNM, with a focus on ARCNM cases. In this study, we compare the histopathological features from patients with X-linked, autosomal recessive, and dominant forms, respectively, mutated in myotubularin (MTM1), amphiphysin 2 (BIN1), and dynamin 2 (DNM2). We further characterize the ultrastructural defects in ARCNM muscles. We demonstrate that the two BIN1 isoforms expressed in skeletal muscle possess the phosphoinositide-binding domain and are specifically targeted to the triads close to the DHPR-RYR1 complex. Cardiac isoforms do not contain this domain, suggesting that splicing of BIN1 regulates its specific function in skeletal muscle. Immunofluorescence analyses of muscles from patients with BIN1 mutations reveal aberrations of BIN1 localization and triad organization. These defects are also observed in X-linked and autosomal dominant forms of CNM and in Mtm1 knockout mice. In addition to previously reported implications of BIN1 in cancer as a tumor suppressor, these findings sustain an important role for BIN1 skeletal muscle isoforms in membrane remodeling and organization of the excitation-contraction machinery. We propose that aberrant BIN1 localization and defects in triad structure are part of a common pathogenetic mechanism shared between the three forms of centronuclear myopathies.

International audience; Duchenne muscular dystrophy (DMD) is a lethal, X-linked disorder associated with dystrophin deficiency that results in chronic inflammation, sarcolemma damage, and severe skeletal muscle degeneration. Recently, the use of L-arginine, the substrate of nitric oxide synthase (nNOS), has been proposed as a pharmacological treatment to attenuate the dystrophic pattern of DMD. However, little is known about signaling events that occur in dystrophic muscle with l-arginine treatment. Considering the implication of inflammation in dystrophic processes, we asked whether L-arginine inhibits inflammatory signaling cascades. We demonstrate that L-arginine decreases inflammation and enhances muscle regeneration in the mdx mouse model. Classic stimulatory signals, such as proinflammatory cytokines interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha, are significantly decreased in mdx mouse muscle, resulting in lower nuclear factor (NF)-kappaB levels and activity. NF-kappaB serves as a pivotal transcription factor with multiple levels of regulation; previous studies have shown perturbation of NF-kappaB signaling in both mdx and DMD muscle. Moreover, L-arginine decreases the activity of metalloproteinase (MMP)-2 and MMP-9, which are transcriptionally activated by NF-kappaB. We show that the inhibitory effect of L-arginine on the NF-kappaB/MMP cascade reduces beta-dystroglycan cleavage and translocates utrophin and nNOS throughout the sarcolemma. Collectively, our results clarify the molecular events by which L-arginine promotes muscle membrane integrity in dystrophic muscle and suggest that NF-kappaB-related signaling cascades could be potential therapeutic targets for DMD management.

International audience; Nucleus positioning is key for intracellular organization, cell differentiation, and organ development and is affected in many diseases, including myopathies due to alteration in amphiphysin-2 (BIN1). The actin and microtubule cytoskeletons are essential for nucleus positioning, but their crosstalk in this process is sparsely characterized. Here, we report that impairment of amphiphysin/BIN1 in Caenorhabditis elegans, mammalian cells, or muscles from patients with centronuclear myopathy alters nuclear position and shape. We show that AMPH-1/BIN1 binds to nesprin and actin, as well as to the microtubule-binding protein CLIP170 in both species. Expression of the microtubule-anchoring CAP-GLY domain of CLIP170 fused to the nuclear-envelope-anchoring KASH domain of nesprin rescues nuclear positioning defects of amph-1 mutants. Amphiphysins thus play a central role in linking the nuclear envelope with the actin and microtubule cytoskeletons. We propose that BIN1 has a direct and evolutionarily conserved role in nuclear positioning, altered in myopathies.

© 2015 The Authors.Desminopathies belong to a family of muscle disorders called myofibrillar myopathies that are caused by Desmin mutations and lead to protein aggregates in muscle fibers. To date, the initial pathological steps of desminopathies and the impact of desmin aggregates in the genesis of the disease are unclear. Using live, high-resolution microscopy, we show that Desmin loss of function and Desmin aggregates promote skeletal muscle defects and alter heart biomechanics. In addition, we show that the calcium dynamics associated with heart contraction are impaired and are associated with sarcoplasmic reticulum dilatation as well as abnormal subcellular distribution of Ryanodine receptors. Our results demonstrate that desminopathies are associated with perturbed excitation-contraction coupling machinery and that aggregates are more detrimental than Desmin loss of function. Additionally, we show that pharmacological inhibition of aggregate formation and Desmin knockdown revert these phenotypes. Our data suggest alternative therapeutic approaches and further our understanding of the molecular determinants modulating Desmin aggregate formation. Desminopathies are myopathies and cardiomyopathies associated with Desmin mutations leading to protein aggregates. Ramspacher et al. demonstrate that altered Desmin function or expression affect the EC coupling machinery and calcium dynamics. They show that aggregates are more toxic than the loss of function and can be rescued by knockdown and pharmacological treatment.

International audience; Phosphoinositides play a central role in many physiological processes by assisting the recruitment of proteins to membranes through specific phosphoinositide-binding motifs. How this recruitment is coordinated in space and time is not well understood. Here we show that BIN1/M-Amphiphysin2, a protein involved in T-tubule biogenesis in muscle cells and frequently mutated in centronuclear myopathies, clusters PtdIns(4,5)P2 to recruit its downstream partner dynamin. By using several mutants associated with centronuclear myopathies, we find that the N-BAR and the SH3 domains of BIN1 control the kinetics and the accumulation of dynamin on membranes, respectively. We show that phosphoinositide clustering is a mechanism shared by other proteins that interact with PtdIns(4,5)P2, but do not contain a BAR domain. Our numerical simulations point out that clustering is a diffusion-driven process in which phosphoinositide molecules are not sequestered. We propose that this mechanism plays a key role in the recruitment of downstream phosphoinositide-binding proteins.

© 2014 The Authors. Published under the terms of the CC BY 4.0 license. This is an open access article under the terms of the Creative Commons Attribution 4.0 License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited., Mutations in amphiphysin-2/BIN1, dynamin 2, and myotubularin are associated with centronuclear myopathy (CNM), a muscle disorder characterized by myofibers with atypical central nuclear positioning and abnormal triads. Mis-splicing of amphiphysin-2/BIN1 is also associated with myotonic dystrophy that shares histopathological hallmarks with CNM. How amphiphysin-2 orchestrates nuclear positioning and triad organization and how CNM-associated mutations lead to muscle dysfunction remains elusive. We find that N-WASP interacts with amphiphysin-2 in myofibers and that this interaction and N-WASP distribution are disrupted by amphiphysin-2 CNM mutations. We establish that N-WASP functions downstream of amphiphysin-2 to drive peripheral nuclear positioning and triad organization during myofiber formation. Peripheral nuclear positioning requires microtubule/Map7/Kif5b-dependent distribution of nuclei along the myofiber and is driven by actin and nesprins. In adult myofibers, N-WASP and amphiphysin-2 are only involved in the maintenance of triad organization but not in the maintenance of peripheral nuclear positioning. Importantly, we confirmed that N-WASP distribution is disrupted in CNM and myotonic dystrophy patients. Our results support a role for N-WASP in amphiphysin-2-dependent nuclear positioning and triad organization and in CNM and myotonic dystrophy pathophysiology.

Myotubularin (MTM1) and amphiphysin 2 (BIN1) are two proteins mutated in different forms of centronuclear myopathy, but the functional and pathological relationship between these two proteins was unknown. Here, we identified MTM1 as a novel binding partner of BIN1, both in vitro and endogenously in skeletal muscle. Moreover, MTM1 enhances BIN1-mediated membrane tubulation, depending on binding and phosphoinositide phosphatase activity. BIN1 patient mutations induce a conformational change in BIN1 and alter its binding and regulation by MTM1. In conclusion, we identified the first molecular and functional link between MTM1 and BIN1, supporting a common pathological mechanism in different forms of centronuclear myopathy.

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., Author Summary Congenital myopathies are a group of heredity diseases characterized by muscle weakness and impaired locomotion that manifest in both children and adults. X-linked myotubular myopathy (XLMTM) is a subtype of congenital myopathy that predominantly affects males and is caused by mutations in the myotubularin (MTM1) gene. To date, more than 200 pathogenic mutations have been identified in MTM1. However, no effective therapy is available to treat patients presenting with XLMTM. This is largely due to a lack of understanding of molecular processes perturbed in the XLMTM disease state, thereby limiting the availability of suitable therapeutic targets. In this study, we show that catalytically inactive MTMR12 interacts with myotubularin in skeletal muscle. This complex formation is required to provide stability to myotubularin in the normal functioning of skeletal muscle and these interactions appear to be disrupted in XLMTM. This work therefore offers a novel direction for therapy development, both in XLMTM and other genetic diseases, by identifying crucial protein interactors of disease-causing proteins whose complexes might be stabilized in the disease state to restore normal function.

The level and turnover of phosphoinositides (PIs) are tightly controlled by a large set of PI-specific enzymes (PI kinases and phosphatases). Mammalian PI phosphatases are conserved through evolution and among this large family the dual-specificity phosphatase (PTP/DSP) are metal-independent enzymes displaying the amino acid signature Cys-X5-Arg-Thr/Ser (CX5RT/S) in their active site. Such catalytic site characterizes the myotubularin 3-phosphatases that dephosphorylate PtdIns3P and PtdIns(3,5)P₂ and produce PtdIns5P. Substrates of myotubularins have been implicated in endocytosis and membrane trafficking while PtdIns5P may have a role in signal transduction. As a paradox, 6 of the 14 members of the myotubularin family lack enzymatic activity and are considered as dead phosphatases. Several myotubularins have been genetically linked to human diseases: MTM1 is mutated in the congenital myopathy X-linked centronuclear or myotubular myopathy (XLCNM) and MTMR14 (JUMPY) has been linked to an autosomal form of such disease, while MTMR2 and MTMR13 are mutated in Charcot-Marie-Tooth (CMT) neuropathies. Furthermore, recent evidences from genetic association studies revealed that several other myotubularins could be associated to chronic disorders such as cancer and obesity, highlighting their importance for human health. Here, we discuss cellular and physiological roles of myotubularins and their implication in human diseases, and we present potential pathological mechanisms affecting specific tissues in myotubularin-associated diseases.

The level and turnover of phosphoinositides (PIs) are tightly controlled by a large set of PI-specific enzymes (PI kinases and phosphatases). Mammalian PI phosphatases are conserved through evolution and among this large family the dual-specificity phosphatase (PTP/DSP) are metal-independent enzymes displaying the amino acid signature Cys-X5-Arg-Thr/Ser (CX5RT/S) in their active site. Such catalytic site characterizes the myotubularin 3-phosphatases that dephosphorylate PtdIns3P and PtdIns(3,5)P 2 and produce PtdIns5P. Substrates of myotubularins have been implicated in endocytosis and membrane trafficking while PtdIns5P may have a role in signal transduction. As a paradox, 6 of the 14 members of the myotubularin family lack enzymatic activity and are considered as dead phosphatases. Several myotubularins have been genetically linked to human diseases: MTM1 is mutated in the congenital myopathy X-linked centronuclear or myotubular myopathy (XLCNM) and MTMR14 (JUMPY) has been linked to an autosomal form of such disease, while MTMR2 and MTMR13 are mutated in Charcot-Marie-Tooth (CMT) neuropathies. Furthermore, recent evidences from genetic association studies revealed that several other myotubularins could be associated to chronic disorders such as cancer and obesity, highlighting their importance for human health. Here, we discuss cellular and physiological roles of myotubularins and their implication in human diseases, and we present potential pathological mechanisms affecting specific tissues in myotubularin-associated diseases.

Myotubularin MTM1 is a phosphoinositide (PPIn) 3-phosphatase mutated in X-linked centronuclear myopathy (XLCNM; myotubular myopathy). We investigated the involvement of MTM1 enzymatic activity on XLCNM phenotypes. Exogenous expression of human MTM1 in yeast resulted in vacuolar enlargement, as a consequence of its phosphatase activity. Expression of mutants from patients with different clinical progression and determination of PtdIns3P and PtdIns5P cellular levels confirmed the link between vacuolar morphology and MTM1 phosphatase activity, and showed that some disease mutants retain phosphatase activity. Viral gene transfer of phosphatase-dead myotubularin mutants (MTM1C375S and MTM1S376N) significantly improved most histological signs of XLCNM displayed by a Mtm1-null mouse, at similar levels as wild-type MTM1. Moreover, the MTM1C375S mutant improved muscle performance and restored the localization of nuclei, triad alignment, and the desmin intermediate filament network, while it did not normalize PtdIns3P levels, supporting phosphatase-independent roles of MTM1 in maintaining normal muscle performance and organelle positioning in skeletal muscle. Among the different XLCNM signs investigated, we identified only triad shape and fiber size distribution as being partially dependent on MTM1 phosphatase activity. In conclusion, this work uncovers MTM1 roles in the structural organization of muscle fibers that are independent of its enzymatic activity. This underlines that removal of enzymes should be used with care to conclude on the physiological importance of their activity., Author Summary X-linked centronuclear myopathy is a muscle disorder characterized by neonatal hypotonia and abnormal organelle positioning in skeletal muscle. This myopathy is due to different mutations in the MTM1 gene encoding the phosphoinositide phosphatase myotubularin. Disease-causing mutations are found all along the protein sequence and not only in the phosphatase catalytic domain. We investigated the link between myotubularin phosphatase activity and disease phenotypes. We used brewer yeast as a simple cellular model to analyze the in vivo phosphatase activity of different disease mutants. Our results show that mutations responsible for severe forms of myopathy are either active or inactive phosphatases. To further question this finding, we used the mice myotubularin knock-out model that reproduces faithfully the histopathological findings from human patients. Expression of phosphatase-dead mutants improved most phenotypes of knock-out mice comparable to wild-type myotubularin. This shows that the maintenance of normal skeletal muscles is largely independent from myotubularin phosphatase activity, while defects in the activity may participate in the onset of the disease. Moreover, it could have important implications in the design of therapeutic approaches aimed at manipulating the phosphoinositide levels in the different diseases linked to myotubularin homologues. Finally, these data call for cautiousness when manipulating such enzymes to conclude on the physiological relevance of their activity.

IntroductionMuscle fibers can efficiently contract to promote body movement and are site of energy storage.These important properties are sustained by their intricate intracellular structure. In particular,membrane remodelling and trafficking play a central role in these processes. The phosphoinositides(PIs)secondmessengersarekeyregulatorofintramembraneorganizationanddynamics.However,theroles of phosphoinositides and membrane organization in muscle functions under normal and path-ological conditions are not well defined.Two families of PI-phosphatases have been implicated in myopathies: myotubularins and Jumpy/MTMR14(Lecompte etal.,2008).Humanmyotubularins(MTM1andMTMR1-13)arephosphoinositide3-phosphatases or phosphatase-like proteins mutated in several neuromuscular disorders (Laporteet al., 2003; Previtali et al., 2007; Taylor and Dixon, 2003; Wishart and Dixon, 2002). MTM1 ismutated in X-linked myotubular (centronuclear) myopathy (XLMTM) (Laporte et al., 1996), whereasMTMR2 and MTMR13 are mutated in demyelinating Charcot–Marie–Tooth neuropathy types 4B1 and4B2,respectively(Azzedineetal.,2003;Bolinoetal.,2000;Sendereketal.,2003).Myotubularinssharehomology tothe catalytic domainsof protein tyrosine phosphatases and dual-specificity phosphatases

International audience; The potential role and function of gastrokine-1 (GNK1) in smooth muscle cells is investigated in this work by first establishing a preparative protocol to obtain this native protein from freshly dissected chicken gizzard. Some unexpected biochemical properties of gastrokine-1 were deduced by producing specific polyclonal antibody against the purified protein. We focused on the F-actin interaction with gastrokine-1 and the potential role and function in smooth muscle contractile properties. BACKGROUND: GNK1 is thought to provide mucosal protection in the superficial gastric epithelium. However, the actual role of gastrokine-1 with regards to its known decreased expression in gastric cancer is still unknown. Recently, trefoil factors (TFF) were reported to have important roles in gastric epithelial regeneration and cell turnover, and could be involved in GNK1 interactions. The aim of this study was to evaluate the role and function of GNK1 in smooth muscle cells. METHODOLOGY/PRINCIPAL FINDINGS: From fresh chicken gizzard smooth muscle, an original purification procedure was used to purify a heat soluble 20 kDa protein that was sequenced and found to correspond to the gastrokine-1 protein sequence containing one BRICHOS domain and at least two or possibly three transmembrane regions. The purified protein was used to produce polyclonal antibody and highlighted the smooth muscle cell distribution and F-actin association of GNK1 through a few different methods. CONCLUSION/SIGNIFICANCE: Altogether our data illustrate a broader distribution of gastrokine-1 in smooth muscle than only in the gastrointestinal epithelium, and the specific interaction with F-actin highlights and suggests a new role and function of GNK1 within smooth muscle cells. A potential role via TFF interaction in cell-cell adhesion and assembly of actin stress fibres is discussed.

Duchenne muscular dystrophy muscles undergo increased oxidative stress and altered calcium homeostasis, which contribute to myofiber loss by trigging both necrosis and apoptosis. Here, we asked whether treatment with free radical scavengers could improve the dystrophic pattern of mdx muscles. Five-week-old mdx mice were treated for 2 weeks with alpha-lipoic acid/l-carnitine. This treatment decreased the plasmatic creatine kinase level, the antioxidant enzyme activity, and lipid peroxidation products in mdx diaphragm. Free radical scavengers also modulated the phosphorylation/activity of some component of the mitogen-activated protein kinase (MAPK) cascades: p38 MAPK, the extracellular signal-related kinase, and the Jun kinase. beta-Dystroglycan (beta-DG), a multifunctional adaptor or scaffold capable of interacting with components of the extracellular signal-related kinase-MAP kinase cascade, was also affected after treatment. In the mdx muscles, beta-DG (43 kd) was cleaved by matrix metalloproteinases into a 30-kd form (beta-DG30). We show that the proinflammatory protein nuclear factor-kappaB activator decreased after the treatment, leading to a significant reduction of matrix metalloproteinase activity in the mdx diaphragm. Our data highlight the implication of oxidative stress and cell signaling defects in dystrophin-deficient muscle via the MAP kinase cascade-beta-DG interaction and nuclear factor-kappaB-mediated inflammation process.

Previous studies have shown a blunted ventilatory response to hypercapnia in mdx mice older than 7 months. We test the hypothesis that in the mdx mice ventilatory response changes with age, concomitantly with the increased functional impairment of the respiratory muscles. We thus studied the ventilatory response to CO(2) in 5 and 16 month-old mdx and C57BL10 mice (n = 8 for each group). Respiratory rate (RR), tidal volume (VT), and minute ventilation (VE) were measured, using whole-body plethysmography, during air breathing and in response to hypercapnia (3, 5 and 8% CO(2)). The ventilatory protocol was completed by histological analysis of the diaphragm and intercostals muscles. During air breathing, the 16 month-old mdx mice showed higher RR and, during hypercapnia (at 8% CO(2) breathing), significantly lower RR (226 +/- 26 vs. 270 +/- 21 breaths/min) and VE (1.81 +/- 0.35 vs. 3.96 +/- 0.59 ml min(-1) g(-1)) (P < 0.001) in comparison to C57BL10 controls. On the other hand, 5 month-old C57BL10 and mdx mice did not present any difference in their ventilatory response to air breathing and to hypercapnia. In conclusion, this study shows similar ventilation during air breathing and in response to hypercapnia in the 5 month-old mdx and control mice, in spite of significant pathological structural changes in the respiratory muscles of the mdx mice. However in the 16 month-old mdx mice we observed altered ventilation under air and blunted ventilation response to hypercapnia compared to age-matched control mice. Ventilatory response to hypercapnia thus changes with age in mdx mice, in line with the increased histological damage of their respiratory muscles.

In the peripheral nervous system, utrophin and the short dystrophin isoform (Dp116) are co-localized at the outermost layer of the myelin sheath of nerve fibers; together with the dystroglycan complex. Dp116 is associated with multiple glycoproteins, i.e. sarcoglycans, and alpha- and beta-dystroglycan, which anchor the cytoplasmic protein subcomplex to the extracellular basal lamina. In peripheral nerve, matrix metalloproteinase activity disrupts the dystroglycan complex by cleaving the extracellular domain of beta-dystroglycan. Metalloproteinase creates a 30 kDa fragment of beta-dystroglycan, leading to a disruption of the link between the extracellular matrix and the cell membrane. Here we asked if the processing of the beta-dystroglycan could influence the anchorage of Dp116 and/or utrophin in normal and mdx Schwann cell membrane. We showed that metalloproteinase-9 was more activated in mdx nerve than in wild-type ones. This activation leads to an accumulation of the 30 kDa beta-dystroglycan isoform and has an impact on the anchorage of Dp116 and utrophin isoforms in mdx Schwann cells membrane. Our results showed that Dp116 had greater affinity to the full length form of beta-dystroglycan than the 30 kDa form. Moreover, we showed for the first time that the short isoform of utrophin (Up71) was over-expressed in mdx Schwann cells compared with wild-type. In addition, this utrophin isoform (Up71) seems to have greater affinity to the 30 kDa beta-dystroglycan which could explain the increased stabilization of this 30 kDa form at the membrane compartment. Our results highlight the potential participation of the short utrophin isoform and the cleaved form of beta-dystroglycan in mdx Schwann cell membrane architecture. We proposed that these two proteins could be implicated in Schwann cell proliferation in response to a microenvironment stress such as mediated by accumulating macrophages in mdx mouse muscle inflammation sites.

Muscle fibers attach to laminin in the basal lamina using two mechanisms, i.e., dystrophin with its associated proteins and alpha7beta1 integrin. In humans, gene-mutation defects in one member of these complexes result in muscular dystrophies. This study revealed changes after L-arginine treatment of utrophin-associated proteins and the alpha7B integrin subunit in mdx mouse, a dystrophin-deficient animal model. In the two studied muscles (cardiac muscle and diaphragm), the alpha7B integrin subunit was increased in 5-week-old treated mice. Interestingly, the diaphragm histopathological appearance was significantly improved by L-arginine administration. These results highlight a possible way to compensate for dystrophin deficiency via alpha7beta1 integrin.

The X-linked centronuclear myopathy, also called myotubular myopathy, is a muscle disorder characterized by neonatal hypotonia and abnormal organelles positioning in skeletal muscle. This myopathy is due to different mutations in the MTM1 gene encoding the phosphoinositide phosphatase myotubularin. Disease-causing mutations are found all along the protein sequence and not only in the phosphatase catalytic domain. We investigated the link between myotubularin phosphatase activity and disease phenotypes. We used brewer yeast as a simple cellular model to analyze the in vivo phosphatase activity of different disease mutants. Our results show that mutations responsible for severe forms of myopathy are either active or inactive phosphatases. To further question this finding, we used the murine myotubularin knock-out model that reproduces faithfully the histopathological findings from human patients. Expression of phosphatase-inactive mutants improves the phenotypes of the knock-out mice comparable to wild-type myotubularin. This shows that the maintenance of normal skeletal muscles is largely independent from myotubularin phosphatase activity. Moreover, it could have important implications in the design of therapeutic approaches aiming at manipulating the phosphoinositide level in the different diseases linked to myotubularin homologues. Finally, this work underlines that removal of enzymes should be used with care to conclude on the physiological importance of their activity.

P1.35 Amphiphysin 2 (BIN1) and triad defects in several forms of centronuclear myopathies A. Toussaint , M. Maurer , B.S. Cowling , K. Hnia , M. Mohr , A. Oldfors , Y. Schwab , U. Yis , T. Maisonobe , T. Stojkovic , V. Laugel , A. Echaniz-Laguna , S. Blot , J.L. Mandel , L. Tiret , I. Nishino , J. Laporte 1 1 IGBMC, Illkirch, France, 2 INRA, UMR955 de Genetique Fonctionnelle et Medicale, Ecole Nationale Veterinaire d’Alfort, Maisons-Alfort, France, Centre Hospitalier Universitaire (CHU) Hautepierre, Strasbourg, France, 4 Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden, Gaziantep Children’s Hospital, Division of Child Neurology, Gaziantep, Turkey, Groupe Hospitalier Pitie-Salpetriere, Paris, France, 7 Institut de Myologie, Groupe Hospitalier Pitie-Salpetriere, Paris, France, Hopital Civil, Strasbourg, France, 9 Ecole Nationale Veterinaire d’Alfort, Maisons-Alfort, France, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan