The cellularisation of mammalian myotubes by myoseverin
Urodele amphibians display a remarkable ability to regenerate and are the only adult vertebrates able to regenerate their limbs. In mammals the capacity for regeneration is greatly curtailed and an important aspect of this is thought to be the plasticity of the differentiated state. On amputation of a newt limb, mature differentiated cell types at the site of injury are able to dedifferentiate to produce a mass of proliferating cells which will eventually re-differentiate to form the tissues of the regenerate. These events are absent in mammals where an inflammatory response predominates. Skeletal muscle is widely used as a model system for the study of dedifferentiation events in limb regeneration. Skeletal muscle myoblasts withdraw from the cell cycle and fuse to form multinucleate myotubes during differentiation, providing a striking differentiated phenotype. These multinucleate cells undergo conversion to mononucleate cells, referred to as cellularisation, during limb regeneration in urodeles. In recent years, a number of experimental tools to reverse differentiation of mammalian myotubes in vitro have been reported, one of which is myoseverin, a small microtubule-binding molecule isolated from a combinatorial chemical library by its ability to induce cellularisation of multinucleate myotubes. The work in this thesis has further investigated the mechanism of action of myoseverin, assessing to what extent the molecule can be said to initiate a programme in mammalian cells characteristic of that involved in urodele regeneration. Time-lapse microscopy has made possible observation of the process of cellularisation at a single cell level, and of the resulting mononucleate progeny, to determine the fate of these cells following cellularisation, while microarray analysis enabled evaluation of'events at a molecular level. These experiments revealed a significant role for microtubules in the mechanism of cellularisation and have highlighted cellularisation as a necessary, but not sufficient, aspect of the complex process of dedifferentiation.