RESUMO
The myotendinous junction (MTJ) is the major site of force transfer in skeletal muscle, and defects in its structure correlate with a subset of muscular dystrophies. Col22a1 encodes the MTJ component collagen XXII, the function of which remains unknown. Here, we have cloned and characterized the zebrafish col22a1 gene and conducted morpholino-based loss-of-function studies in developing embryos. We showed that col22a1 transcripts localize at muscle ends when the MTJ forms and that COLXXII protein integrates the junctional extracellular matrix. Knockdown of COLXXII expression resulted in muscular dystrophy-like phenotype, including swimming impairment, curvature of embryo trunk/tail, strong reduction of twitch-contraction amplitude and contraction-induced muscle fiber detachment, and provoked significant activation of the survival factor Akt. Electron microscopy and immunofluorescence studies revealed that absence of COLXXII caused a strong reduction of MTJ folds and defects in myoseptal structure. These defects resulted in reduced contractile force and susceptibility of junctional extracellular matrix to rupture when subjected to repeated mechanical stress. Co-injection of sub-phenotypic doses of morpholinos against col22a1 and genes of the major muscle linkage systems showed a synergistic gene interaction between col22a1 and itga7 (α7ß1 integrin) that was not observed with dag1 (dystroglycan). Finally, pertinent to a conserved role in humans, the dystrophic phenotype was rescued by microinjection of recombinant human COLXXII. Our findings indicate that COLXXII contributes to the stabilization of myotendinous junctions and strengthens skeletal muscle attachments during contractile activity.
Assuntos
Colágeno/genética , Técnicas de Silenciamento de Genes , Distrofia Muscular Animal/patologia , Tendões/patologia , Peixe-Zebra/genética , Animais , Sobrevivência Celular/efeitos dos fármacos , Colágeno/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Embrião não Mamífero/ultraestrutura , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Imunofluorescência , Humanos , Integrinas/metabolismo , Mamíferos , Microinjeções , Morfolinos/farmacologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Debilidade Muscular/metabolismo , Debilidade Muscular/patologia , Distrofia Muscular Animal/embriologia , Distrofia Muscular Animal/genética , Fenótipo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Tendões/efeitos dos fármacos , Tendões/metabolismo , Tendões/ultraestruturaRESUMO
We found that zebrafish has two differentially expressed col14a1 paralogs. col14a1a expression peaked between 18-somite stage and 24 hours postfertilization (hpf), whereas col14a1b was first expressed at 32 hpf. To uncover functions of collagen XIV (COLXIV) during early embryogenesis, we focused our study on col14a1a. We characterized the α1 (XIV-A) chain as a collagenase-sensitive 200-kDa protein that formed dimer that could be reduced at high pH. As observed for the transcript, COLXIV-A protein expression peaked between 24 and 48 hpf. Using antisense probes and polyclonal antibodies, we show that col14a1a and its protein product COLXIV-A are transiently expressed in several epithelia, including epithelia undergoing shape changes, such as the fin folds. In contrast, anti-COLXII antibodies stained only connective tissues. COLXIV-A was also detected in the basement membrane (BM), where it co-localized with COLXII. At later developmental stages, COLXIV-A was not expressed in epithelia anymore but persisted in the BM. Morpholino knockdown of COLXIV-A provoked a skin detachment phenotype. Electron microscopy analysis revealed that morpholino-injected embryos lacked a lamina densa and lamina lucida at 24 hpf, and BM defects, such as gaps in the adepidermal granules, were still detected at 48 hpf. These BM defects were accompanied by a rupture of the dermis and detachment of the epidermis. Taken together, these data suggest an unexpected role of COLXIV-A in undifferentiated epithelia and in the formation of embryonic basement membranes.
Assuntos
Colágeno/genética , Epitélio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Nadadeiras de Animais/embriologia , Nadadeiras de Animais/metabolismo , Animais , Membrana Basal/embriologia , Membrana Basal/metabolismo , Western Blotting , Colágeno/metabolismo , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Embrião não Mamífero/ultraestrutura , Epitélio/embriologia , Feminino , Técnicas de Silenciamento de Genes , Hibridização In Situ , Masculino , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Multinucleated muscle fibres arise by fusion of precursor cells called myoblasts. We previously showed that CKIP-1 ectopic expression in C2C12 myoblasts increased cell fusion. In this work, we report that CKIP-1 depletion drastically impairs C2C12 myoblast fusion in vitro and in vivo during zebrafish muscle development. Within developing fast-twich myotome, Ckip-1 localises at the periphery of fast precursor cells, closed to the plasma membrane. Unlike wild-type myoblasts that form spatially arrayed multinucleated fast myofibres, Ckip-1-deficient myoblasts show a drastic reduction in fusion capacity. A search for CKIP-1 binding partners identified the ARPC1 subunit of Arp2/3 actin nucleation complex essential for myoblast fusion. We demonstrate that CKIP-1, through binding to plasma membrane phosphoinositides via its PH domain, regulates cell morphology and lamellipodia formation by recruiting the Arp2/3 complex at the plasma membrane. These results establish CKIP-1 as a regulator of cortical actin that recruits the Arp2/3 complex at the plasma membrane essential for muscle precursor elongation and fusion.
Assuntos
Proteínas de Transporte/fisiologia , Fusão de Membrana/fisiologia , Mioblastos/citologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Comunicação Celular/fisiologia , Diferenciação Celular/fisiologia , Fusão Celular , Linhagem Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Mamíferos , Camundongos , Mioblastos/metabolismo , Transfecção , Peixe-ZebraRESUMO
The myotendinous junction (MTJ) is essential for the integrity of the musculoskeletal unit. Here, we show that gene ablation of the MTJ marker col22a1 in zebrafish results in MTJ dysfunction but with variable degrees of expression and distinct phenotypic classes. While most individuals reach adulthood with no overt muscle phenotype (class 1), a subset of the progeny displays severe movement impairment and die before metamorphosis (class 2). Yet all mutants display muscle weakness due to ineffective muscle force transmission that is ultimately detrimental for class-specific locomotion-related functions. Movement impairment at the critical stage of swimming postural learning causes class 2 larval death by compromising food intake. In class 1 adults, intensive exercise is required to uncover a decline in muscle performance, accompanied by higher energy demand and mitochondrial adaptation. This study underscores COL22A1 as a candidate gene for myopathies associated with dysfunctional force transmission and anticipates a phenotypically heterogeneous disease.
Assuntos
Tendões , Peixe-Zebra , Animais , Locomoção , Músculo Esquelético , Fenótipo , Postura , Peixe-Zebra/genéticaRESUMO
Skeletal muscles are composed of hundreds of multinucleated muscle fibers (myofibers) whose myonuclei are regularly positioned all along the myofiber's periphery except the few ones clustered underneath the neuromuscular junction (NMJ) at the synaptic zone. This precise myonuclei organization is altered in different types of muscle disease, including centronuclear myopathies (CNMs). However, the molecular machinery regulating myonuclei position and organization in mature myofibers remains largely unknown. Conversely, it is also unclear how peripheral myonuclei positioning is lost in the related muscle diseases. Here, we describe the microtubule-associated protein, MACF1, as an essential and evolutionary conserved regulator of myonuclei positioning and maintenance, in cultured mammalian myotubes, in Drosophila muscle, and in adult mammalian muscle using a conditional muscle-specific knockout mouse model. In vitro, we show that MACF1 controls microtubules dynamics and contributes to microtubule stabilization during myofiber's maturation. In addition, we demonstrate that MACF1 regulates the microtubules density specifically around myonuclei, and, as a consequence, governs myonuclei motion. Our in vivo studies show that MACF1 deficiency is associated with alteration of extra-synaptic myonuclei positioning and microtubules network organization, both preceding NMJ fragmentation. Accordingly, MACF1 deficiency results in reduced muscle excitability and disorganized triads, leaving voltage-activated sarcoplasmic reticulum Ca2+ release and maximal muscle force unchanged. Finally, adult MACF1-KO mice present an improved resistance to fatigue correlated with a strong increase in mitochondria biogenesis.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microtúbulos/metabolismo , Mitocôndrias Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Mioblastos Esqueléticos/metabolismo , Junção Neuromuscular/metabolismo , Biogênese de Organelas , Animais , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/ultraestrutura , Acoplamento Excitação-Contração , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Microtúbulos/genética , Microtúbulos/ultraestrutura , Mitocôndrias Musculares/genética , Mitocôndrias Musculares/ultraestrutura , Fadiga Muscular , Fibras Musculares Esqueléticas/ultraestrutura , Força Muscular , Mioblastos Esqueléticos/ultraestrutura , Junção Neuromuscular/genética , Junção Neuromuscular/ultraestrutura , Fatores de TempoRESUMO
How some animals regenerate missing body parts is not well understood. Taking advantage of the zebrafish caudal fin model, we performed a global unbiased time-course transcriptomic analysis of fin regeneration. Biostatistics analyses identified extracellular matrix (ECM) as the most enriched gene sets. Basement membranes (BMs) are specialized ECM structures that provide tissues with structural cohesion and serve as a major extracellular signaling platform. While the embryonic formation of BM has been extensively investigated, its regeneration in adults remains poorly studied. We therefore focused on BM gene expression kinetics and showed that it recapitulates many aspects of development. As such, the re-expression of the embryonic col14a1a gene indicated that col14a1a is part of the regeneration-specific program. We showed that laminins and col14a1a genes display similar kinetics and that the corresponding proteins are spatially and temporally controlled during regeneration. Analysis of our CRISPR/Cas9-mediated col14a1a knockout fish showed that collagen XIV-A contributes to timely deposition of laminins. As changes in ECM organization can affect tissue mechanical properties, we analyzed the biomechanics of col14a1a-/- regenerative BM using atomic force microscopy (AFM). Our data revealed a thinner BM accompanied by a substantial increase of the stiffness when compared to controls. Further AFM 3D-reconstructions showed that BM is organized as a checkerboard made of alternation of soft and rigid regions that is compromised in mutants leading to a more compact structure. We conclude that collagen XIV-A transiently acts as a molecular spacer responsible for BM structure and biomechanics possibly by helping laminins integration within regenerative BM.