Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 25
Filtrar
Mais filtros

Base de dados
Tipo de documento
Intervalo de ano de publicação
1.
Hum Mutat ; 41(3): 619-631, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31765060

RESUMO

MUSK encodes the muscle-specific receptor tyrosine kinase (MuSK), a key component of the agrin-LRP4-MuSK-DOK7 signaling pathway, which is essential for the formation and maintenance of highly specialized synapses between motor neurons and muscle fibers. We report a patient with severe early-onset congenital myasthenic syndrome and two novel missense mutations in MUSK (p.C317R and p.A617V). Functional studies show that MUSK p.C317R, located at the frizzled-like cysteine-rich domain of MuSK, disrupts an integral part of MuSK architecture resulting in ablated MuSK phosphorylation and acetylcholine receptor (AChR) cluster formation. MUSK p.A617V, located at the kinase domain of MuSK, enhances MuSK phosphorylation resulting in anomalous AChR cluster formation. The identification and evidence for pathogenicity of MUSK mutations supported the initiation of treatment with ß2-adrenergic agonists with a dramatic improvement of muscle strength in the patient. This work suggests uncharacterized mechanisms in which control of the precise level of MuSK phosphorylation is crucial in governing synaptic structure.


Assuntos
Mutação , Síndromes Miastênicas Congênitas/diagnóstico , Síndromes Miastênicas Congênitas/genética , Receptores Proteína Tirosina Quinases/genética , Receptores Colinérgicos/genética , Sinapses/genética , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Agonistas de Receptores Adrenérgicos beta 2/uso terapêutico , Alelos , Substituição de Aminoácidos , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Análise Mutacional de DNA , Feminino , Marcação de Genes , Humanos , Camundongos , Modelos Moleculares , Conformação Molecular , Proteínas Musculares/metabolismo , Síndromes Miastênicas Congênitas/tratamento farmacológico , Síndromes Miastênicas Congênitas/metabolismo , Linhagem , Fosforilação , Receptores Proteína Tirosina Quinases/química , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Colinérgicos/química , Receptores Colinérgicos/metabolismo , Relação Estrutura-Atividade , Sinapses/metabolismo
2.
Hum Mol Genet ; 26(13): 2377-2385, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28379354

RESUMO

Spinal muscular atrophy (SMA) is a common and often fatal neuromuscular disorder caused by low levels of the Survival Motor Neuron (SMN) protein. Amongst the earliest detectable consequences of SMN deficiency are profound defects of the neuromuscular junctions (NMJs). In model mice these synapses appear disorganized, fail to mature and are characterized by poorly arborized nerve terminals. Given one role of the SMN protein in orchestrating the assembly of spliceosomal snRNP particles and subsequently regulating the alternative splicing of pre-mRNAs, a plausible link between SMN function and the distal neuromuscular SMA phenotype is an incorrectly spliced transcript or transcripts involved in establishing or maintaining NMJ structure. In this study, we explore the effects of one such transcript-Z+Agrin-known to be a critical organizer of the NMJ. We confirm that low SMN protein reduces motor neuronal levels of Z+Agrin. Repletion of this isoform of Agrin in the motor neurons of SMA model mice increases muscle fiber size, enhances the post-synaptic NMJ area, reduces the abnormal accumulation of intermediate filaments in nerve terminals of the neuromuscular synapse and improves the innervation of muscles. While these effects are independent of changes in SMN levels or increases in motor neuron numbers they nevertheless have a significant effect on the overall disease phenotype, enhancing mean survival in severely affected SMA model mice by ∼40%. We conclude that Agrin is an important target of the SMN protein and that mitigating NMJ defects may be one strategy in treating human spinal muscular atrophy.


Assuntos
Agrina/genética , Junção Neuromuscular/metabolismo , Agrina/metabolismo , Processamento Alternativo , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular Espinal/genética , Proteínas do Tecido Nervoso/genética , Doenças Neuromusculares/genética , Doenças Neuromusculares/metabolismo , Junção Neuromuscular/genética , Isoformas de Proteínas/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Sinapses/metabolismo
3.
Nature ; 495(7442): 474-80, 2013 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-23474986

RESUMO

CLP1 was the first mammalian RNA kinase to be identified. However, determining its in vivo function has been elusive. Here we generated kinase-dead Clp1 (Clp1(K/K)) mice that show a progressive loss of spinal motor neurons associated with axonal degeneration in the peripheral nerves and denervation of neuromuscular junctions, resulting in impaired motor function, muscle weakness, paralysis and fatal respiratory failure. Transgenic rescue experiments show that CLP1 functions in motor neurons. Mechanistically, loss of CLP1 activity results in accumulation of a novel set of small RNA fragments, derived from aberrant processing of tyrosine pre-transfer RNA. These tRNA fragments sensitize cells to oxidative-stress-induced p53 (also known as TRP53) activation and p53-dependent cell death. Genetic inactivation of p53 rescues Clp1(K/K) mice from the motor neuron loss, muscle denervation and respiratory failure. Our experiments uncover a mechanistic link between tRNA processing, formation of a new RNA species and progressive loss of lower motor neurons regulated by p53.


Assuntos
Neurônios Motores/metabolismo , Neurônios Motores/patologia , RNA de Transferência de Tirosina/metabolismo , Fatores de Transcrição/metabolismo , Esclerose Lateral Amiotrófica , Animais , Animais Recém-Nascidos , Axônios/metabolismo , Axônios/patologia , Morte Celular , Diafragma/inervação , Perda do Embrião , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Éxons/genética , Feminino , Fibroblastos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Atrofia Muscular Espinal , Doenças Neuromusculares/metabolismo , Doenças Neuromusculares/patologia , Estresse Oxidativo , Processamento Pós-Transcricional do RNA , RNA de Transferência de Tirosina/genética , Proteínas de Ligação a RNA , Respiração , Nervos Espinhais/citologia , Fatores de Transcrição/deficiência , Proteína Supressora de Tumor p53/metabolismo , Tirosina/genética , Tirosina/metabolismo
4.
J Neurochem ; 143(5): 569-583, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28555889

RESUMO

Inherited deficiency in ether lipids, a subgroup of phospholipids whose biosynthesis needs peroxisomes, causes the fatal human disorder rhizomelic chondrodysplasia punctata. The exact roles of ether lipids in the mammalian organism and, therefore, the molecular mechanisms underlying the disease are still largely enigmatic. Here, we used glyceronephosphate O-acyltransferase knockout (Gnpat KO) mice to study the consequences of complete inactivation of ether lipid biosynthesis and documented substantial deficits in motor performance and muscle strength of these mice. We hypothesized that, probably in addition to previously described cerebellar abnormalities and myelination defects in the peripheral nervous system, an impairment of neuromuscular transmission contributes to the compromised motor abilities. Structurally, a morphologic examination of the neuromuscular junction (NMJ) in diaphragm muscle at different developmental stages revealed aberrant axonal branching and a strongly increased area of nerve innervation in Gnpat KO mice. Post-synaptically, acetylcholine receptor (AChR) clusters colocalized with nerve terminals within a widened endplate zone. In addition, we detected atypical AChR clustering, as indicated by decreased size and number of clusters following stimulation with agrin, in vitro. The turnover of AChRs was unaffected in ether lipid-deficient mice. Electrophysiological evaluation of the adult diaphragm indicated that although evoked potentials were unaltered in Gnpat KO mice, ether lipid deficiency leads to fewer spontaneous synaptic vesicle fusion events but, conversely, an increased post-synaptic response to spontaneous vesicle exocytosis. We conclude from our findings that ether lipids are essential for proper development and function of the NMJ and may, therefore, contribute to motor performance. Read the Editorial Highlight for this article on page 463.


Assuntos
Força Muscular/fisiologia , Debilidade Muscular/fisiopatologia , Junção Neuromuscular/fisiopatologia , Fosfolipídeos/deficiência , Animais , Diafragma/metabolismo , Modelos Animais de Doenças , Camundongos Knockout , Debilidade Muscular/metabolismo , Junção Neuromuscular/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Colinérgicos/metabolismo , Transmissão Sináptica/fisiologia
5.
Mol Cell Proteomics ; 13(8): 1993-2003, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24899341

RESUMO

The development of the neuromuscular synapse depends on signaling processes that involve protein phosphorylation as a crucial regulatory event. Muscle-specific kinase (MuSK) is the key signaling molecule at the neuromuscular synapse whose activity is required for the formation of a mature and functional synapse. However, the signaling cascade downstream of MuSK and the regulation of the different components are still poorly understood. In this study we used a quantitative phosphoproteomics approach to study the phosphorylation events and their temporal regulation downstream of MuSK. We identified a total of 10,183 phosphopeptides, of which 203 were significantly up- or down-regulated. Regulated phosphopeptides were classified into four different clusters according to their temporal profiles. Within these clusters we found an overrepresentation of specific protein classes associated with different cellular functions. In particular, we found an enrichment of regulated phosphoproteins involved in posttranscriptional mechanisms and in cytoskeletal organization. These findings provide novel insights into the complex signaling network downstream of MuSK and form the basis for future mechanistic studies.


Assuntos
Músculo Esquelético/metabolismo , Fosfopeptídeos/isolamento & purificação , Proteômica/métodos , Receptores Proteína Tirosina Quinases/metabolismo , Agrina/farmacologia , Animais , Linhagem Celular , Citoesqueleto/metabolismo , Regulação da Expressão Gênica , Camundongos , Fosfopeptídeos/metabolismo , Processamento Pós-Transcricional do RNA , Transdução de Sinais
6.
Artigo em Inglês | MEDLINE | ID: mdl-38697654

RESUMO

A coordinated and complex interplay of signals between motor neurons, skeletal muscle cells, and Schwann cells controls the formation and maintenance of neuromuscular synapses. Deficits in the signaling pathway for building synapses, caused by mutations in critical genes or autoantibodies against key proteins, are responsible for several neuromuscular diseases, which cause muscle weakness and fatigue. Here, we describe the role that four key genes, Agrin, Lrp4, MuSK, and Dok7, play in this signaling pathway, how an understanding of their mechanisms of action has led to an understanding of several neuromuscular diseases, and how this knowledge has contributed to emerging therapies for treating neuromuscular diseases.


Assuntos
Junção Neuromuscular , Transdução de Sinais , Humanos , Animais , Agrina/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Musculares/metabolismo , Doenças Neuromusculares , Receptores Colinérgicos/metabolismo , Sinapses/fisiologia , Sinapses/metabolismo , Neurônios Motores/fisiologia , Neurônios Motores/metabolismo
7.
Mol Cell Neurosci ; 49(4): 475-86, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22210232

RESUMO

Efficient synaptic transmission at the neuromuscular junction (NMJ) requires the topological maturation of the postsynaptic apparatus from an oval acetylcholine receptor (AChR)-rich plaque into a complex pretzel-shaped array of branches. However, compared to NMJ formation very little is known about the mechanisms that regulate NMJ maturation. Recently the process of in vivo transformation from plaque into pretzel has been reproduced in vitro by culturing myotubes aneurally on laminin-coated substrate. It was proposed that the formation of complex AChR clusters is regulated by a MuSK-dependent muscle intrinsic program. To elucidate the structure-function role of MuSK in the aneural maturation of AChR pretzels, we used muscle cell lines expressing MuSK mutant and chimeric proteins. Here we report, that besides its role during agrin-induced AChR clustering, MuSK kinase activity is also necessary for substrate-dependent cluster formation. Constitutive-active MuSK induces larger AChR clusters, a faster cluster maturation on laminin and increases the anchorage of AChRs to the cytoskeleton compared to MuSK wild-type. In addition, we find that the juxtamembrane region of MuSK, which has previously been shown to regulate agrin-induced AChR clustering, is unable to induce complex AChR clusters on laminin substrate. Most interestingly, MuSK kinase activity is not sufficient for laminin-dependent AChR cluster formation since the MuSK ectodomain is also required suggesting a so far undiscovered instructive role for the extracellular domain of MuSK.


Assuntos
Junção Neuromuscular/metabolismo , Receptores Proteína Tirosina Quinases/química , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Colinérgicos/química , Receptores Colinérgicos/metabolismo , Animais , Linhagem Celular , Humanos , Immunoblotting , Junção Neuromuscular/química , Estrutura Terciária de Proteína , Ratos
8.
Cell Signal ; 104: 110584, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36608736

RESUMO

Muscle-specific kinase (MuSK) is the key regulator of neuromuscular junction development. MuSK acts via several distinct pathways and is responsible for pre- and postsynaptic differentiation. MuSK is unique among receptor tyrosine kinases as activation and signaling are particularly tightly regulated. Initiation of kinase activity requires Agrin, a heparan sulphate proteoglycan derived from motor neurons, the low-density lipoprotein receptor-related protein-4 (Lrp4) and the intracellular adaptor protein Dok-7. There is a great knowledge gap between MuSK activation and downstream signaling. Recent studies using omics techniques have addressed this knowledge gap, thereby greatly contributing to a better understanding of MuSK signaling. Impaired MuSK signaling causes severe muscle weakness as described in congenital myasthenic syndromes or myasthenia gravis but the underlying pathophysiology is often unclear. This review focuses on recent advances in deciphering MuSK activation and downstream signaling. We further highlight latest break-throughs in understanding and treatment of MuSK-related disorders and discuss the role of MuSK in non-muscle tissue.


Assuntos
Junção Neuromuscular , Receptores Colinérgicos , Fosforilação , Junção Neuromuscular/genética , Junção Neuromuscular/metabolismo , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas Musculares/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo
9.
J Exp Med ; 220(11)2023 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-37703004

RESUMO

T follicular helper (Tfh) cells are essential for the development of germinal center B cells and high-affinity antibody-producing B cells in humans and mice. Here, we identify the guanine nucleotide exchange factor (GEF) Rin-like (Rinl) as a negative regulator of Tfh generation. Loss of Rinl leads to an increase of Tfh in aging, upon in vivo immunization and acute LCMV Armstrong infection in mice, and in human CD4+ T cell in vitro cultures. Mechanistically, adoptive transfer experiments using WT and Rinl-KO naïve CD4+ T cells unraveled T cell-intrinsic GEF-dependent functions of Rinl. Further, Rinl regulates CD28 internalization and signaling, thereby shaping CD4+ T cell activation and differentiation. Thus, our results identify the GEF Rinl as a negative regulator of global Tfh differentiation in an immunological context and species-independent manner, and furthermore, connect Rinl with CD28 internalization and signaling pathways in CD4+ T cells, demonstrating for the first time the importance of endocytic processes for Tfh differentiation.


Assuntos
Antígenos CD28 , Fatores de Troca do Nucleotídeo Guanina , Humanos , Animais , Camundongos , Transdução de Sinais , Diferenciação Celular , Transferência Adotiva
10.
Biochim Biophys Acta ; 1813(6): 1198-210, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21419809

RESUMO

RIN proteins serve as guanine nucleotide exchange factors for Rab5a. They are characterized by the presence of a RIN homology domain and a C-terminal Vps9 domain. Currently three family members have been described and analyzed. Here we report the identification of a novel RIN family member, Rin-like (Rinl), that represents a new interaction partner of the receptor tyrosine kinase MuSK, which is an essential key regulator of neuromuscular synapse development. Rinl is localized to neuromuscular synapses but shows the highest expression in thymus and spleen. Rinl preferentially binds to nucleotide-free Rab5a and catalyzes the exchange of GDP for GTP. Moreover, Rinl also binds GDP-bound Rab22 and increases the GDP/GTP exchange implicating Rinl in endocytotic processes regulated by Rab5a and Rab22. Interestingly, Rinl shows a higher catalytic rate for Rab22 compared to Rab5a. Rinl is closely associated with the cytoskeleton and thus contributes to the spatial control of Rab5a and Rab22 signaling at actin-positive compartments. Most importantly, overexpression of Rinl affects fluid-phase as well as EGFR endocytosis.


Assuntos
Endocitose , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Animais , Células COS , Chlorocebus aethiops , Citoesqueleto/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Microscopia Confocal , Ligação Proteica , Ratos , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas rab de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/genética
11.
Proc Natl Acad Sci U S A ; 106(9): 3513-8, 2009 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-19221030

RESUMO

Synapse formation at the neuromuscular junction (NMJ) requires an alternatively spliced variant of agrin (Z(+) agrin) that is produced only by neurons. Here, we show that Nova1 and Nova2, neuron-specific splicing factors identified as targets in autoimmune motor disease, are essential regulators of Z(+) agrin. Nova1/Nova2 double knockout mice are paralyzed and fail to cluster AChRs at the NMJ, and breeding them with transgenic mice constitutively expressing Z(+) agrin in motor neurons rescued AChR clustering. Surprisingly, however, these rescued mice remained paralyzed, while electrophysiologic studies demonstrated that the motor axon and synapse were functional-spontaneous and evoked recordings revealed synaptic transmission and muscle contraction. These results point to a proximal defect in motor neuron firing in the absence of Nova and reveal a previously unsuspected role for RNA regulation in the physiologic activation of motor neurons.


Assuntos
Agrina/metabolismo , Processamento Alternativo/genética , Antígenos de Neoplasias/metabolismo , Doença dos Neurônios Motores/metabolismo , Doença dos Neurônios Motores/fisiopatologia , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sinapses/metabolismo , Agrina/química , Agrina/genética , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/genética , Eletrofisiologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Doença dos Neurônios Motores/genética , Proteínas do Tecido Nervoso/genética , Antígeno Neuro-Oncológico Ventral , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Ligação a RNA/genética
12.
Front Mol Neurosci ; 15: 780659, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35370548

RESUMO

Muscle-specific kinase (MuSK) is a receptor tyrosine kinase absolutely required for neuromuscular junction formation. MuSK is activated by binding of motor neuron-derived Agrin to low-density lipoprotein receptor related protein 4 (Lrp4), which forms a complex with MuSK. MuSK activation and downstream signaling are critical events during the development of the neuromuscular junction. Receptor tyrosine kinases are commonly internalized upon ligand binding and crosstalk between endocytosis and signaling has been implicated. To extend our knowledge about endocytosis of synaptic proteins and its role during postsynaptic differentiation at the neuromuscular junction, we studied the stability and internalization of Lrp4, MuSK and acetylcholine receptors (AChRs) in response to Agrin. We provide evidence that MuSK but not Lrp4 internalization is increased by Agrin stimulation. MuSK kinase-activity is not sufficient to induce MuSK internalization and the absence of Lrp4 has no effect on MuSK endocytosis. Moreover, MuSK internalization and signaling are unaffected by the inhibition of Dynamin suggesting that MuSK endocytosis uses a non-conventional pathway and is not required for MuSK-dependent downstream signaling.

13.
Neurosci Lett ; 716: 134676, 2020 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-31811897

RESUMO

The receptor tyrosine kinase MuSK (muscle-specific kinase) is the key signaling molecule during the formation of a mature and functional neuromuscular junction (NMJ). Signal transduction events downstream of MuSK activation induce both pre- and postsynaptic differentiation, which, most prominently, includes the clustering of acetylcholine receptors (AChRs) at synaptic sites. MuSK activation requires a complex interplay between its co-receptor Lrp4 (low-density lipoprotein receptor-related protein-4), the motor neuron-derived heparan-sulfate proteoglycan Agrin and the intracellular adaptor protein Dok-7. A tight regulation of MuSK kinase activity is crucial for proper NMJ development. Defects in MuSK signaling are the cause of muscle weakness as reported in congenital myasthenic syndromes and myasthenia gravis. This review focuses on recent structure-based analyses of MuSK, Agrin, Lrp4 and Dok-7 interactions and their function during MuSK activation. Conclusions about the regulation of the MuSK kinase that were derived from molecular structures will be highlighted. In addition, the role of MuSK during development and disease will be discussed.


Assuntos
Doenças da Junção Neuromuscular/metabolismo , Junção Neuromuscular/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Colinérgicos/metabolismo , Agrina/metabolismo , Animais , Humanos , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteínas Musculares/metabolismo
14.
Cells ; 8(7)2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31269763

RESUMO

Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction (NMJ). Autoantibodies target key molecules at the NMJ, such as the nicotinic acetylcholine receptor (AChR), muscle-specific kinase (MuSK), and low-density lipoprotein receptor-related protein 4 (Lrp4), that lead by a range of different pathogenic mechanisms to altered tissue architecture and reduced densities or functionality of AChRs, reduced neuromuscular transmission, and therefore a severe fatigable skeletal muscle weakness. In this review, we give an overview of the history and clinical aspects of MG, with a focus on the structure and function of myasthenic autoantigens at the NMJ and how they are affected by the autoantibodies' pathogenic mechanisms. Furthermore, we give a short overview of the cells that are implicated in the production of the autoantibodies and briefly discuss diagnostic challenges and treatment strategies.


Assuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Músculo Esquelético/patologia , Miastenia Gravis/imunologia , Junção Neuromuscular/patologia , Agrina/imunologia , Agrina/metabolismo , Animais , Autoantígenos/metabolismo , Humanos , Proteínas Relacionadas a Receptor de LDL/imunologia , Proteínas Relacionadas a Receptor de LDL/metabolismo , Músculo Esquelético/imunologia , Músculo Esquelético/ultraestrutura , Miastenia Gravis/patologia , Junção Neuromuscular/imunologia , Junção Neuromuscular/ultraestrutura , Receptores Proteína Tirosina Quinases/imunologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Colinérgicos/imunologia , Receptores Colinérgicos/metabolismo , Receptores Nicotínicos/imunologia , Receptores Nicotínicos/metabolismo
15.
Biotechnol J ; 12(1)2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27420480

RESUMO

The question whether two proteins interact with each other or whether a protein localizes to a certain region of the cell is often addressed with fluorescence microscopy and analysis of a potential colocalization of fluorescence markers. Since a mere visual estimation does not allow quantification of the degree of colocalization, different statistical methods of pixel-intensity correlation are commonly used to score it. We observed that these correlation coefficients are prone to false positive results and tend to show high values even for molecules that reside in different organelles. Our aim was to improve this type of analysis and we developed a novel method combining object-recognition based colocalization analysis with pixel-intensity correlation to calculate an object-corrected Pearson coefficient. We designed a macro for the Fiji-version of the software ImageJ and tested the performance systematically with various organelle markers revealing an improved robustness of our approach over classical methods. In order to prove that colocalization does not necessarily mean a physical interaction, we performed FRET (fluorescence resonance energy transfer) microscopy. This confirmed that non-interacting molecules can exhibit a nearly complete colocalization, but that they do not show any significant FRET signal in contrast to proteins that are bound to each other.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Compartimento Celular , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal/métodos , Imagem Molecular/métodos , Software
17.
Mol Biol Cell ; 25(25): 4130-49, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25318670

RESUMO

Mutations in the cytolinker protein plectin lead to grossly distorted morphology of neuromuscular junctions (NMJs) in patients suffering from epidermolysis bullosa simplex (EBS)-muscular dystrophy (MS) with myasthenic syndrome (MyS). Here we investigated whether plectin contributes to the structural integrity of NMJs by linking them to the postsynaptic intermediate filament (IF) network. Live imaging of acetylcholine receptors (AChRs) in cultured myotubes differentiated ex vivo from immortalized plectin-deficient myoblasts revealed them to be highly mobile and unable to coalesce into stable clusters, in contrast to wild-type cells. We found plectin isoform 1f (P1f) to bridge AChRs and IFs via direct interaction with the AChR-scaffolding protein rapsyn in an isoform-specific manner; forced expression of P1f in plectin-deficient cells rescued both compromised AChR clustering and IF network anchoring. In conditional plectin knockout mice with gene disruption in muscle precursor/satellite cells (Pax7-Cre/cKO), uncoupling of AChRs from IFs was shown to lead to loss of postsynaptic membrane infoldings and disorganization of the NMJ microenvironment, including its invasion by microtubules. In their phenotypic behavior, mutant mice closely mimicked EBS-MD-MyS patients, including impaired body balance, severe muscle weakness, and reduced life span. Our study demonstrates that linkage to desmin IF networks via plectin is crucial for formation and maintenance of AChR clusters, postsynaptic NMJ organization, and body locomotion.


Assuntos
Filamentos Intermediários/metabolismo , Proteínas Musculares/metabolismo , Junção Neuromuscular/metabolismo , Plectina/metabolismo , Animais , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Knockout , Atividade Motora , Fibras Musculares Esqueléticas/metabolismo , Força Muscular , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/metabolismo , Transporte Proteico
18.
FEBS J ; 280(14): 3281-97, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23621612

RESUMO

Muscle-specific kinase (MuSK), a receptor tyrosine kinase, is the key player during the formation of the neuromuscular junction. Signal transduction events downstream of MuSK activation induce both pre- and postsynaptic differentiation, which, most prominently, includes the clustering of acetylcholine receptors at synaptic sites. More recently, regulated MuSK endocytosis and degradation have been implicated as crucial events for MuSK signalling activity, implicating a cross-talk between signalling and endocytosis. In the present study, we use a live imaging approach to study MuSK endocytosis. We find that MuSK is internalized via a clathrin-, dynamin-dependent pathway. MuSK is transported to Rab7-positive endosomes for degradation and recycled via Rab4- and Rab11-positive vesicles. MuSK activation by Dok7 mildly affects the localization of MuSK on the cell surface but has no effect on the rate of MuSK internalization. Interestingly, MuSK colocalizes with actin and Arf6 at the cell surface and during endosomal trafficking. Disruption of the actin cytoskeleton or the proper function of Arf6 concentrates MuSK in cell protrusions. Moreover, inhibition of Arf6 or cytoskeletal rearrangements impairs acetylcholine receptor clustering and phosphorylation. These results suggest that MuSK uses both classical and nonclassical endosomal pathways that involve a variety of different components of the endosomal machinery.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Actinas/metabolismo , Clatrina/metabolismo , Endocitose , Receptores Proteína Tirosina Quinases/metabolismo , Fator 6 de Ribosilação do ADP , Agrina/metabolismo , Animais , Células COS , Chlorocebus aethiops , Citoesqueleto/metabolismo , Dinaminas/metabolismo , Endossomos/enzimologia , Camundongos , Proteínas Musculares/metabolismo , Transporte Proteico , Proteólise , Receptores Colinérgicos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab4 de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7
19.
PLoS One ; 8(1): e53826, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23326516

RESUMO

Congenital myasthenic syndromes (CMSs) are a heterogeneous group of genetic disorders affecting neuromuscular transmission. The agrin/muscle-specific kinase (MuSK) pathway is critical for proper development and maintenance of the neuromuscular junction (NMJ). We report here an Iranian patient in whom CMS was diagnosed since he presented with congenital and fluctuating bilateral symmetric ptosis, upward gaze palsy and slowly progressive muscle weakness leading to loss of ambulation. Genetic analysis of the patient revealed a homozygous missense mutation c.2503A>G in the coding sequence of MUSK leading to the p.Met835Val substitution. The mutation was inherited from the two parents who were heterozygous according to the notion of consanguinity. Immunocytochemical and electron microscopy studies of biopsied deltoid muscle showed dramatic changes in pre- and post-synaptic elements of the NMJs. These changes induced a process of denervation/reinnervation in native NMJs and the formation, by an adaptive mechanism, of newly formed and ectopic NMJs. Aberrant axonal outgrowth, decreased nerve terminal ramification and nodal axonal sprouting were also noted. In vivo electroporation of the mutated MuSK in a mouse model showed disorganized NMJs and aberrant axonal growth reproducing a phenotype similar to that observed in the patient's biopsy specimen. In vitro experiments showed that the mutation alters agrin-dependent acetylcholine receptor aggregation, causes a constitutive activation of MuSK and a decrease in its agrin- and Dok-7-dependent phosphorylation.


Assuntos
Debilidade Muscular , Músculo Esquelético , Síndromes Miastênicas Congênitas , Junção Neuromuscular , Receptores Proteína Tirosina Quinases/genética , Receptores Colinérgicos/genética , Agrina/metabolismo , Animais , Criança , Células HEK293 , Humanos , Masculino , Camundongos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Debilidade Muscular/genética , Debilidade Muscular/metabolismo , Debilidade Muscular/fisiopatologia , Músculo Esquelético/inervação , Músculo Esquelético/fisiopatologia , Mutação de Sentido Incorreto , Síndromes Miastênicas Congênitas/genética , Síndromes Miastênicas Congênitas/metabolismo , Síndromes Miastênicas Congênitas/fisiopatologia , Junção Neuromuscular/genética , Junção Neuromuscular/metabolismo , Junção Neuromuscular/fisiopatologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Colinérgicos/metabolismo , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento/metabolismo , Transdução de Sinais , Transmissão Sináptica/genética
20.
Neuromuscul Disord ; 19(5): 366-78, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19346129

RESUMO

Mice deficient in the glycosyltransferase Large are characterized by severe muscle and central nervous system abnormalities. In this study, we show that the formation and maintenance of neuromuscular junctions in Large(myd) mice are greatly compromised. Neuromuscular junctions are not confined to the muscle endplate zone but are widely spread and are frequently accompanied by exuberant nerve sprouting. Nerve terminals are highly fragmented and binding of alpha-bungarotoxin to postsynaptic acetylcholine receptors (AChRs) is greatly reduced. In vitro, Large(myd) myotubes are responsive to agrin but produce aberrant AChR clusters, which are larger in area and less densely packed with AChRs. In addition, AChR expression on the cell surface is diminished suggesting that AChR assembly or transport is defective. These results together with the finding that O-linked glycosylation at neuromuscular junctions of Large(myd) mice is compromised indicate that the action of Large is necessary for proper neuromuscular junction development.


Assuntos
Predisposição Genética para Doença/genética , N-Acetilglucosaminiltransferases/genética , Doenças da Junção Neuromuscular/genética , Doenças da Junção Neuromuscular/metabolismo , Junção Neuromuscular/genética , Junção Neuromuscular/metabolismo , Agrina/metabolismo , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Mutantes , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/inervação , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Mutação/genética , Junção Neuromuscular/fisiopatologia , Doenças da Junção Neuromuscular/fisiopatologia , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/patologia , Receptores Nicotínicos/metabolismo , Membranas Sinápticas/metabolismo , Membranas Sinápticas/patologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA