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1.
Nat Commun ; 14(1): 7506, 2023 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-37980413

RESUMO

Apical extracellular matrices (aECMs) are complex extracellular compartments that form important interfaces between animals and their environment. In the adult C. elegans cuticle, layers are connected by regularly spaced columnar structures known as struts. Defects in struts result in swelling of the fluid-filled medial cuticle layer ('blistering', Bli). Here we show that three cuticle collagens BLI-1, BLI-2, and BLI-6, play key roles in struts. BLI-1 and BLI-2 are essential for strut formation whereas activating mutations in BLI-6 disrupt strut formation. BLI-1, BLI-2, and BLI-6 precisely colocalize to arrays of puncta in the adult cuticle, corresponding to struts, initially deposited in diffuse stripes adjacent to cuticle furrows. They eventually exhibit tube-like morphology, with the basal ends of BLI-containing struts contact regularly spaced holes in the cuticle. Genetic interaction studies indicate that BLI strut patterning involves interactions with other cuticle components. Our results reveal strut formation as a tractable example of precise aECM patterning at the nanoscale.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Colágeno/genética , Matriz Extracelular/genética
2.
Methods Mol Biol ; 798: 77-84, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22130832

RESUMO

Secondary skeletal muscle fiber phenotype is dependent upon depolarization from motor neuron innervation. To study the effects of depolarization on muscle fiber type development, several in vivo and in vitro model systems exist. We have developed a relatively simple-to-use in vitro model system in which differentiated muscle cells are directly electrically stimulated at precise frequencies. This allows for single cell analysis as well as biochemical and molecular analyses of the mechanisms that control skeletal muscle phenotype.


Assuntos
Estimulação Elétrica/métodos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/fisiologia , Separação Celular/métodos , Imuno-Histoquímica/métodos , Cultura Primária de Células
3.
Exp Cell Res ; 316(6): 1039-49, 2010 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-20070941

RESUMO

Vertebrate skeletal muscle fiber types are defined by a broad array of differentially expressed contractile and metabolic protein genes. The mechanisms that establish and maintain these different fiber types vary throughout development and with changing functional demand. Chicken skeletal muscle fibers can be generally categorized as fast and fast/slow based on expression of the slow myosin heavy chain 2 (MyHC2) gene in fast/slow muscle fibers. To investigate the cellular and molecular mechanisms that control fiber type formation in secondary or fetal muscle fibers, myoblasts from the fast pectoralis major (PM) and fast/slow medial adductor (MA) muscles were isolated, allowed to differentiate in vitro, and electrically stimulated. MA muscle fibers were induced to express the slow MyHC2 gene by electrical stimulation, whereas PM muscle fibers did not express the slow MyHC2 gene under identical stimulation conditions. However, PM muscle fibers did express the slow MyHC2 gene when electrical stimulation was combined with inhibition of inositol triphosphate receptor (IP3R) activity. Electrical stimulation was sufficient to increase nuclear localization of expressed nuclear-factor-of-activated-T-cells (NFAT), NFAT-mediated transcription, and slow MyHC2 promoter activity in MA muscle fibers. In contrast, both electrical stimulation and inhibitors of IP3R activity were required for these effects in PM muscle fibers. Electrical stimulation also increased levels of peroxisome-proliferator-activated receptor-gamma co-activator-1 (PGC-1alpha) protein in PM and MA muscle fibers. These results indicate that MA muscle fibers can be induced by electrical stimulation to express the slow MyHC2 gene and that fast PM muscle fibers are refractory to stimulation-induced slow MyHC2 gene expression due to fast PM muscle fiber specific cellular mechanisms involving IP3R activity.


Assuntos
Regulação da Expressão Gênica , Fibras Musculares Esqueléticas/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Animais , Compostos de Boro/metabolismo , Células Cultivadas , Embrião de Galinha , Estimulação Elétrica , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Fibras Musculares Esqueléticas/citologia , Mioblastos/citologia , Mioblastos/metabolismo , Cadeias Pesadas de Miosina/genética , Fatores de Transcrição NFATC/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Transcrição Gênica
4.
J Neurosci ; 23(9): 3577-87, 2003 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-12736328

RESUMO

Vertebrate neuromuscular junctions (NMJs) contain specialized basal laminas enriched for proteins not found at high concentrations extrasynaptically. Alterations in NMJ basement membrane components can result in loss of NMJ structural integrity and lead to muscular dystrophies. We demonstrate here that the conserved Caenorhabditis elegans basement membrane-associated molecules nidogen/entactin (NID-1) and type XVIII collagen (CLE-1) are associated with axons and particularly enriched near synaptic contacts. NID-1 is concentrated laterally, between the nerve cord and muscles, whereas CLE-1 is concentrated dorsal to the ventral nerve cord and ventral to the dorsal nerve cord, above the regions where synapses form. Mutations in these molecules cause specific and distinct defects in the organization of neuromuscular junctions. The mutant animals exhibit mild movement defects and altered responses to an inhibitor of acetylcholinesterase and a cholinergic agonist, indicating altered synaptic function. Our results provide the first demonstration that basement membrane molecules are important for NMJ formation and/or maintenance in C. elegans and that collagen XVIII and nidogen can have important roles in synapse organization.


Assuntos
Membrana Basal/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação ao Cálcio , Colágeno/metabolismo , Glicoproteínas de Membrana/metabolismo , Junção Neuromuscular/metabolismo , Alelos , Animais , Caenorhabditis elegans , Agonistas Colinérgicos/farmacologia , Inibidores da Colinesterase/farmacologia , Colágeno/genética , Colágeno Tipo XVIII , Genes Reporter , Proteínas de Fluorescência Verde , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Transtornos dos Movimentos/genética , Mutação , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/biossíntese , Sistema Nervoso/fisiopatologia , Malformações do Sistema Nervoso/genética , Sinapses/metabolismo , Sinaptotagminas , Ácido gama-Aminobutírico/metabolismo
5.
Dev Genes Evol ; 213(3): 107-19, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12690448

RESUMO

Mutations in the lozenge gene of Drosophila melanogaster elicit a pleiotropic set of adult phenotypes, including severe compound eye perturbations resulting from the defective recruitment of photoreceptors R1/6 and R7, cone and pigment cells. In this study, we show that excessive patterned apoptosis is evident at the same developmental stage in these lozenge mutants. In lozenge null mutants, apoptosis occurs prior to lozenge-dependent cell fate specification. A second gene, D-Pax2, genetically interacts with lozenge. Interestingly, D-Pax2 mutants also exhibit increased cell death, but slightly later in development than that in lozenge mutants. Although expression of the caspase inhibitor p35 eliminates death in both lozenge and D-Pax2 mutants, the lozenge mutant eye phenotypes persist because other normal Lozenge functions are still lacking. D-Pax2 eye phenotypes, in contrast, are dramatically altered in a p35 background, because cells that normally differentiate as cone and primary pigment cells are subsequently transformed into secondary pigment cells. This study leads us to propose that Lozenge, aside from its known role in gene regulation of cell-specific transcription factors, is required to contribute to the repression of cell death mechanisms, creating a permissive environment for the survival of undifferentiated cells in early eye development. Lack of lozenge expression increases the likelihood that an undifferentiated cell will initiate its default death program and die prematurely. The ectopic cell death evident in D-Pax2 mutants appears to arise from the cell fate transformation of cone cells into secondary pigment cells, either autonomously or as a result of defective signalling.


Assuntos
Apoptose/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/genética , Olho/embriologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Animais , Mapeamento Cromossômico , Primers do DNA , Imuno-Histoquímica , Hibridização In Situ , Modelos Biológicos , Células Fotorreceptoras Retinianas Cones/embriologia , Análise de Sequência de DNA
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