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.
| Identifer | oai:union.ndltd.org:ADTP/238133 |
| Date | January 2006 |
| Creators | Davies, Todd John |
| Publisher | The University of Waikato |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://www.waikato.ac.nz/library/research_commons/rc_about.shtml#copyright |
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