Mechanisms of myoblast fusion
Cell-cell fusion is critical for a wide array of cellular processes, including sperm-egg fertilization, macrophage function, bone and placental development, and muscle formation. The detailed mechanisms underlying plasma membrane fusion is an area of mammalian cell biology that is nearly entirely undefined despite its importance in development and physiology. The main reason for this deficiency is because the proteins that function directly in the fusion of plasma membranes have not been identified before the discovery of Myomaker during Millay’s post-doctoral training.
In the Millay lab, we identified a second myogenic fusion protein (Myomerger) that together with Myomaker can induce fusion of normally non-fusing cells. This is the first time cell fusion has been reconstituted with mammalian proteins. We then discovered that the Myomaker/Myomerger system drives fusion through a novel mechanism. They do not work like traditional membrane fusion proteins (such as SNAREs), but instead they independently govern distinct points of the fusion pathway. Our next steps are to: (i) identify the biochemical activity of Myomaker (ii) determine mechanisms by which the helical regions of Myomerger drive pore formation (iii) identify novel ancillary factors that cooperate with Myomaker/Myomerger during myoblast fusion.
Physiological role of muscle stem cell fusion
Muscle stem cell fusion has a well-recognized role during regeneration, however there is currently no consensus in the field as to whether fusion is required for adult muscle growth, homeostasis, and during aging. Using our unique Myomaker targeted alleles we have shown that muscle stem cells are indeed required for overload-induced muscle growth in the adult. We then developed a novel exercise protocol and showed that muscle undergoes various adaptations to exercise, each having a different requirement for muscle stem cell fusion. Collectively, these data lead to the novel paradigm that newly added myonuclei are distinct from existing myonuclei. We are now optimizing novel tools to genetically mark newly added myonuclei and, through unique sequencing approaches, determine what they are contributing to the myofiber. Using single nuclear RNA-sequencing we are discovering a maturation pathway for myonuclei, which has the potential to be harnessed to augment aging-induced muscle loss.
Design of novel gene delivery vehicles for muscle
Delivery of any therapeutic modality to skeletal muscle represents a unique problem due to its distribution throughout the body in inaccessible locations. The bottleneck for treatment of genetic muscle diseases, such as the muscular dystrophies, is an ability to re-introduce a normal copy of the mutated gene through gene therapy approaches. Our goal is to improve the efficiency of correcting genetic muscle diseases by generating new technologies that redirect and enhance tropism of delivery vehicles to muscle.
The central idea is to harness our discoveries of the muscle cell fusion machinery (Myomaker/Myomerger) to empower viruses and non-viral particles to be more trophic for skeletal muscle. We are engineering the muscle fusogens (Myomaker and Myomerger) on the surface of various delivery vehicles including cells, enveloped viruses, and non-viral particles (exosomes) with the goal of enhancing the transduction of muscle. We have already shown that Myomaker expression in mesenchymal stromal cells induces their fusion with muscle, providing proof-of-concept data that this delivery system is functional. We will continue to optimize this system while engineering new classes of delivery vehicles that contain the muscle fusogenic proteins.