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81.
3D analysis of founder cell and fusion competent myoblast arrangements outlines a new model of myoblast fusion 总被引:2,自引:0,他引:2
Formation of the Drosophila larval body wall muscles requires the specification, coordinated cellular behaviors and fusion of two cell types: Founder Cells (FCs) that control the identity of the individual muscle and Fusion Competent Myoblasts (FCMs) that provide mass. These two cell types come together to control the final size, shape and attachment of individual muscles. However, the spatial arrangement of these cells over time, the sequence of fusion events and the contribution of these cellular relationships to the fusion process have not been addressed. We analyzed the three-dimensional arrangements of FCs and FCMs over the course of myoblast fusion and assayed whether these issues impact the process of myoblast fusion. We examined the timing of the fusion process by analyzing the fusion profile of individual muscles in wild type and fusion mutants. We showed that there are two temporal phases of myoblast fusion in wild type embryos. Limited fusion events occur during the first 3 h of fusion, while the majority of fusion events occur in the remaining 2.5 h. Altogether, our data have led us to propose a new model of myoblast fusion where the frequency of myoblast fusion events may be influenced by the spatial arrangements of FCs and FCMs. 相似文献
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83.
Dedieu S Dourdin N Dargelos E Poussard S Veschambre P Cottin P Brustis JJ 《Biology of the cell / under the auspices of the European Cell Biology Organization》2002,94(2):65-76
Previous studies have led us to hypothesize that m-calpain plays a pivotal role in myoblast fusion through its involvement in cell membrane and cytoskeleton component reorganization. To support this hypothesis, a convenient and simple myoblast culture model using frozen embryonic myoblasts was developed, which resolved a number of problems inherent to cell primary culture. Biological assays on cultured myoblasts using different media to define the characteristics of the fusion process were first conducted. Proteinase was detectable before the initiation of the fusion process and was closely correlated to the phenomenon of fusion under each culture condition studied. In addition, the study of calpastatin showed that the initiation of fusion does not require a decrease in the level of this endogenous inhibitor of calpains and also confirmed that calpastatin may be implicated in the determination of the end of fusion. On the other hand, analysis of the evolution of myogenic factors revealed that myogenins, MyoD and Myf5, increase very significantly during the formation of multinucleated myotubes. Moreover, the antisense technique against myogenin is capable of preventing the process of fusion by 50%, confirming the pivotal role of this factor in the early stages of differentiation. The possible role of myogenic regulator factors on m-calpain gene expression is discussed. 相似文献
84.
Nicole Stupka Christopher Kintakas Jason D. White Fiona W. Fraser Michael Hanciu Noriko Aramaki-Hattori Sheree Martin Chantal Coles Fiona Collier Alister C. Ward Suneel S. Apte Daniel R. McCulloch 《The Journal of biological chemistry》2013,288(3):1907-1917
Skeletal muscle development and regeneration requires the fusion of myoblasts into multinucleated myotubes. Because the enzymatic proteolysis of a hyaluronan and versican-rich matrix by ADAMTS versicanases is required for developmental morphogenesis, we hypothesized that the clearance of versican may facilitate the fusion of myoblasts during myogenesis. Here, we used transgenic mice and an in vitro model of myoblast fusion, C2C12 cells, to determine a potential role for ADAMTS versicanases. Versican processing was observed during in vivo myogenesis at the time when myoblasts were fusing to form multinucleated myotubes. Relevant ADAMTS genes, chief among them Adamts5 and Adamts15, were expressed both in developing embryonic muscle and differentiating C2C12 cells. Reducing the levels of Adamts5 mRNA in vitro impaired myoblast fusion, which could be rescued with catalytically active but not the inactive forms of ADAMTS5 or ADAMTS15. The addition of inactive ADAMTS5, ADAMTS15, or full-length V1 versican effectively impaired myoblast fusion. Finally, the expansion of a hyaluronan and versican-rich matrix was observed upon reducing the levels of Adamts5 mRNA in myoblasts. These data indicate that these ADAMTS proteinases contribute to the formation of multinucleated myotubes such as is necessary for both skeletal muscle development and during regeneration, by remodeling a versican-rich pericellular matrix of myoblasts. Our study identifies a possible pathway to target for the improvement of myogenesis in a plethora of diseases including cancer cachexia, sarcopenia, and muscular dystrophy. 相似文献
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Skeletal muscle increases in size due to weight bearing loads or passive stretch. This growth response is dependent in part upon myoblast proliferation. Although skeletal muscles are responsive to mechanical forces, the effect on myoblast proliferation remains unknown. To investigate the effects of mechanical stretch on myoblast proliferation, primary myoblasts isolated from Balb/c mice were subjected to 25% cyclical uniaxial stretch for 5 h at 0.5 Hz. Stretch stimulated myoblast proliferation by 32% and increased cell number by 41% 24 and 48 h after stretch, respectively. COX2 mRNA increased 3.5-fold immediately poststretch. Prostaglandin E2 and F2alpha increased 2.4- and 1.6-fold 6 h after stretch, respectively. Because COX2 has been implicated in regulating muscle growth and regeneration, we hypothesized that stretched myoblasts may proliferate via a COX2-dependent mechanism. We employed two different models to disrupt COX2 activity: (1) treatment with a COX2-selective drug, and (2) transgenic mice null for COX2. Treating myoblasts with a COX2-specific inhibitor blocked stretch-induced proliferation. Likewise, stretched COX2-/- myoblasts failed to proliferate compared to controls. However, supplementing stretched, COX2-/- myoblasts with prostaglandin E2 or fluprostenol increased proliferation. These data suggest that the COX2 pathway is critical for myoblast proliferation in response to stretch. 相似文献
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According to previous research, integrin β1 and ILK play an important role in the extracellular matrix (ECM)–integrin–cytoskeleton pathway for mechanotransduction. The aim of this study was to investigate strain induced integrin β1 and ILK expression in three-dimensional (3D) and in two-dimensional (2D) cultured rat skeletal myoblasts. Sprague–Dawley (SD) rat skeletal myoblasts were isolated and seeded on the PLGA-collagen composite scaffolds. The 3D cultured and the conventionally 2D monolayer cultured myoblasts were loaded 2000 μstrain tensile strain at 0.5 Hz for 2 h, 4 h, 8 h, 12 h and 24 h, respectively with the self-made four-point bending system. The expressions of integrin β1 and ILK mRNA were measured by RT-PCR and the different changes between the 3D and 2D cultures were compared. The mRNA expression levels of both integrin β1 and ILK were up regulated after mechanical loading (P < 0.05), meanwhile, it was higher and peaked faster in 3D cultures than in the 2D cultures. It can be concluded that the ECM–integrin–cytoskeleton pathway responds to tensile strain by elevated expression of integrin β1 and ILK, and the response is stronger in 3D cultures than in conventional 2D monolayer cultures. 相似文献
89.
Ewa Nurowska Andrew Constanti Beata Dworakowska Vincent Mouly Denis Furling Paola Lorenzon Tiziana Pietrangelo Krzysztof Dołowy Fabio Ruzzier 《Cellular & molecular biology letters》2009,14(2):336-346
The whole-cell patch clamp technique was used to record potassium currents in in vitro differentiating myoblasts isolated from healthy and myotonic dystrophy type 1 (DM1) foetuses carrying 2000 CTG repeats. The
fusion of the DM1 myoblasts was reduced in comparison to that of the control cells. The dystrophic muscle cells expressed
less voltage-activated K+ (delayed rectifier and non-inactivating delayed rectifier) and inward rectifier channels than the age-matched control cells.
However, the resting membrane potential was not significantly different between the control and the DM1 cells. After four
days in a differentiation medium, the dystrophic cells expressed the fast-inactivating transient outward K+ channels, which were not observed in healthy cells. We suggest that the low level of potassium currents measured in differentiated
DM1 cells could be related to their impaired fusion. 相似文献
90.
Stretch activation of GTP-binding proteins in C2C12 myoblasts 总被引:1,自引:0,他引:1
Mechanical stimulation has been proposed as a fundamental determinant of muscle physiology. The mechanotransduction of strain and strain rate in C2C12 myoblasts were investigated utilizing a radiolabeled GTP analogue to detect stretch-induced GTP-binding protein activation. Cyclic uniaxial strains of 10% and 20% at a strain rate of 20% s(-1) rapidly (within 1 min) activated a 25-kDa GTPase (183 +/- 17% and 186 +/- 19%, respectively), while 2% strain failed to elicit a response (109 +/- 11%) relative to controls. One, five, and sixty cycles of 10% strain elicited 187 +/- 20%, 183 +/- 17%, and 276 +/- 38% increases in activation. A single 10% stretch at 20% s(-1), but not 0.3% s(-1), resulted in activation. Insulin activated the same 25-kDa band in a dose-dependent manner. Western blot analysis revealed a panel of GTP-binding proteins in C2C12 myoblasts, and tentatively identified the 25-kDa GTPase as rab5. In separate experiments, a 40-kDa protein tentatively identified as Galpha(i) was activated (240 +/- 16%) by 10% strain at 1 Hz for 15 min. These results demonstrate the rapid activation of GTP-binding proteins by mechanical strain in myoblasts in both a strain magnitude- and strain rate-dependent manner. 相似文献