Structure–function analysis of myomaker domains required for myoblast fusion

Abstract

During skeletal muscle development, myoblasts fuse to form multinucleated myofibers. Myomaker [Transmembrane protein 8c (TMEM8c)] is a muscle-specific protein that is essential for myoblast fusion and sufficient to promote fusion of fibroblasts with muscle cells; however, the structure and biochemical properties of this membrane protein have not been explored. Here, we used CRISPR/Cas9 mutagenesis to disrupt myomaker expression in the C2C12 muscle cell line, which resulted in complete blockade to fusion. To define the functional domains of myomaker required to direct fusion, we established a heterologous cell-cell fusion system, in which fibroblasts expressing mutant versions of myomaker were mixed with WT myoblasts. Our data indicate that the majority of myomaker is embedded in the plasma membrane with seven membrane-spanning regions and a required intracellular C-terminal tail. We show that myomaker function is conserved in other mammalian orthologs; however, related family members (TMEM8a and TMEM8b) do not exhibit fusogenic activity. These findings represent an important step toward deciphering the cellular components and mechanisms that control myoblast fusion and muscle formation.P lasma membrane fusion is a fundamental cellular process required for the conception, development, and physiology of multicellular organisms. Membrane fusion occurs during viral infection of a host cell, between intracellular membranes, and between two plasma membranes to form syncytial tissues (1). Cell-cell fusion is critical for a wide array of cellular processes, including sperm-egg fertilization, macrophage function, bone and placental development, and skeletal muscle formation. This form of fusion must be precisely controlled to prevent inappropriate cellular mixing.The fusion of myoblasts requires cell recognition, migration, adhesion, signaling, and finally, membrane coalescence (2). Much of our knowledge about myoblast fusion has originated from studies performed in Drosophila. In this system, intracellular signaling results in cytoskeletal alterations and actin polymerization, which drive the formation of cellular projections that invade neighboring cells to cause fusion (3). Recent evidence also indicates a critical function for branched actin polymerization during Drosophila indirect flight muscle fusion (4). The essential role of the cytoskeleton in fusion is conserved in mammals, in which the actin regulators Rac1, cdc42, and N-WASp are required for muscle development (5-8). In addition to actin dynamics, numerous proteins that regulate diverse cellular processes have been associated with myoblast fusion (9-13).We recently discovered a muscle-specific membrane protein named myomaker [annotated as Transmembrane protein 8c (TMEM8c)] that is absolutely required for skeletal myocyte fusion in the mouse (14,15). To begin to decipher the mechanisms whereby myomaker controls cell-cell fusion, we designed a heterologous cell-cell fusion assay to monitor the ability of a series of myomaker mutants to direct the fusion of lab...

Keep Your Friends Close: Cell–Cell Contact and Skeletal Myogenesis

“…It is expressed exclusively in the skeletal muscle lineage and at the times during muscle development and regeneration when fusion is occurring. Significantly, ectopic expression of myomaker in fibroblasts allows them to fuse with myoblasts, an activity not seen previously with any other protein (Millay et al 2013(Millay et al , 2016Landemaine et al 2014). Whether myomaker is the long sought direct fusogen, or somehow regulates the process in another fundamental way, is not yet clear.…”

“…The identity of a true fusogen in myoblast fusion is, therefore, of very high interest. One possibility here is the recently discovered myomaker, a cell-surface protein with seven membrane-spanning regions (Millay et al 2013(Millay et al , 2016. Myomaker (also called TMEM8c) is essential for myoblast fusion in both mice and zebrafish (Millay et al 2013(Millay et al , 2014Landemaine et al 2014).…”

Muscle cell communication in development and repair

“…Myomaker (also known as TMEM8C) is enriched in developing muscle; the myomaker protein contains seven transmembrane domains and is localized to the plasma membrane [36]. Consistent with the reactivation of developmental gene programs in response to muscle injury, myomaker is re-expressed after muscle insult while it is minimally expressed in mature uninjured muscle.…”

Control of muscle formation by the fusogenic micropeptide myomixer

“…The muscle phenotype of Myomixer mutant mice is reminiscent of that seen in mice lacking Myomaker, a fusogenic transmembrane muscle protein (18, 19), suggesting a functional relationship between these two regulators of myoblast fusion. To assess the functional relationship between Myomixer and Myomaker in myoblast fusion, we retrovirally expressed each protein in C2C12 myoblasts and monitored myotube formation.…”

“…Myomaker can induce fusion of 10T1/2 mouse fibroblasts with myoblasts (18, 19). In contrast, Myomixer expressing 10T1/2 cells did not fuse with C2C12 cells (Fig.…”