No description available.

Myogenesis, or skeletal muscle formation, begins during embryogenesis and involves the proliferation of myoblasts followed by their exit from the cell-cycle to differentiate and form myotubes. This formation of skeletal muscle is a complex process involving many genes and various signalling pathways. Mighty is a novel myogenic gene discovered at AgResearch by the Functional Muscle Genomics (FMG) group in a genetic screen performed on the muscle of myostatin null and wild-type mice. It was found that heavily muscled mice, lacking myostatin, had increased expression of the mighty gene. This gene was found to be conserved, with cognates found in mammals, amphibians, teleosts, and arthropods. Mighty was found to be expressed in a variety of tissues, but only skeletal muscle showed increased mighty mRNA expression in myostatin null mice, indicating the specific regulation of mighty by myostatin in skeletal muscle (Marshall, 2005). The aim of this study was to characterise the mighty protein and examine its role in myogenesis to elucidate mighty's function. To undertake this study, antibodies specific for the full-length mighty protein and antibodies specific for a peptide region of mighty were characterised. Results using these antibodies, showed endogenous mighty, from myoblasts, to be a low-abundant, nuclear protein which shows a mobility of ~52 kDa in SDS gels, different to that of recombinant mighty protein. The mobility difference of endogenous mighty compared to recombinant mighty appears to be due to phosphorylation and may involve other post-translational modifications. In agreement, the determined isoelectric point (~5.7) of endogenous mighty also appears to be the result of phosphorylation. Interestingly, 52 kDa mighty was not detected in muscle extracts, but a ~30 kDa protein was specifically detected, indicating multiple forms, and subsequent roles, for mighty protein. Mass spectrometry (MS) was also performed for further characterisation of the mighty protein and possible post-translational modifications. Although hits were achieved with both recombinant mighty proteins, endogenous mighty MS analysis was not accomplished due to its low-abundance. The function of the mighty protein in myoblasts was investigated during proliferation and differentiation. The results indicate that proliferating myoblasts have low levels of mighty in G0 and increased levels in G1/S during the cell cycle. This differential expression of mighty may involve cell cycle exit at the G1/S phase. Differentiation results showed mighty to be upregulated before MyoD during differentiation, placing mighty very early in the differentiation hierarchy. This agrees with previous results by Marshall (2005) which showed mighty to upregulate MyoD through IGF-II expression. Enhanced differentiation was also seen in double muscle bovine myoblasts concomitantly with increased mighty expression. In conclusion, mighty appears to be a post-translationally modified protein that plays an early role in myogenic differentiation. This role in differentiation appears to be upstream of MyoD through the upregulation of IGF-II and may be linked to cell cycle exit in the G1 phase of the cell cycle.

Staufen1 is a double-stranded RNA-binding protein known to be involved in the transport, localization, decay and increased translation of some mRNAs. The goal of the present study is to determine the role of Staufen1 during myogenic differentiation by characterizing the effects of Staufen1 over-expression in C2C12 cells. Immunofluorescence experiments revealed that Staufen1 over-expression causes a decrease in the fusion and differentiation indices and leads to the formation of myotubes with significantly fewer nuclei. We show, by western blot and qRT-PCR, that the protein expression of MyoD, myogenin and MyHC and the mRNA expression of MyoD, myogenin, Mef2A, Mef2C and p35 are significantly decreased during differentiation when Staufen1 is over-expressed. We then found that c-myc protein expression was increased during proliferation but that its mRNA expression remained unchanged. In this study we propose that Staufen1 negatively regulates skeletal muscle differentiation through the posttranscriptional regulation of c-myc, Mef2A, Mef2C and p35 transcripts.

A-type lamin mutations are associated with degenerative disorders causing dilated cardiomyopathy, Charcot-Marie-Tooth neuropathy and Limb-Girdle Muscular Dystrophy. Our lab has identified MLIP; a novel protein that interacts with lamin A/C. Knocked down MLIP expression in C2C12 myoblasts down regulates myogenic regulatory factors, MyoD and Myogenin, which delays myogenic differentiation. We hypothesize that MLIP is essential for myogenic differentiation. Our goal is to define the MLIP associated pathways involved in myogenic programming. Gene expression profiling of MLIP stably knocked down C2C12 cells, identified 30 genes implicated in human disease. Mutations in five of those genes (DMPK, HSPB8, LMNB2, NEFL and SGCD) cause muscular dystrophy, neuropathies, and lipodystrophies that have phenotypic overlap with laminopathies. Further studies involving the MLIP knocked down cell lines demonstrated that in the absence of puromycin, MLIP protein expression returns to normal. This in turn affects the interpretation of the gene expression data and attempted MLIP recovery experiments.

Graduation date: 1991

Staufen1 is a double-stranded RNA-binding protein known to be involved in the transport, localization, decay and increased translation of some mRNAs. The goal of the present study is to determine the role of Staufen1 during myogenic differentiation by characterizing the effects of Staufen1 over-expression in C2C12 cells. Immunofluorescence experiments revealed that Staufen1 over-expression causes a decrease in the fusion and differentiation indices and leads to the formation of myotubes with significantly fewer nuclei. We show, by western blot and qRT-PCR, that the protein expression of MyoD, myogenin and MyHC and the mRNA expression of MyoD, myogenin, Mef2A, Mef2C and p35 are significantly decreased during differentiation when Staufen1 is over-expressed. We then found that c-myc protein expression was increased during proliferation but that its mRNA expression remained unchanged. In this study we propose that Staufen1 negatively regulates skeletal muscle differentiation through the posttranscriptional regulation of c-myc, Mef2A, Mef2C and p35 transcripts.

A-type lamin mutations are associated with degenerative disorders causing dilated cardiomyopathy, Charcot-Marie-Tooth neuropathy and Limb-Girdle Muscular Dystrophy. Our lab has identified MLIP; a novel protein that interacts with lamin A/C. Knocked down MLIP expression in C2C12 myoblasts down regulates myogenic regulatory factors, MyoD and Myogenin, which delays myogenic differentiation. We hypothesize that MLIP is essential for myogenic differentiation. Our goal is to define the MLIP associated pathways involved in myogenic programming. Gene expression profiling of MLIP stably knocked down C2C12 cells, identified 30 genes implicated in human disease. Mutations in five of those genes (DMPK, HSPB8, LMNB2, NEFL and SGCD) cause muscular dystrophy, neuropathies, and lipodystrophies that have phenotypic overlap with laminopathies. Further studies involving the MLIP knocked down cell lines demonstrated that in the absence of puromycin, MLIP protein expression returns to normal. This in turn affects the interpretation of the gene expression data and attempted MLIP recovery experiments.

No description available.

Effects of homologous extracts prepared from mature avian skeletal muscle on the development of isolated myoblasts from the thigh muscle of 11-12 day old chick embryoswere studied in vitro under previously unpublished culture conditions. Results of these experiments indicate that:1) fusion of myoblasts can occur in a predictable manner under the culture conditions described in this report2) some factor(s) present in extracts of homologous adult organ is able to partially inhibit this fusion3) such inhibition occurs maximally between 12 and 24 hours after myoblasts are placed in an in vitro environment, and is only partially reversible by the re-establishment of optimal culture conditions4) the inhibitory factor(s) is apparently long lived, non-dialyzablQ, heat labile, and subject to inactivation by proteolytic enzymes.

Staufen1 is a double-stranded RNA-binding protein known to be involved in the transport, localization, decay and increased translation of some mRNAs. The goal of the present study is to determine the role of Staufen1 during myogenic differentiation by characterizing the effects of Staufen1 over-expression in C2C12 cells. Immunofluorescence experiments revealed that Staufen1 over-expression causes a decrease in the fusion and differentiation indices and leads to the formation of myotubes with significantly fewer nuclei. We show, by western blot and qRT-PCR, that the protein expression of MyoD, myogenin and MyHC and the mRNA expression of MyoD, myogenin, Mef2A, Mef2C and p35 are significantly decreased during differentiation when Staufen1 is over-expressed. We then found that c-myc protein expression was increased during proliferation but that its mRNA expression remained unchanged. In this study we propose that Staufen1 negatively regulates skeletal muscle differentiation through the posttranscriptional regulation of c-myc, Mef2A, Mef2C and p35 transcripts.