Tracking Nuclear Envelope Protein Movement within Mosaic Myotubes

Muscular dystrophies are an inherited group of disorders, where aberrant proteins lead to necrosis, and subsequent loss of muscle mass and strength. A primary goal of therapies for muscular dystrophy are to improve muscle function; one mechanism through which this can be accomplished this is to transfer functional proteins to affected muscle. To this effect, cell grafting therapies have been researched in their efficacy in being able to contribute to repair and regeneration of dystrophic muscle.;Mutations in the nuclear envelope proteins, lamin A/C and emerin, are implicated in Emery-Dreifuss muscular dystrophy as well as other disorders classified as nuclear envelopathies. Lamin A/C are type V intermediate filament proteins that comprise the nuclear lamina, a scaffold structure on the nuclear side of the inner nuclear membrane that provides structural support as well as interacting with chromatin and transcriptional regulators. Emerin is an integral nuclear membrane protein and is a key binding partner of lamin. It also provides an anchoring point for structural proteins as well as interacting with gene expression and chromatin structure regulators. The ability of healthy human or mouse muscle to contribute to regeneration of dystrophic muscle in hybrid mouse-human muscle fibers has observed to be successful. In both human muscle precursor cell implantation, as well as xenografting of human muscle tissue into mice host, healthy myonuclei have been incorporated into the dystrophic host, but in both cases it was observed that human lamin A is not transported into neighboring mouse nuclei.;We set out to investigate whether this restriction of lamin A diffusion is a matter of the inability of the protein to be transported between species, or if it is a characteristic of the protein itself. We also chose to examine in parallel the movement of emerin, a key binding partner of lamin A in the nuclear envelope, which shares in common the characteristic of being mutated in Emery-Dreifuss Muscular Dystrophy. We first verified, in vitro, that human lamin A will not transfer to mouse nuclei in a hybrid muscle fiber. We then moved to a mouse only system using lamin A/C-knockout and emerin-knockout immortalized and primary myoblasts. In both of our mouse systems, we see that hybrid myotubes formed from these myoblasts show that lamin A is not transported between adjacent nuclei, while emerin is able to be shuttled between neighboring nuclei. We also observed low efficiency in hybrid myotube formation, which suggests that cell grafting therapies for nuclear envelopathies may not be an efficient treatment.;Future research should investigate the mechanisms that are responsible for lamin A/C synthesis and transport. Having verified this phenomenon, we gain a useful tool for tracking nuclear origin in cell grafting therapies.