RESUMO
Proper muscle function depends on the neuromuscular junctions (NMJs), which mature postnatally to complex "pretzel-like" structures, allowing for effective synaptic transmission. Postsynaptic acetylcholine receptors (AChRs) at NMJs are anchored in the actin cytoskeleton and clustered by the scaffold protein rapsyn, recruiting various actin-organizing proteins. Mechanisms driving the maturation of the postsynaptic machinery and regulating rapsyn interactions with the cytoskeleton are still poorly understood. Drebrin is an actin and microtubule cross-linker essential for the functioning of the synapses in the brain, but its role at NMJs remains elusive. We used immunohistochemistry, RNA interference, drebrin inhibitor 3,5-bis-trifluoromethyl pyrazole (BTP2) and co-immunopreciptation to explore the role of this protein at the postsynaptic machinery. We identify drebrin as a postsynaptic protein colocalizing with the AChRs both in vitro and in vivo. We also show that drebrin is enriched at synaptic podosomes. Downregulation of drebrin or blocking its interaction with actin in cultured myotubes impairs the organization of AChR clusters and the cluster-associated microtubule network. Finally, we demonstrate that drebrin interacts with rapsyn and a drebrin interactor, plus-end-tracking protein EB3. Our results reveal an interplay between drebrin and cluster-stabilizing machinery involving rapsyn, actin cytoskeleton, and microtubules.
Assuntos
Acetilcolina/metabolismo , Microtúbulos/fisiologia , Mioblastos/fisiologia , Junção Neuromuscular/fisiologia , Neuropeptídeos/farmacologia , Receptores Colinérgicos/metabolismo , Sinapses/fisiologia , Citoesqueleto de Actina/metabolismo , Animais , Células Cultivadas , Camundongos , Microtúbulos/efeitos dos fármacos , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Junção Neuromuscular/efeitos dos fármacos , Receptores Colinérgicos/genética , Transmissão SinápticaRESUMO
The neuromuscular junctions (NMJs) connect muscle fibers with motor neurons and enable the coordinated contraction of skeletal muscles. The dystrophin-associated glycoprotein complex (DGC) is an essential component of the postsynaptic machinery of the NMJ and is important for the maintenance of NMJ structural integrity. To identify novel proteins that are important for NMJ organization, we performed a mass spectrometry-based screen for interactors of α-dystrobrevin 1 (aDB1), one of the components of the DGC. The guanidine nucleotide exchange factor (GEF) Arhgef5 was found to be one of the aDB1 binding partners that is recruited to Tyr-713 in a phospho-dependent manner. We show here that Arhgef5 localizes to the NMJ and that its genetic depletion in the muscle causes the fragmentation of the synapses in conditional knockout mice. Arhgef5 loss in vivo is associated with a reduction in the levels of active GTP-bound RhoA and Cdc42 GTPases, highlighting the importance of actin dynamics regulation for the maintenance of NMJ integrity.
RESUMO
Neuromuscular junctions (NMJs) are specialized synapses in the peripheral nervous system that allow the transmission of neuronal impulses to skeletal muscles for their contraction. Due to its size and accessibility, the NMJ is a commonly used model for studying basic principles of synapse organization and function. Similar to synapses in the central nervous system, NMJs are composed of presynaptic axonal terminals, the postsynaptic machinery formed at the membrane of the muscle fibers, and the synapse-associated glial cells. The special glial cells at the NMJs are called terminal Schwann cells or perisynaptic Schwann cells (PSCs). Decades of studies on the NMJ, as well as the most recent discoveries, have revealed multiple functions for PSCs at different stages of synaptic formation, maintenance, and disassembly. This review summarizes major observations in the field.
Assuntos
Junção Neuromuscular/metabolismo , Terminações Pré-Sinápticas/metabolismo , Células de Schwann/metabolismo , Animais , Modelos Biológicos , Junção Neuromuscular/citologia , Células de Schwann/citologiaRESUMO
The proper function of skeletal muscles relies on their ability to process signals derived from motor neurons, transmit stimuli along the muscle fibers, contract, and regenerate efficiently after injury. The dystrophin-glycoprotein complex (DGC; also called the dystrophin-associated protein complex) plays a central role in all of these processes. It acts as a transmembrane platform that anchors the extracellular matrix (ECM) to the intracellular cytoskeleton and makes muscle fibers more resistant to injury. The DGC also contributes to the transmission of contraction-evoked force from the sarcomere to the ECM. The dysfunction of DGC-associated proteins can lead to myopathies, including Duchenne's muscular dystrophy, manifested by progressive muscle damage and impairments in regeneration. The DGC also plays a pivotal role in the organization of neuromuscular junctions (NMJs), where it stabilizes postsynaptic machinery, including receptors for the neurotransmitter acetylcholine (AChRs). Here, we focus on the role of the DGC complex in NMJ and skeletal muscle physiology and discuss the novel components that are associated with the complex.
Assuntos
Distrofina/metabolismo , Glicoproteínas/metabolismo , Junção Neuromuscular/metabolismo , Animais , Proteínas Associadas à Distrofina/metabolismo , Humanos , Modelos Neurológicos , Complexos Multiproteicos/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/etiologia , Distrofia Muscular de Duchenne/metabolismo , Receptores Colinérgicos/metabolismoRESUMO
Skeletal muscle growth and regeneration relies on the activation of muscle specific stem cells, that is, satellite cells. The activation and differentiation of satellite cells into myoblasts, as well as their migration, proliferation, and fusion of mononuclear myoblasts into a functional multi-nucleated muscle fiber, are associated with extracellular matrix (ECM) protein synthesis and degradation. The extracellular environment is dynamically adapting to the changes accompanying skeletal muscle growth or repair. Enzymes engaged in many biological processes that involve ECM remodeling are matrix metalloproteinases (MMPs). Among metalloproteinases crucial for skeletal muscles are two gelatinases-MMP-9 and MMP-2. In the current study we test the effect of silencing the MMP-9 and MMP-2 expression on the proliferation and differentiation of in vitro cultured skeletal muscle myoblasts. We show that downregulating gelatinase MMP-9 expression results in a delayed myoblast differentiation.
Assuntos
Gelatinases/genética , Gelatinases/metabolismo , Mioblastos Esqueléticos/citologia , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Matriz Extracelular/metabolismo , Inativação Gênica , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/fisiologia , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/fisiologia , Metaloproteinases da Matriz/metabolismo , Desenvolvimento Muscular/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Mioblastos Esqueléticos/patologia , Cultura Primária de Células , Ratos , Regeneração/fisiologia , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/fisiologiaRESUMO
Neuromuscular junctions (NMJs) are specialized synapses that connect motor neurons to skeletal muscle fibers and orchestrate proper signal transmission from the nervous system to muscles. The efficient formation and maintenance of the postsynaptic machinery that contains acetylcholine receptors (AChR) are indispensable for proper NMJ function. Abnormalities in the organization of synaptic components often cause severe neuromuscular disorders, such as muscular dystrophy. The dystrophin-associated glycoprotein complex (DGC) was shown to play an important role in NMJ development. We recently identified liprin-α-1 as a novel binding partner for one of the cytoplasmic DGC components, α-dystrobrevin-1. In the present study, we performed a detailed analysis of localization and function of liprin-α-1 at the murine NMJ. We showed that liprin-α-1 localizes to both pre- and postsynaptic compartments at the NMJ, and its synaptic enrichment depends on the presence of the nerve. Using cultured muscle cells, we found that liprin-α-1 plays an important role in AChR clustering and the organization of cortical microtubules. Our studies provide novel insights into the function of liprin-α-1 at vertebrate neuromuscular synapses.
Assuntos
Junção Neuromuscular/metabolismo , Proteínas/metabolismo , Transmissão Sináptica , Proteínas Adaptadoras de Transdução de Sinal , Animais , Linhagem Celular , Expressão Gênica , Camundongos , Microtúbulos/metabolismo , Músculos/metabolismo , Transporte Proteico , Proteínas/genética , Receptores Colinérgicos/metabolismoRESUMO
Neuromuscular junctions (NMJs), the synapses made by motor neurons on muscle fibers, form during embryonic development but undergo substantial remodeling postnatally. Several lines of evidence suggest that α-dystrobrevin, a component of the dystrophin-associated glycoprotein complex (DGC), is a crucial regulator of the remodeling process and that tyrosine phosphorylation of one isoform, α-dystrobrevin-1, is required for its function at synapses. We identified a functionally important phosphorylation site on α-dystrobrevin-1, generated phosphorylation-specific antibodies to it and used them to demonstrate dramatic increases in phosphorylation during the remodeling period, as well as in nerve-dependent regulation in adults. We then identified proteins that bind to this site in a phosphorylation-dependent manner and others that bind to α-dystrobrevin-1 in a phosphorylation-independent manner. They include multiple members of the DGC, as well as α-catulin, liprin-α1, Usp9x, PI3K, Arhgef5 and Grb2. Finally, we show that two interactors, α-catulin (phosphorylation independent) and Grb2 (phosphorylation dependent) are localized to NMJs in vivo, and that they are required for proper organization of neurotransmitter receptors on myotubes.
Assuntos
Proteínas Associadas à Distrofina/metabolismo , Proteína Adaptadora GRB2/metabolismo , Junção Neuromuscular/metabolismo , Neuropeptídeos/metabolismo , Receptores Colinérgicos/metabolismo , alfa Catenina/metabolismo , Animais , Linhagem Celular , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Mapas de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Transporte Proteico , Transmissão SinápticaRESUMO
Enoxaparin sodium is a low-molecular-weight heparin. It is not clear whether the risk of development of osteoporosis after administration of low-molecular-weight heparins is lower than after administration of standard heparin. The aim of the present study was to investigate the effects of enoxaparin on histomorphometric parameters of bones in female Wistar rats (13-15 weeks old at the beginning of the experiment). Enoxaparin was administered at doses of 1000 anti-Xa IU/kg sc daily or 2000 anti-Xa IU/kg sc daily for 4 weeks. Bone mass, mineral and calcium content (in the tibia, femur and L-4 vertebra), length and diameter in the tibia and femur, and histomorphometric parameters of the tibia (periosteal and endosteal transverse growth, width of periosteal and endosteal osteoid, area of the transverse cross-section of the cortical bone in the diaphysis and area of the transverse cross-section of the marrow cavity) and the femur (width of epiphyseal and metaphyseal trabeculae, width of epiphyseal cartilage) were examined. Enoxaparin, administered at doses of 1000 anti-Xa IU/kg sc daily or 2000 anti-Xa IU/kg sc daily for 28 days, induced osteopenic changes in the rat skeletal system. The changes observed in bone histomorphometric parameters indicate that enoxaparin caused the inhibition of bone formation and intensification of bone resorption.