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1.
Food Chem ; 308: 125576, 2020 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-31648092

RESUMO

This study investigated the effects of cold storage at different temperatures (4, -0.5, -3, and -20 °C) on protein degradation and its relationship to structural changes of black carp muscle. At -0.5 and 4 °C, major structural changes occurred, including the formation of gaps between myofibers and myofibrils, breakage of myofibrils and myofibers, and degradation of sarcoplasmic reticulum. Gel-based proteomic analysis showed that these structural changes were accompanied by degradation of a series of myofibrillar proteins, including titin, nebulin, troponin, myosin, myomesin, myosin-binding protein, and α-actinin. Loss of extractable gelatinolytic and caseinolytic protease activities was also observed. At -3 and -20 °C, formation of ice crystals was the most noticeable change. The major proteins were degraded at different locations in the black carp muscle, and gelatinolytic and caseinolytic proteases appear to contribute to the degradation of those proteins.


Assuntos
Carpas/metabolismo , Actinina/metabolismo , Animais , Proteínas de Transporte , Temperatura Baixa , Conectina/metabolismo , Cyprinidae/metabolismo , Proteínas Musculares/metabolismo , Miofibrilas/metabolismo , Miosinas/metabolismo , Proteólise , Proteômica
2.
Invest Ophthalmol Vis Sci ; 60(14): 4583-4595, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31675075

RESUMO

Purpose: Trabecular meshwork (TM) cells detect and coordinate responses to intraocular pressure (IOP) in the eye. TM cells become dysfunctional in glaucoma where IOP is often elevated. Recently, we showed that normal TM (NTM) cells communicate by forming tubular connections called tunneling nanotubes (TNTs). Here, we investigated TNTs in glaucomatous TM (GTM) cells. Methods: Primary GTM and NTM cells were established from cadaver eyes. Transfer of Vybrant DiO and DiD-labeled vesicles via TNT connections was measured. Imaris software measured the number and length of cell protrusions from immunofluorescent confocal images. Live-cell imaging of the actin cytoskeleton was performed. The distribution of myosin-X, a regulator of TNTs/filopodia, was investigated in TM cells and tissue. Results: GTM cells contained significantly more transferred fluorescent vesicles than NTM cells (49.6% vs. 35%). Although NTM cells had more protrusions at the cell surface than GTM cells (7.61 vs. 4.65 protrusions/cell), GTM protrusions were significantly longer (12.1 µm vs. 9.76 µm). Live-cell imaging demonstrated that the GTM actin cytoskeleton was less dynamic, and vesicle transfer between cells was significantly slower than NTM cells. Furthermore, rearrangement of the actin cortex adjacent to the TNT may influence TNT formation. Myosin-X immunostaining was punctate and disorganized in GTM cells and tissue compared to age-matched NTM controls. Conclusions: Together, our data demonstrate that GTM cells have phenotypic and functional differences in their TNTs. Significantly slower vesicle transfer via TNTs in GTM cells may delay the timely propagation of cellular signals when pressures become elevated in glaucoma.


Assuntos
Citoesqueleto de Actina/metabolismo , Glaucoma de Ângulo Aberto/patologia , Miosinas/metabolismo , Nanotubos , Pseudópodes/metabolismo , Malha Trabecular/patologia , Western Blotting , Tamanho Celular , Células Cultivadas , Senescência Celular/fisiologia , Densitometria , Glaucoma de Ângulo Aberto/metabolismo , Humanos , Microscopia Confocal , Fagocitose/fisiologia , Fenótipo , Transdução de Sinais/fisiologia , Malha Trabecular/metabolismo
3.
Nat Commun ; 10(1): 3593, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399564

RESUMO

Filopodia, dynamic membrane protrusions driven by polymerization of an actin filament core, can adhere to the extracellular matrix and experience both external and cell-generated pulling forces. The role of such forces in filopodia adhesion is however insufficiently understood. Here, we study filopodia induced by overexpression of myosin X, typical for cancer cells. The lifetime of such filopodia positively correlates with the presence of myosin IIA filaments at the filopodia bases. Application of pulling forces to the filopodia tips through attached fibronectin-coated laser-trapped beads results in sustained growth of the filopodia. Pharmacological inhibition or knockdown of myosin IIA abolishes the filopodia adhesion to the beads. Formin inhibitor SMIFH2, which causes detachment of actin filaments from formin molecules, produces similar effect. Thus, centripetal force generated by myosin IIA filaments at the base of filopodium and transmitted to the tip through actin core in a formin-dependent fashion is required for filopodia adhesion.


Assuntos
/metabolismo , Miosinas/metabolismo , Neoplasias/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Pseudópodes/fisiologia , Citoesqueleto de Actina , Animais , Células COS , /genética , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Proteínas dos Microfilamentos , Miosina não Muscular Tipo IIA/antagonistas & inibidores , Miosina não Muscular Tipo IIA/genética , Miosina não Muscular Tipo IIA/ultraestrutura , Pseudópodes/patologia , Tionas/farmacologia , Uracila/análogos & derivados , Uracila/farmacologia
4.
Int J Mol Sci ; 20(16)2019 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-31408934

RESUMO

Recent findings have revealed the role of membrane traffic in the signaling of transforming growth factor-ß (TGF-ß). These findings originate from the pivotal function of TGF-ß in development, cell proliferation, tumor metastasis, and many other processes essential in malignancy. Actin and unconventional myosin have crucial roles in subcellular trafficking of receptors; research has also revealed a growing number of unconventional myosins that have crucial roles in TGF-ß signaling. Unconventional myosins modulate the spatial organization of endocytic trafficking and tether membranes or transport them along the actin cytoskeletons. Current models do not fully explain how membrane traffic forms a bridge between TGF-ß and the downstream effectors that produce its functional responsiveness, such as cell migration. In this review, we present a brief overview of the current knowledge of the TGF-ß signaling pathway and the molecular components that comprise the core pathway as follows: ligands, receptors, and Smad mediators. Second, we highlight key role(s) of myosin motor-mediated protein trafficking and membrane domain segregation in the modulation of the TGF-ß signaling pathway. Finally, we review future challenges and provide future prospects in this field.


Assuntos
Miosinas/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Vesículas Revestidas por Clatrina/metabolismo , Endocitose , Humanos , Microdomínios da Membrana/metabolismo , Transporte Proteico
5.
Biochim Biophys Acta Mol Cell Res ; 1866(11): 118510, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31319111

RESUMO

Discoidin Domain Receptor (DDR) genes and their homologues have been identified in sponges, worms and flies. These genes code for proteins that are implicated in cell adhesion to matrix proteins. DDRs are now recognized as playing central regulatory roles in several high prevalence human diseases, including invasive cancers, atherosclerosis, and organ fibrosis. While the mechanisms by which DDRs contribute to these diseases are just now being delineated, one of the common themes involves cell adhesion to collagen and the assembly and organization of collagen fibers in the extracellular matrix. In mammals, the multi-functional roles of DDRs in promoting cell adhesion to collagen fibers and in mediating collagen-dependent signaling, suggest that DDRs contribute to multiple pathways of extracellular matrix remodeling, which are centrally important processes in health and disease. In this review we consider that interactions of the cytoplasmic domains of DDR1 with cytoskeletal motor proteins may contribute to matrix remodeling by promoting collagen fiber alignment and compaction. Poorly controlled collagen remodeling with excessive compaction of matrix proteins is a hallmark of fibrotic lesions in many organs and tissues that are affected by infectious, traumatic or chemical-mediated injury. An improved understanding of the mechanisms by which DDRs mediate collagen remodeling and collagen-dependent signaling could suggest new drug targets for treatment of fibrotic diseases.


Assuntos
Colágeno/química , Receptor com Domínio Discoidina 1/química , Receptor com Domínio Discoidina 1/metabolismo , Fibrose/metabolismo , Miosinas/química , Domínios e Motivos de Interação entre Proteínas , Animais , Adesão Celular/genética , Colágeno/metabolismo , Receptor com Domínio Discoidina 1/genética , Matriz Extracelular/metabolismo , Humanos , Miosinas/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas , Transdução de Sinais
6.
Nat Cell Biol ; 21(8): 933-939, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31358965

RESUMO

Actomyosin networks, the cell's major force production machineries, remodel cellular membranes during myriad dynamic processes1,2 by assembling into various architectures with distinct force generation properties3,4. While linear and branched actomyosin architectures are well characterized in cell-culture and cell-free systems3, it is not known how actin and myosin networks form and function to remodel membranes in complex three-dimensional mammalian tissues. Here, we use four-dimensional spinning-disc confocal microscopy with image deconvolution to acquire macromolecular-scale detail of dynamic actomyosin networks in exocrine glands of live mice. We address how actin and myosin organize around large membrane-bound secretory vesicles and generate the forces required to complete exocytosis5-7. We find that actin and non-muscle myosin II (NMII) assemble into previously undescribed polyhedral-like lattices around the vesicle membrane. The NMII lattice comprises bipolar minifilaments8-10 as well as non-canonical three-legged configurations. Using photobleaching and pharmacological perturbations in vivo, we show that actomyosin contractility and actin polymerization together push on the underlying vesicle membrane to overcome the energy barrier and complete exocytosis7. Our imaging approach thus unveils a force-generating actomyosin lattice that regulates secretion in the exocrine organs of live animals.


Assuntos
Actomiosina/metabolismo , Exocitose/fisiologia , Contração Muscular/fisiologia , Miosinas/metabolismo , Citoesqueleto de Actina/metabolismo , Actomiosina/genética , Animais , Membrana Celular/metabolismo , Exocitose/genética , Camundongos Transgênicos , Microscopia Confocal/métodos , Miosinas/genética , Vesículas Secretórias/metabolismo
7.
Biomed Pharmacother ; 117: 109053, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31176169

RESUMO

Chronic obstructive pulmonary disease (COPD) often causes diaphragm dysfunction, which is the main contributor to neuro-muscular respiratory failure and associated with the prognosis of COPD. However, the morphological changes in diaphragm during the development of COPD are complicated and underlying mechanisms have not been absolutely elucidated. Considering smoking is one of the most leading and important risk factors for development of COPD, we set out to investigate the effects of smoking on muscle fibre remodeling and underlying mechanisms in diaphragms. Rats were randomly exposed to cigarette smoke (CS) for one of three durations of 4, 8, and 12 weeks. CS exposure resulted in increased percentage of type I muscle fibres, enhanced apoptosis index, endoplasmic reticulum (ER) dilation, elevated expression of glucose-regulated protein 78, C/EBP homologous protein, caspase-12, peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), cytochrome c oxidase 4, Sdhb, p53 and a reduction in fibre diameters in rat diaphragms. The results indicated that CS exposure induced a shift to muscle fibres with aerobic metabolism in predominance in rat diaphragms, which may be dependent on the regulation of PGC-1α and p53. Additionally, ER stress (ERS) associated apoptosis may contribute to the pathogenesis of diaphragmatic muscle atrophy induced by CS exposure.


Assuntos
Diafragma/patologia , Fibras Musculares Esqueléticas/patologia , Fumar/efeitos adversos , Animais , Apoptose , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Enfisema/complicações , Enfisema/patologia , Retículo Endoplasmático/ultraestrutura , Estresse do Retículo Endoplasmático , Feminino , Mitocôndrias/metabolismo , Atrofia Muscular/patologia , Miosinas/metabolismo , Oxirredução , Pneumonia/complicações , Pneumonia/patologia , Ratos Sprague-Dawley , Fatores de Tempo , Proteína Supressora de Tumor p53/metabolismo
8.
Food Chem ; 295: 320-326, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31174764

RESUMO

This research focused on the effects of l-arginine (l-Arg) and l-histidine (l-His) on the heat-induced aggregation of fish myosin. l-Arg/l-His increased the pH of the myosin solution from 6.82 to 8.74 and 7.24, respectively, and decreased the turbidity, aggregate size, shear modulus, and breaking force. The incorporation of l-Arg/l-His decreased the surface hydrophobicity during setting, but increased it during the two-step heating. The heat-induced aggregation of myosin was suppressed by both amino acids, with the inhibitory effect being greater for l-Arg than l-His. On one hand, the change in the pH played a critical role in suppressing the heat-induced aggregation of myosin. On the other hand, the characteristics of l-Arg/l-His themselves, such as net charges and particular R-groups, were another main contributor to aggregation suppression. Particularly, l-Arg/l-His could interact with exposed aromatic residues of myosin, and the interactions may dominate and overwhelm the burial of aromatic residues during two-step heating.


Assuntos
Arginina/química , Proteínas de Peixes da Dieta/química , Histidina/química , Miosinas/química , Animais , Cyprinidae , Proteínas de Peixes da Dieta/metabolismo , Géis/química , Temperatura Alta , Interações Hidrofóbicas e Hidrofílicas , Miosinas/metabolismo
9.
Int J Mol Sci ; 20(12)2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31238505

RESUMO

The relation between the force (load) and the velocity of shortening (V) in contracting skeletal muscle is part of a rectangular hyperbola: (P + a) V = b(Po - P); where Po is the maximum isometric force and a and b are constants. The force-velocity (P-V) relation suggests that muscle can regulate its energy output depending on the load imposed on it (Hill, 1938). After the establishment of the sliding filament mechanism (H.E. Huxley and Hanson, 1954), the P-V relation has been regarded to reflect the cyclic interaction between myosin heads in myosin filaments and the corresponding myosin-binding sites in actin filaments, coupled with ATP hydrolysis (A.F. Huxley, 1957). In single skeletal muscle fibers, however, the P-V relation deviates from the hyperbola at the high force region, indicating complicated characteristics of the cyclic actin-myosin interaction. To correlate the P-V relation with kinetics of actin-myosin interaction, skinned muscle fibers have been developed, in which the surface membrane is removed to control chemical and ionic conditions around the 3D lattice of actin and myosin filaments. This article also deals with experimental methods with which the structural instability of skinned fibers can be overcome by applying parabolic decreases in fiber length.


Assuntos
Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiologia , Animais , Humanos , Contração Isotônica , Cinética , Modelos Biológicos , Fibras Musculares Esqueléticas/ultraestrutura , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Força Muscular , Músculo Esquelético/ultraestrutura , Fenômenos Fisiológicos Musculoesqueléticos , Miosinas/química , Miosinas/metabolismo , Relação Estrutura-Atividade
10.
Bull Exp Biol Med ; 167(1): 65-68, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31177460

RESUMO

Tropomyosin (Tpm) is one of the main regulatory proteins in the myocardium. In some heart pathologies, interchain disulfide crosslinking in the Tpm molecule occurs. In the ventricle, this change in the structural properties of the Tpm molecule affects calcium regulation of the actin-myosin interaction. Using an in vitro motility assay, we found that Tpm crosslinking does not affect the actin-myosin interaction in the atria. We assume that the intramolecular crosslinking of Tpm in the atrium does not play such a crucial role in the pathogenesis of heart failure as it plays in the heart ventricles.


Assuntos
Actinas/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Miosinas/metabolismo , Tropomiosina/química , Tropomiosina/metabolismo , Actinas/química , Animais , Cálcio/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Miosinas/química , Ligação Proteica , Coelhos
11.
J Biol Chem ; 294(18): 7219-7220, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-31053679

RESUMO

"Myosin" is famous as a component of muscle fibrils, but the majority of myosin family members act elsewhere with roles unrelated to muscle contraction. The biological functions of a relatively new family of these unconventional myosins, myosins 18A and 18B, are poorly understood. New research from Horsthemke et al. describes a new isoform (Myo18Aγ) that is essential for heart function and viability in mice. Their findings both support and contradict other work in the field and raise new questions about the roles of myosin 18 proteins in vivo.


Assuntos
Miofibrilas/metabolismo , Miosinas/metabolismo , Animais , Citoesqueleto/metabolismo , Camundongos
12.
PLoS One ; 14(5): e0216228, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31141508

RESUMO

The African spiny mouse, Acomys spp., is capable of scar-free dermal wound healing. Here, we have performed a comprehensive analysis of gene expression throughout wound healing following full-thickness excisional dermal wounds in both Acomys cahirinus and Mus musculus. Additionally, we provide an annotated, de novo transcriptome assembly of A. cahirinus skin and skin wounds. Using a novel computational comparative RNA-Seq approach along with pathway and co-expression analyses, we identify enrichment of regeneration associated genes as well as upregulation of genes directly related to muscle development or function. Our RT-qPCR data reveals induction of the myogenic regulatory factors, as well as upregulation of embryonic myosin, starting between days 14 and 18 post-wounding in A. cahirinus. In contrast, the myogenic regulatory factors remain downregulated, embryonic myosin is only modestly upregulated, and no new muscle fibers of the panniculus carnosus are generated in M. musculus wounds. Additionally, we show that Col6a1, a key component of the satellite cell niche, is upregulated in A. cahirinus compared to M. musculus. Our data also demonstrate that the macrophage profile and inflammatory response is different between species, with A. cahirinus expressing significantly higher levels of Il10. We also demonstrate differential expression of the upstream regulators Wnt7a, Wnt2 and Wnt6 during wound healing. Our analyses demonstrate that A. cahirinus is capable of de novo skeletal muscle regeneration of the panniculus carnosus following removal of the extracellular matrix. We believe this study represents the first detailed analysis of de novo skeletal muscle regeneration observed in an adult mammal.


Assuntos
Murinae/fisiologia , Músculo Esquelético/fisiologia , Regeneração , Pele , Transcriptoma , Cicatrização , Animais , Camundongos , Murinae/genética , Desenvolvimento Muscular/genética , Miosinas/metabolismo , Regeneração/genética , Proteínas Wnt/metabolismo
13.
PLoS One ; 14(5): e0214494, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31095594

RESUMO

We introduce a Mueller-matrix imaging polarization-based approach for the quantitative digital screening of the polycrystalline structure of fibrillary-based biological tissues in vitro. The morphometric evaluation of histological sections of myocardium was performed utilizing the high-order statistical moments calculated based on the spatial distribution of linear and circular birefringence and dichroism obtained experimentally. We demonstrate that spatial distributions of phase of light and optical anisotropy of scattering inherent to fibrillar networks of myocardium at different necrotic stages can be effectively used as a quantitative marker of stages of myosin fibril degradation. Processing the images of phase of light scattered in biological tissues with high order statistical analysis provides a functional tool for the quantitative characterization of necrotic conditions of the myocardium.


Assuntos
Anisotropia , Miocárdio/metabolismo , Algoritmos , Birrefringência , Microscopia de Polarização/métodos , Miosinas/metabolismo
14.
Can J Vet Res ; 83(2): 142-148, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31097876

RESUMO

Bilateral deafness with concurrent vestibular dysfunction was first reported in the Doberman pinscher in 1980. Here, we identify a coding mutation in the MYO7A gene that is perfectly associated with the disorder. The lack of visual deficits in affected dogs suggests that, like rodents but unlike humans, MYO7A is not required for retinal function. DNA testing of the mutation will enable dog breeders to manage the incidence of this genetic defect.


Assuntos
Surdez/veterinária , Doenças do Cão/genética , Predisposição Genética para Doença , Mutação de Sentido Incorreto , Miosinas/genética , Doenças Vestibulares/veterinária , Animais , Estudos de Casos e Controles , DNA/genética , Surdez/genética , Cães , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Genótipo , Miosinas/metabolismo , Doenças Vestibulares/genética , Sequenciamento Completo do Genoma
15.
PLoS Genet ; 15(5): e1008083, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31116733

RESUMO

How biochemical and mechanical information are integrated during tissue development is a central question in morphogenesis. In many biological systems, the PIX-GIT complex localises to focal adhesions and integrates both physical and chemical information. We used Drosophila melanogaster egg chamber formation to study the function of PIX and GIT orthologues (dPix and Git, respectively), and discovered a central role for this complex in controlling myosin activity and epithelial monolayering. We found that Git's focal adhesion targeting domain mediates basal localisation of this complex to filament structures and the leading edge of migrating cells. In the absence of dpix and git, tissue disruption is driven by contractile forces, as reduction of myosin activators restores egg production and morphology. Further, dpix and git mutant eggs closely phenocopy defects previously reported in pak mutant epithelia. Together, these results indicate that the dPix-Git complex controls egg chamber morphogenesis by controlling myosin contractility and Pak kinase downstream of focal adhesions.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Ativadoras de GTPase/genética , Morfogênese/genética , Miosinas/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Movimento Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Adesões Focais/metabolismo , Adesões Focais/ultraestrutura , Proteínas Ativadoras de GTPase/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Mecanotransdução Celular , Miosinas/metabolismo , Zigoto/citologia , Zigoto/crescimento & desenvolvimento , Zigoto/metabolismo , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismo
16.
Anim Sci J ; 90(7): 801-807, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31134719

RESUMO

Skeletal muscle consists of bundles of myofibers containing millions of myofibrils, each of which is formed of longitudinally aligned sarcomere structures. Sarcomeres are the minimum contractile unit, which mainly consists of four components: Z-bands, thin filaments, thick filaments, and connectin/titin. The size and shape of the sarcomere component is strictly controlled. Surprisingly, skeletal muscle cells not only synthesize a series of myofibrillar proteins but also regulate the assembly of those proteins into the sarcomere structures. However, authentic sarcomere structures cannot be reconstituted by combining purified myofibrillar proteins in vitro, therefore there must be an elaborate mechanism ensuring the correct formation of myofibril structure in skeletal muscle cells. This review discusses the role of myosin, a main component of the thick filament, in thick filament formation and the dynamics of myosin in skeletal muscle cells. Changes in the number of myofibrils in myofibers can cause muscle hypertrophy or atrophy. Therefore, it is important to understand the fundamental mechanisms by which myofibers control myofibril formation at the molecular level to develop approaches that effectively enhance muscle growth in animals.


Assuntos
Citoesqueleto/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Miosinas/fisiologia , Animais , Atrofia , Hipertrofia , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/patologia , Miofibrilas/metabolismo , Miofibrilas/patologia , Miosinas/metabolismo , Sarcômeros/metabolismo
17.
Int J Mol Sci ; 20(7)2019 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-30959804

RESUMO

Much has been learned about the interaction between myosin and actin through biochemistry, in vitro motility assays and cryo-electron microscopy (cryoEM) of F-actin, decorated with myosin heads. Comparatively less is known about actin-myosin interactions within the filament lattice of muscle, where myosin heads function as independent force generators and thus most measurements report an average signal from multiple biochemical and mechanical states. All of the 3D imaging by electron microscopy (EM) that has revealed the interplay of the regular array of actin subunits and myosin heads within the filament lattice has been accomplished using the flight muscle of the large water bug Lethocerus sp. The Lethocerus flight muscle possesses a particularly favorable filament arrangement that enables all the myosin cross-bridges contacting the actin filament to be visualized in a thin section. This review covers the history of this effort and the progress toward visualizing the complex set of conformational changes that myosin heads make when binding to actin in several static states, as well as the fast frozen actively contracting muscle. The efforts have revealed a consistent pattern of changes to the myosin head structures as determined by X-ray crystallography needed to explain the structure of the different actomyosin interactions observed in situ.


Assuntos
Actinas/metabolismo , Microscopia Crioeletrônica , Imagem Tridimensional , Músculos/metabolismo , Músculos/ultraestrutura , Miosinas/metabolismo , Animais , Secções Congeladas
18.
Res Vet Sci ; 124: 270-279, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31003009

RESUMO

In ungulates the stability of the fetlock joint is dependent on several muscles, which are exposed to high stress and strain. Among those muscles, the proximal sesamoidean ligament or PSL (also known as the suspensory ligament or Ruini's elasto-tendinous organ) is organized at birth in layers of muscle fibres alternated with abundant tendinous tissue that, during the postnatal development, becomes the predominant tissue. In this study we analysed the PSL of the sheep at the age of 1, 30 and 180 days and determined the expression of several genes which either (a) are markers of muscle fibre growth and maturation, or (b) play a role as signal molecules. We observed an accelerated maturation, as indicated by the transition of MyHC isoform expression towards the slow isoforms and a reduced regenerative potential indicated by the low Pax7 expression and the altered Wnt signalling. We also found a specific myogenic expression pattern of MyoD, Myf5 and Myogenin in the developing PSL and high mRNA levels of specific fibrogenic factors, as TGF-ß1, that, undoubtedly, stimulate the growth of connective tissue. Our observations confirmed, at molecular level, the peculiarity of the fast involution observed in PSL a muscle that undergoes a very specific active differentiation process during early development, which implies myofibres involution and their replacement with connective tissue.


Assuntos
Ligamentos/crescimento & desenvolvimento , Desenvolvimento Muscular/genética , Miosinas/genética , Carneiro Doméstico/genética , Fatores Etários , Animais , Diferenciação Celular , Fatores de Regulação Miogênica , Miosinas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ossos Sesamoides , Carneiro Doméstico/crescimento & desenvolvimento
19.
PLoS Biol ; 17(4): e3000211, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30990821

RESUMO

During spermatogenesis, interconnected haploid spermatids segregate undesired cellular contents into residual bodies (RBs) before detaching from RBs. It is unclear how this differentiation process is controlled to produce individual spermatids or motile spermatozoa. Here, we developed a live imaging system to visualize and investigate this process in C. elegans. We found that non-muscle myosin 2 (NMY-2)/myosin II drives incomplete cytokinesis to generate connected haploid spermatids, which are then polarized to segregate undesired cellular contents into RBs under the control of myosin II and myosin VI. NMY-2/myosin II extends from the pseudo-cleavage furrow formed between two haploid spermatids to the spermatid poles, thus promoting RB expansion. In the meantime, defective spermatogenesis 15 (SPE-15)/myosin VI migrates from spermatids towards the expanding RB to promote spermatid budding. Loss of myosin II or myosin VI causes distinct cytoplasm segregation defects, while loss of both myosins completely blocks RB formation. We found that the final separation of spermatids from RBs is achieved through myosin VI-mediated cytokinesis, while myosin II is dispensable at this step. SPE-15/myosin VI and F-actin form a detergent-resistant actomyosin VI ring that undergoes continuous contraction to promote membrane constriction between spermatid and RB. We further identified that RGS-GAIP-interacting protein C terminus (GIPC)-1 and GIPC-2 cooperate with myosin VI to regulate contractile ring formation and spermatid release. Our study reveals distinct roles of myosin II and myosin VI in spermatid differentiation and uncovers a novel myosin VI-mediated cytokinesis process that controls spermatid release.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Miosinas/metabolismo , Espermátides/metabolismo , Actinas/metabolismo , Animais , Caenorhabditis elegans , Diferenciação Celular/fisiologia , Citoplasma/metabolismo , Masculino , Cadeias Pesadas de Miosina/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Espermatogênese/fisiologia , Espermatozoides/metabolismo
20.
Dev Cell ; 49(2): 189-205.e6, 2019 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-31014479

RESUMO

Efficient chemotaxis requires rapid coordination between different parts of the cell in response to changing directional cues. Here, we investigate the mechanism of front-rear coordination in chemotactic neutrophils. We find that changes in the protrusion rate at the cell front are instantaneously coupled to changes in retraction at the cell rear, while myosin II accumulation at the rear exhibits a reproducible 9-15-s lag. In turning cells, myosin II exhibits dynamic side-to-side relocalization at the cell rear in response to turning of the leading edge and facilitates efficient turning by rapidly re-orienting the rear. These manifestations of front-rear coupling can be explained by a simple quantitative model incorporating reversible actin-myosin interactions with a rearward-flowing actin network. Finally, the system can be tuned by the degree of myosin regulatory light chain (MRLC) phosphorylation, which appears to be set in an optimal range to balance persistence of movement and turning ability.


Assuntos
Quimiotaxia/fisiologia , Miosina Tipo II/fisiologia , Neutrófilos/fisiologia , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular , Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Extensões da Superfície Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Feminino , Humanos , Miosina Tipo II/metabolismo , Miosinas/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
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