Assuntos
Distrofia Muscular de Emery-Dreifuss/complicações , Distrofia Muscular de Emery-Dreifuss/genética , Síndromes Miastênicas Congênitas/tratamento farmacológico , Síndromes Miastênicas Congênitas/genética , Plectina/genética , Adrenérgicos/uso terapêutico , Adulto , Efedrina/uso terapêutico , Mutação da Fase de Leitura , Humanos , Masculino , Distrofia Muscular de Emery-Dreifuss/patologiaRESUMO
AIMS: Plectin, a universally expressed multi-functional cytolinker protein, is crucial for intermediate filament networking, including crosstalk with actomyosin and microtubules. In addition to its involvement in a number of diseases affecting skin, skeletal muscle, heart, and other stress-exposed tissues, indications for a neuropathological role of plectin have emerged. Having identified P1c as the major isoform expressed in neural tissues in previous studies, our aim for the present work was to investigate whether, and by which mechanism(s), the targeted deletion of this isoform affects neuritogenesis and proper nerve cell functioning. METHODS: For ex vivo phenotyping, we used dorsal root ganglion and hippocampal neurons derived from isoform P1c-deficient and plectin-null mice, complemented by in vitro experiments using purified proteins and cell fractions. To assess the physiological significance of the phenotypic alterations observed in P1c-deficient neurons, P1c-deficient and wild-type littermate mice were subjected to standard behavioural tests. RESULTS: We demonstrate that P1c affects axonal microtubule dynamics by isoform-specific interaction with tubulin. P1c deficiency in neurons leads to altered dynamics of microtubules and excessive association with tau protein, affecting neuritogenesis, neurite branching, growth cone morphology, and translocation and directionality of movement of vesicles and mitochondria. On the organismal level, we found P1c deficiency manifesting as impaired pain sensitivity, diminished learning capabilities and reduced long-term memory of mice. CONCLUSIONS: Revealing a regulatory role of plectin scaffolds in microtubule-dependent nerve cell functions, our results have potential implications for cytoskeleton-related neuropathies.
Assuntos
Memória/fisiologia , Neurônios/metabolismo , Organelas/metabolismo , Dor/metabolismo , Proteínas tau/metabolismo , Animais , Filamentos Intermediários/metabolismo , Camundongos , Microtúbulos/metabolismo , Dor/fisiopatologia , Plectina/deficiênciaRESUMO
Epidermolysis bullosa simplex with muscular dystrophy (EBS-MD, MIM 226670) is caused by plectin defects. We performed mutational analysis and immunohistochemistry using EBS-MD (n = 3 cases) and control skeletal muscle to determine pathogenesis. Mutational analysis revealed a novel homozygous plectin-exon32 rod domain mutation (R2465X). All plectin/HD1-121 antibodies stained the control skeletal muscle membrane. However, plectin antibodies stained the cytoplasm of type II control muscle fibers (as confirmed by ATPase staining), whereas HD1-121 stained the cytoplasm of type I fibers. EBS-MD samples lacked membrane (n = 3) but retained cytoplasmic HD1-121 (n = 1) and plectin staining in type II fibers (n = 3). Ultrastructurally, EBS-MD demonstrated widening and vacuolization adjacent to the membrane and disorganization of Z-lines (n = 2 of 3) compared to controls (n = 5). Control muscle immunogold labeling colocalized plectin and desmin to filamentous bridges between Z-lines and the membrane that were disrupted in EBS-MD muscle. We conclude that fiber-specific plectin expression is associated with the desmin-cytoskeleton, Z-lines, and crucially myocyte membrane linkage, analogous to hemidesmosomes in skin.
Assuntos
Epidermólise Bolhosa Simples/metabolismo , Predisposição Genética para Doença/genética , Músculo Esquelético/metabolismo , Distrofias Musculares/metabolismo , Plectina/genética , Plectina/metabolismo , Adulto , Membrana Celular/metabolismo , Membrana Celular/patologia , Membrana Celular/ultraestrutura , Criança , Citoplasma/metabolismo , Citoplasma/patologia , Citoplasma/ultraestrutura , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Citoesqueleto/ultraestrutura , Análise Mutacional de DNA , Desmossomos/metabolismo , Desmossomos/patologia , Desmossomos/ultraestrutura , Epidermólise Bolhosa Simples/complicações , Epidermólise Bolhosa Simples/patologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Microscopia Imunoeletrônica , Pessoa de Meia-Idade , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Rápida/patologia , Fibras Musculares de Contração Rápida/ultraestrutura , Fibras Musculares de Contração Lenta/metabolismo , Fibras Musculares de Contração Lenta/patologia , Fibras Musculares de Contração Lenta/ultraestrutura , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Distrofias Musculares/complicações , Distrofias Musculares/patologia , Mutação/genética , Plectina/análise , Estrutura Terciária de Proteína/genéticaRESUMO
Plectin is one of the largest and most versatile cytolinker proteins known. Cloned and sequenced in 1991, it was later shown to have nonsense mutations in recessive epidermolysis bullosa with muscular dystrophy. A dominant mutation in the gene was found to cause epidermolysis bullosa simplex Ogna without muscular dystrophy. Here we report the DNA sequencing of the plectin gene (PLEC1) in a Dutch family originally described in 1972 as having epidermolysis bullosa with muscular dystrophy. The results revealed homozygosity for a new plectin nonsense mutation at position 13187 and its specific 8q24 marker haplotype profile. Western blotting of cultured fibroblasts and immunofluorescence microscopy of skin biopsy confirm that the plectin protein expression is grossly reduced or absent. A summary of the life-long clinical course of the two affected brothers homozygous for the new E1914X mutation is given.
Assuntos
Epidermólise Bolhosa Simples/genética , Genes Dominantes , Predisposição Genética para Doença , Proteínas de Filamentos Intermediários/genética , Distrofias Musculares/genética , Mutação Puntual , Sequência de Bases , Western Blotting , Doença Crônica , Epidermólise Bolhosa Simples/complicações , Epidermólise Bolhosa Simples/patologia , Imunofluorescência , Ligação Genética , Humanos , Masculino , Dados de Sequência Molecular , Distrofias Musculares/complicações , Distrofias Musculares/patologia , Noruega , Linhagem , Fenótipo , Plectina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estudos de Amostragem , Índice de Gravidade de Doença , IrmãosRESUMO
Rapid freezing of cell monolayers at ambient pressure is limited regarding the thickness of ice crystal damage-free freezing. The specific freezing conditions of the cells under investigation are decisive for the success of such methods. Improved reproducibility of results could be expected by cryoimmobilization at high pressure because this achieves a greater thickness of adequate freezing. In a novel approach, we tested the suitability of sapphire discs as cell substrata for high-pressure freezing. Frozen samples on sapphire were subjected to freeze-substitution while in the same flat sample holders as used for high-pressure freezing. We obtained cells that displayed an excellent preservation of fine structure. Because sapphire is a tissue culture substratum suitable for light microscopy, its use in combination with high-pressure freezing could become a powerful tool in correlative studies of cell dynamics at light and electron microscopic levels.
Assuntos
Células Epiteliais/fisiologia , Congelamento , Pressão , Óxido de Alumínio , Animais , Células Cultivadas , Criopreservação/métodos , Substituição ao Congelamento , Vidro , Microscopia Eletrônica/métodosRESUMO
Rapid cryofixation of cells cultured on coverslips without the use of chemical fixatives has proved advantageous for the immunolocalization of antigens by electron microscopy. Here, we demonstrate the application of sapphire-attached tissue culture cells (PtK2 epithelial cells and mouse myoblasts) to metal-mirror impact freezing. The potential of the Leica EM-CPC cryoworkstation for routine freezing and for safe transfer of the cryofrozen samples into a sapphire disc magazine for freeze-substitution (SD-FS unit) has been exploited. Subsequently, the SD-FS unit has been tested for its use in methanol freeze-substitution and low temperature embedding for immunoelectron microscopy. The structural preservation of Lowicryl HM20-embedded cells has been assessed as being free of damage by large ice crystals.
Assuntos
Óxido de Alumínio/química , Criopreservação/métodos , Células Epiteliais/ultraestrutura , Substituição ao Congelamento , Animais , Células Cultivadas , Epitélio/ultraestrutura , Técnicas Histológicas , Macropodidae , Camundongos , Modelos Estruturais , Ratos , Fatores de TempoRESUMO
Using both monoclonal and polyclonal antibodies against mammalian plectin (multifunctional protein cross-linking cytoskeletal structures, mainly intermediate filaments, in mammalian cells), several putative isoforms of plectin-like proteins were found in protein extracts from the green alga Chlamydomonas eugametos (Volvocales). Immunofluorescence and immunoblotting revealed that some of the plectin-like proteins were present in perinuclear region or localized near the cell wall, probably being attached to the cytoplasmic membrane.
Assuntos
Proteínas de Algas/metabolismo , Chlamydomonas/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Immunoblotting , Plectina , Coelhos , Frações Subcelulares/metabolismoRESUMO
Plectin, a member of the cytolinkers protein family, plays a crucial role in cells as a stabilizing element of cells against mechanical stress. Its absence results in muscular dystrophy, skin blistering, and signs of neuropathy. The C-terminal domain of plectin contains several highly homologous repeat domains that also occur in other cytolinkers. Secondary structure analysis revealed that the building block of these domains, the PLEC repeat, is similar to the ankyrin repeat. We present a model that attempts to explain how the C-terminal domain, which comprises approximately 1900 amino acid, could be stabilized to maintain its structural integrity even under extensive mechanical stress. In this model, larger solenoid modules formed from PLEC repeats can be disulfide-bridged via conserved cysteines. Our hypothesis suggests that this process could be mediated by cytoplasmic NOS-generated products, such as the radical peroxynitrite. Reinforcement of molecular structure would provide a rationale why during exercising or physical stress radicals are formed without necessarily being deleterious. This article contains supplementary material that may be viewed at the BioEssays website at http://www.interscience.wiley.com/jpages/0265-9247/suppmat/23/v23_11.1064.html.
Assuntos
Anquirinas/química , Cisteína/química , Proteínas de Filamentos Intermediários/química , Sequências Repetitivas de Aminoácidos/fisiologia , Sequência de Aminoácidos , Animais , Anquirinas/fisiologia , Cisteína/fisiologia , Humanos , Proteínas de Filamentos Intermediários/fisiologia , Modelos Moleculares , Dados de Sequência Molecular , Oxidantes/fisiologia , Plectina , Estrutura Terciária de ProteínaRESUMO
Plectin is a cytoskeleton linker protein expressed in a variety of tissues including skin, muscle, and nerves. Mutations in its gene are associated with epidermolysis bullosa simplex with late-onset muscular dystrophy. Whereas in most of these patients the pathogenic events are mediated by nonsense-mediated mRNA decay, the consequences of an in-frame mutation are less clear. We analyzed a patient with compound heterozygosity for a 3-bp insertion at position 1287 leading to the insertion of leucine as well as the missense mutation Q1518X leading to a stop codon. The presence of plectin mRNA was demonstrated by a RNase protection assay. However, a marked reduction of plectin protein was found using immunofluorescence microscopy of the patient's skin and Western blot analysis of the patient's cultured keratinocytes. The loss of plectin protein was associated with morphological alterations in plectin-containing structures of the dermo-epidermal junction, in skeletal muscle, and in nerves as detected by electron microscopy. In an in vitro overlay assay using recombinant plectin peptides spanning exons 2 to 15 the insertion of leucine resulted in markedly increased self-aggregation of plectin peptides. These results describe for the first time the functional consequences of an in-frame insertion mutation in humans.
Assuntos
Epidermólise Bolhosa Simples/genética , Proteínas de Filamentos Intermediários/genética , Leucina/genética , Sequência de Bases , Pré-Escolar , Códon sem Sentido , DNA/química , DNA/genética , Análise Mutacional de DNA , Epidermólise Bolhosa Simples/patologia , Saúde da Família , Feminino , Heterozigoto , Humanos , Proteínas de Filamentos Intermediários/deficiência , Masculino , Microscopia Eletrônica , Mutagênese Insercional , Núcleo Familiar , Linhagem , Plectina , Pele/metabolismo , Pele/patologia , Pele/ultraestruturaRESUMO
Microtubule-associated proteins such as MAP1B have long been suspected to play an important role in neuronal differentiation, but proof has been lacking. Previous MAP1B gene targeting studies yielded contradictory and inconclusive results and did not reveal MAP1B function. In contrast to two earlier efforts, we now describe generation of a complete MAP1B null allele. Mice heterozygous for this MAP1B deletion were not affected. Homozygous mutants were viable but displayed a striking developmental defect in the brain, the selective absence of the corpus callosum, and the concomitant formation of myelinated fiber bundles consisting of misguided cortical axons. In addition, peripheral nerves of MAP1B-deficient mice had a reduced number of large myelinated axons. The myelin sheaths of the remaining axons were of reduced thickness, resulting in a decrease of nerve conduction velocity in the adult sciatic nerve. On the other hand, the anticipated involvement of MAP1B in retinal development and gamma-aminobutyric acid C receptor clustering was not substantiated. Our results demonstrate an essential role of MAP1B in development and function of the nervous system and resolve a previous controversy over its importance.
Assuntos
Encéfalo/patologia , Comunicação Celular/fisiologia , Corpo Caloso/patologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas do Tecido Nervoso/genética , Sistema Nervoso Periférico/patologia , Animais , Axônios/fisiologia , Encéfalo/embriologia , Corpo Caloso/embriologia , Genes Letais , Heterozigoto , Homozigoto , Camundongos , Camundongos Mutantes , Bainha de Mielina/patologia , Sistema Nervoso Periférico/embriologia , Receptores de GABA/isolamento & purificação , Retina/embriologia , Deleção de SequênciaRESUMO
Mice lacking the gene encoding for the intermediate filament protein desmin have a surprisingly normal myofibrillar organization in skeletal muscle fibers, although myopathy develops in highly used muscles. In the present study we examined how synemin, paranemin, and plectin, three key cytoskeletal proteins related to desmin, are organized in normal and desmin knock-out (K/O) mice. We show that in wild-type mice, synemin, paranemin, and plectin were colocalized with desmin in Z-disc-associated striations and at the sarcolemma. All three proteins were also present at the myotendinous junctions and in the postsynaptic area of motor endplates. In the desmin K/O mice the distribution of plectin was unaffected, whereas synemin and paranemin were partly affected. The Z-disc-associated striations were in general no longer present in between the myofibrils. In contrast, at the myotendinous and neuromuscular junctions synemin and paranemin were still present. Our study shows that plectin differs from synemin and paranemin in its binding properties to the myofibrillar Z-discs and that the cytoskeleton in junctional areas is particularly complex in its organization.
Assuntos
Desmina/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Envelhecimento , Animais , Proteínas Aviárias , Citoesqueleto/metabolismo , Desmina/genética , Técnica Indireta de Fluorescência para Anticorpo , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Microscopia Imunoeletrônica , Músculo Esquelético/ultraestrutura , Junção Neuromuscular/metabolismo , Junção Neuromuscular/ultraestrutura , Plectina , Sarcolema/metabolismo , Sarcolema/ultraestrutura , Tendões/metabolismo , Tendões/ultraestruturaRESUMO
Caspase 8 plays an essential role in the execution of death receptor-mediated apoptosis. To determine the localization of endogenous caspase 8, we used a panel of subunit-specific anti-caspase 8 monoclonal antibodies in confocal immunofluorescence microscopy. In the human breast carcinoma cell line MCF7, caspase 8 predominantly colocalized with and bound to mitochondria. After induction of apoptosis through CD95 or tumor necrosis factor receptor I, active caspase 8 translocated to plectin, a major cross-linking protein of the three main cytoplasmic filament systems, whereas the caspase 8 prodomain remained bound to mitochondria. Plectin was quantitatively cleaved by caspase 8 at Asp 2395 in the center of the molecule in all cells tested. Cleavage of plectin clearly preceded that of other caspase substrates such as poly(ADP-ribose) polymerase, gelsolin, cytokeratins, or lamin B. In primary fibroblasts from plectin-deficient mice, apoptosis-induced reorganization of the actin cytoskeleton, as seen in wild-type cells, was severely impaired, suggesting that during apoptosis, plectin is required for the reorganization of the microfilament system.
Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Receptor fas/metabolismo , Actinas/metabolismo , Actinas/ultraestrutura , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Transporte Biológico , Neoplasias da Mama , Carcinoma , Caspase 8 , Caspase 9 , Caspases/imunologia , Citoplasma/metabolismo , Precursores Enzimáticos/metabolismo , Fibroblastos/metabolismo , Gelsolina/metabolismo , Humanos , Proteínas de Filamentos Intermediários/genética , Queratinas/metabolismo , Lamina Tipo B , Laminas , Camundongos , Camundongos Mutantes , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Plectina , Especificidade por Substrato , Células Tumorais CultivadasRESUMO
We report a novel case of epidermolysis bullosa simplex with severe mucous membrane involvement and mutations in the plectin gene (PLEC1). The patient suffered from extensive blistering of the skin and oral and laryngeal mucous membranes. Electron microscopy of a lesional skin biopsy showed cleft formation within the basal cell layer of the epidermis. Antigen mapping displayed entirely negative staining for plectin, a large (>500 kDa) multifunctional adhesion protein present in hemidesmosomes of the basal keratinocytes. Mutation analysis revealed compound heterozygous, previously undisclosed nonsense mutations, Q1713X and R2351X, of paternal and maternal origin, respectively, within exon 32 of PLEC1. Based on earlier reports, plectin deficiency is associated with late onset muscular dystrophy in patients with epidermolysis bullosa. No signs of muscle weakness have been observed during the 4 y follow-up of our patient. This case illustrates the fact that molecular pathological analyses have prognostic implications in identification and evaluation of patients who appear to be at risk for development of muscular dystrophy later in life.
Assuntos
Epidermólise Bolhosa Simples/genética , Proteínas de Filamentos Intermediários/genética , Biópsia , Criança , Éxons , Humanos , Proteínas de Filamentos Intermediários/deficiência , Masculino , Mucosa , Linhagem , Plectina , Mutação Puntual , Reação em Cadeia da Polimerase , Pele/patologiaRESUMO
Plectin, the largest and most versatile member of the cytolinker/plakin family of proteins characterized to date, has a tripartite structure comprising a central 200 nm-long (&agr;)-helical rod domain flanked by large globular domains. The C-terminal domain comprises a short tail region preceded by six highly conserved repeats (each 28-39 kDa), one of which (repeat 5) contains plectin's intermediate filament (IF)-binding site. We used recombinant and native proteins to assess the effects of plectin repeat 5-binding to IF proteins of different types. Quantitative Eu(3+)-based overlay assays showed that plectin's repeat 5 domain bound to type III IF proteins (vimentin) with preference over type I and II cytokeratins 5 and 14. The ability of both types of IF proteins to self-assemble into filaments in vitro was impaired by plectin's repeat 5 domain in a concentration-dependent manner, as revealed by negative staining and rotary shadowing electron microscopy. This effect was much more pronounced in the case of vimentin compared to cytokeratins 5/14. Preassembled filaments of both types became more and more crosslinked upon incubation with increasing concentrations of plectin repeat 5. However, at high proportions of plectin to IF proteins, disassembly of filaments occurred. Again, vimentin filaments proved considerably more sensitive towards disassembly than those composed of cytokeratins 5 and 14. In general, IFs formed from recombinant proteins were found to be slightly more responsive towards plectin influences than their native counterparts. A dose-dependent plectin-inflicted collapse and putative disruption of IFs was also observed in vivo after ectopic expression of vimentin and plectin's repeat 5 domain in cotransfected vimentin-deficient SW13 (vim(-)) cells. Our results suggest an involvement of plectin not only in crosslinking and stabilization of cytoskeletal IF networks, but also in regulation of their dynamics.
Assuntos
Proteínas de Filamentos Intermediários/metabolismo , Filamentos Intermediários/metabolismo , Queratinas/metabolismo , Vimentina/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Relação Dose-Resposta a Droga , Glioma/patologia , Proteínas de Filamentos Intermediários/química , Filamentos Intermediários/ultraestrutura , Queratinas/química , Camundongos , Morfogênese , Coloração Negativa , Proteínas de Neoplasias/metabolismo , Plectina , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
Plectin, the most versatile cytolinker identified to date, has essential functions in maintaining the mechanical integrity of skin, skeletal muscle and heart, as indicated by analyses of plectin-deficient mice and humans. Expression of plectin in a vast variety of tissues and cell types, combined with a large number of different binding partners identified at the molecular level, calls for complex mechanisms regulating gene transcription and expression of the protein. To investigate these mechanisms, we analyzed the transcript diversity and genomic organization of the murine plectin gene and found a remarkable complexity of its 5'-end structure. An unusually high number of 14 alternatively spliced exons, 11 of them directly splicing into plectin exon 2, were identified. Analysis of their tissue distribution revealed that expression of a few of them is restricted to tissues such as brain, or skeletal muscle and heart. In addition, we found two short exons tissue-specifically spliced into a highly conserved set of exons encoding the N-terminal actin binding domain (ABD), common to plectin and the superfamily of spectrin/dystrophin-type actin binding proteins. Using recombinant proteins we show that a novel ABD version contained in the muscle-specific isoform of plectin exhibits significantly higher actin binding activity than other splice forms. This fine tuning mechanism based on alternative splicing is likely to optimize the proposed biological role of plectin as a cytolinker opposing intense mechanical forces in tissues like striated muscle.
Assuntos
Actinas/metabolismo , Processamento Alternativo , Proteínas de Filamentos Intermediários/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Mapeamento Cromossômico , Éxons , Feto , Proteínas de Filamentos Intermediários/genética , Íntrons , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Especificidade de Órgãos , Plectina , Ligação Proteica , RNA Mensageiro/análise , Homologia de Sequência de AminoácidosRESUMO
Plectin, a widespread cytoskeletal linker protein, is prominently expressed in basal keratinocytes of the epidermis. HD1, originally identified as a hemidesmosomal protein, has been suggested to be an isoform of or closely related to plectin, but the exact relationship between these proteins is unknown. Plectin has recently been identified as the gene/protein system at fault in epidermolysis bullosa simplex associated with muscular dystrophy (EBS-MD; OMIM# 226670). In this study, we examined the expression patterns of plectin and HD1 epitopes in the skin of four unrelated patients with EBS-MD confirmed to be caused by plectin gene mutations. By indirect immunofluorescence, all monoclonal antibodies (mAbs) to plectin (5B3, 10F6) or to HD1 (121, E2, K15, 156) bound to the epidermal basement membrane zone (BMZ) of normal human skin. In addition, immunostaining along the periphery of keratinocytes was detected with mAbs 5B3, 10F6 (antiplectin), K15 and 156 (anti-HD1), but not with mAbs 121 and E2 (anti-HD1). Immunolabeling for mAbs 5B3 and 10F6 (antiplectin) was absent in the skin of three patients who had premature termination codon mutations in the plectin gene in both alleles. In contrast, labeling was only slightly reduced in a patient who was homozygous for a 9-bp in-frame deletion mutation in the same gene. Interestingly, peripheral labeling of keratinocytes using mAbs K15 and 156 (anti-HD1) was clearly present in all the patients despite the disappearance of BMZ labeling. Quantitative analysis by postembedding immunoelectron microscopy demonstrated that both plectin and HD1 epitopes were localized in the inner plaque of hemidesmosomes with a mean distance of 110 and 120 nm from the plasma membrane, respectively. These results confirm the molecular heterogeneity of EBS-MD in terms of the expression patterns of plectin and HD1 epitopes which correlate with clinical severity, the pattern of plectin gene mutations and their consequences.
Assuntos
Epidermólise Bolhosa Simples/complicações , Epidermólise Bolhosa Simples/metabolismo , Epitopos/metabolismo , Proteínas de Filamentos Intermediários/imunologia , Proteínas de Filamentos Intermediários/metabolismo , Distrofias Musculares/complicações , Adulto , Membrana Basal/metabolismo , Membrana Basal/patologia , Criança , Epidermólise Bolhosa Simples/patologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Masculino , Microscopia Imunoeletrônica , Pessoa de Meia-Idade , Plectina , Pele/metabolismo , Pele/patologiaRESUMO
Plectin (M(r) > 500,000) is a versatile and widely expressed cytolinker protein. In striated muscle it is predominantly found at the Z-disc level where it colocalizes with the intermediate filament protein desmin. Both proteins show altered labeling patterns in tissues of muscular dystrophy patients. Moreover, mutations in the plectin gene lead to the autosomal recessive human disorder epidermolysis bullosa simplex with muscular dystrophy, and defects in the desmin gene have been shown to cause familiar cardiac and skeletal myopathy. Since intermediate filaments (IFs) in striated muscle tissue have been found to be intimately associated with mitochondria, we investigated whether plectin is involved in this association. Using postembedding immunogold labeling of Lowicryl sections and immunogold labeling of ultrathin cryosections, we show that plectin is associated with desmin IFs linking myofibrils to mitochondria at the level of the Z-disc and along the entire length of the sarcomere. The localization of plectin label at the mitochondrial membrane itself was consistent with a putative linker function of plectin between desmin IFs and the mitochondrial surface. In mitochondrion-rich muscle fibers, both plectin and desmin were part of an ordered arrangement of mitochondrial side branches, which wound around myofibrils adjacent to the Z-discs and were anchored into a filamentous network transversing from one fibril to the other. The association of mitochondria with plectin and IFs was seen also in tissues without regular distribution patterns of mitochondria, such as heart muscle and neonatal skeletal muscle tissues. These data were supplemented with in vitro binding assays showing direct interaction of plectin with desmin via its carboxy-terminal IF-binding domain. As a cytolinker protein associated with mitochondria and desmin IFs, plectin could play an important role in the positioning and shape formation, in particular branching, of mitochondrial organelles in striated muscle tissues.
Assuntos
Desmina/ultraestrutura , Proteínas de Filamentos Intermediários/ultraestrutura , Filamentos Intermediários/ultraestrutura , Mitocôndrias/ultraestrutura , Músculo Esquelético/ultraestrutura , Animais , Desmina/metabolismo , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Filamentos Intermediários/metabolismo , Microscopia Eletrônica , Músculo Esquelético/metabolismo , Plectina , Ligação Proteica , RatosRESUMO
The integrin alpha6beta4 is an essential component of hemidesmosomes but it also plays a dynamic role in invasive carcinoma cells. The cytoplasmic tail of the beta4 subunit is uniquely large among integrins and includes two pairs of fibronectin type III domains separated by a connecting segment. Here we describe the crystal structure of the first tandem domain pair, a module that is critical for alpha6beta4 function. The structure reveals a novel interdomain interface and candidate protein-binding sites, including a large acidic cleft formed from the surfaces of both domains and a prominent loop that is reminiscent of the RGD integrin-binding loop of fibronectin. This is the first crystal structure of either a hemidesmosome component or an integrin cytoplasmic domain, and it will enable the intracellular functions of alpha6beta4 to be dissected at the atomic level.
Assuntos
Antígenos de Superfície/química , Citoplasma/química , Fibronectinas/química , Integrinas/química , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Cristalografia por Raios X , Primers do DNA , Integrina alfa6beta4 , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
The microtubule-associated proteins 1B and 1A are synthesized as polyprotein precursors which are rapidly cleaved to give rise to heavy and light chains constituting the respective microtubule-associated protein 1B or microtubule-associated protein 1A complex. To identify domains necessary for precursor processing, we expressed microtubule-associated protein 1B deletion mutants in fibroblasts and monitored proteolytic cleavage of the precursor proteins by immunoblot analysis. We found that a novel hydrophilic, proline-rich 45 amino acid domain containing the cleavage site is necessary and sufficient for processing. This domain is conserved in microtubule-associated protein 1A. Additional sequences in the N-terminal half of the heavy chain contribute to the efficiency of cleavage.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Sítios de Ligação , Endopeptidases/metabolismo , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Precursores de Proteínas/genética , Proteínas/genética , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de AminoácidosRESUMO
We determined the previously unknown 3' end of MAP1B mRNA. We found an unusually long and highly conserved 3' untranslated region (3'UTR) of 4.3 kb and detected a polymorphism in the 3' flanking region probably due to the integration of an endogenous retroviral MuERV-L element. Furthermore, we found that MAP1B 3'UTR overlapped with the 5' end of the cDNA encoding DBI-1. However, further analysis suggested that the published structure of DBI-1 cDNA is most likely the result of fortuitous joining of unrelated cDNA fragments during cloning.