Discover the Boosters of Myoblast Fusion

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Project Summary/Abstract Myoblast fusion unites the strength from otherwise mononucleate cellular compartments, thus representing a milestone of muscle evolution. Despite the clear significance, the genetic mechanism that drives the emergence of myoblast fusion has remained largely unknown. The central goal of this proposal is to understand the biochemical and genetic mechanisms of myoblast fusion. We recently uncovered the key roles and regulations of two muscle-specific membrane-coalescing agents Myomaker and Myomixer in human myoblasts. Our complementation tests suggested that although this duo is sufficient to induce cell fusion, it only occurs at low efficiency. As such, at least a third factor is required to constitute the highly efficient fusion system that is normally observed during human development. Our preliminary studies have delineated the expected roles of these mysterious factors that can be summarized as 1) like Myomixer, the unknown factor(s) should be able to induce cell fusion together with Myomaker; 2) When expressed together with Myomixer and Myomaker, the unknown factor(s) should induce more efficient fusion than by any two-factor combinations; 3) like Myomixer and Myomaker, the expression of the unknown factor(s) should also be controlled by MyoD the master regulator of myogenesis. These features provided a clear roadmap to streamline our experiment design that will ultimately identify such factors that we named Boosters. We propose to first unbiasedly identify the interactors of Myomixer and Myomaker given their close functional relationships with the Boosters. In parallel, we will also perform a genome-wide CRISPR knockout screen in unique models of human myoblasts to systematically identify the Booster genes. The fusogenic activity of the Booster candidates will be examined through complementary gain/loss-of-function tests in various cell types. Therefore, our study will integrate the strengths from a broad range of approaches including gene discovery, comparative proteomics, and gene function study. Our plan to systematically identify the unknown myoblast fusion factors exactly from human myoblasts also represents an innovation considering that previous studies have primarily focused on the mouse and invertebrate models. Ultimately, knowledge gained from this study will provide new insights into the mechanisms of intercellular fusion in general and may provide new strategies for improving human muscle development and growth.