Myoblast Fusion Confusion: the Resolution Begins Srihari C

Sampath et al. Skeletal Muscle (2018) 8:3 DOI 10.1186/s13395-017-0149-3 REVIEW Open Access Myoblast fusion confusion: the resolution begins Srihari C. Sampath1,2*, Srinath C. Sampath1,2* and Douglas P. Millay3,4* Abstract The fusion of muscle precursor cells is a required event for proper skeletal muscle development and regeneration. Numerous proteins have been implicated to function in myoblast fusion; however, the majority are expressed in diverse tissues and regulate numerous cellular processes. How myoblast fusion is triggered and coordinated in a muscle-specific manner has remained a mystery for decades. Through the discovery of two muscle-specific fusion proteins, Myomaker and Myomerger–Minion, we are now primed to make significant advances in our knowledge of myoblast fusion. This article reviews the latest findings regarding the biology of Myomaker and Minion–Myomerger, places these findings in the context of known pathways in mammalian myoblast fusion, and highlights areas that require further investigation. As our understanding of myoblast fusion matures so does our potential ability to manipulate cell fusion for therapeutic purposes. Keywords: Myoblast fusion, Myomaker, Myomerger, Minion, Muscle development Background and therefore, methods to dominantly reconstitute fusion Muscle formation occurs during several stages of an organ- and dissect its mechanisms were nonexistent. ism’s life, including embryonic development, and growth Plasma membrane fusion is a complex process that re- and regeneration in the adult. Proper myogenesis begins quires recognition, adhesion, cell signaling, cytoskeletal al- through specification of precursor cells to the myoblast terations, and membrane rearrangements. Current lineage, followed by differentiation, both of which are evidence, reviewed elsewhere, implicates numerous pro- accomplished by the muscle-specific transcription factors teins and regulatory pathways in the recognition, adhesion, MyoD and Myogenin [1, 2]. A critical event in myogenesis and the cell signaling phases of myoblast fusion [3]. How- is the fusion of myoblasts either with one another to gener- ever, none of these proteins have been shown to serve as a ate new multi-nucleated myofibers or with an existing nodal regulator of myoblast membrane fusion. Such a regu- myofiber, thereby increasing the pool of myonuclei and lator would be expected to be expressed specifically in the allowing muscle growth. There has been a relative lack of muscle, be genetically required for muscle formation, and understanding about the mechanism and regulation of function to dominantly induce fusion. In addition, fusogens myoblast fusion, when compared to other events that pre- from other systems are characteristically able to render nor- cede it, such as lineage specification and differentiation. mally non-fusing cells fusogenic. This is the case, for Furthermore, it has historically been difficult to uncouple example, of the H and F proteins of paramyxoviruses such defects in differentiation from those affecting fusion. This as measles virus [4] and the cellular fusogen epithelial fu- is principally because until recently, proteins directly acti- sion failure (Eff-1) in Caenorhabditis elegans [5]. vating fusion but not differentiation remained unidentified, Until very recently, skeletal muscle fusogens fulfilling these criteria had not been identified in any species. It was in fact a major conundrum whether muscle-specific * Correspondence: [email protected]; [email protected]; proteins that directly governed myoblast fusion actually [email protected] existed. The discovery of the multi-pass transmembrane 1 Genomics Institute of the Novartis Research Foundation, 10675 John Jay protein Myomaker as the first muscle-specific fusion fac- Hopkins Drive, San Diego, CA 92121, USA 3Department of Molecular Cardiovascular Biology, Cincinnati Children’s tor was the first step toward resolving the confusion Hospital Medical Center, 240 Albert Sabin Way, Cincinnati, OH 45229, USA surrounding the question of vertebrate myoblast fusion Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sampath et al. Skeletal Muscle (2018) 8:3 Page 2 of 10 [6]. Notably, Myomaker could induce fusion of fibroblasts homologous proteins perform a similar function in other with muscle cells, but not between fibroblasts themselves, mammalian fusogenic cells and to what extent com- highlighting the likelihood that additional myogenic fusion pletely distinct pathways (e.g., syncytin in placental factors existed. Indeed, three independent groups recently trophoblast) activate fusion in the various cell types. identified a second muscle-specific fusion protein named Myomaker function is conserved to zebrafish where it Myomerger–Minion–Myomixer, which when co-expressed is expressed only in the fusion competent fast myocytes with Myomaker is sufficient to induce fusion in non- but not in the fusion-incompetent slow myocytes, and fusogenic fibroblasts [7–9]. There is now little doubt as to this expression is functional as myomaker deletion the existence of muscle-specific fusion proteins. However, results in a lack of fast myocyte fusion [12, 13]. Myo- confusion remains about the mechanisms by which maker function also appears conserved to humans, and Myomaker and Minion–Myomerger coordinate and mutation of the human Myomaker homolog results in accomplish membrane coalescence. The overall goals of defective myoblast fusion and congenital myopathy [14]. this review are to highlight the recent advances in the iden- Expression of the zebrafish Myomerger–Minion ortho- tification of the myoblast fusion machinery, discuss areas log in cell-based assays indicates similar function, and that are currently unresolved, and suggest areas for future indeed, a recent study has shown that it is required for research as well as translational application. myoblast fusion in vivo [9, 15]. In contrast to the conservation of Myomaker and Minion–Myomerger in zebra- Expression and conservation of Myomaker and fish, whether they are also present in Drosophila and Minion–Myomerger in myogenesis other invertebrates is currently unknown. It is unlikely Both Myomaker and Myomerger–Minion were identi- but formally possible that sequence-diverged homologs fied through bioinformatic in silico searches. It is sur- of Myomaker and Minion–Myomerger remain to be dis- prising that prior to their discovery, the numerous covered in invertebrates. The more likely scenario is that genetic screens performed in Drosophila, zebrafish, and invertebrates rely on functional homologs that lack C2C12 myoblasts did not yield muscle-specific proteins sequence similarity. In this case, functional screening of directly governing myoblast fusion. One possibility for Drosophila cDNAs in mammalian cells (for instance the elusiveness of these genes could be their inadequate using the reconstitution system described below) may annotation, particularly as Minion–Myomerger repre- allow identification of such proteins. Alternatively, the sents a small ORF (smORF) encoded microprotein basic biophysical mechanisms for fusion could be differ- which is below the 100 amino acid thresholds normally ent between the species and thus require different used to bioinformatically predict open reading frames factors to accomplish membrane coalescence. [10]. Another complicating factor is that both genes are dynamically expressed, with no detectable expression in Function of Myomaker and Myomerger–Minion in proliferating myoblast cultures but strong transcriptional fusion, and reconstitution in non-fusogenic cells induction upon differentiation. As expected based on In the mouse, genetic loss-of-function experiments for this pattern, numerous E-box elements have been found Myomaker and Minion–Myomerger have revealed that upstream of both Myomaker and Myomerger–Minion these proteins are necessary for muscle formation. In- indicating a role for the myogenic regulatory factors deed, loss of either gene results in death at birth due to (MRFs), MyoD and Myogenin, in the activation of these a lack of skeletal muscle, and defects are seen in muscles genes [11]. of both somitic and non-somitic origin. Of note, mono- In vitro, these genes are expressed in both myocytes nucleated myosin+ cells were detected in both strains of (differentiated mono-nucleated myoblasts) and myo- mice during development, indicating an ability to differ- tubes. This contrasts with what is found in vivo where entiate and thus demonstrating that loss of these genes Myomaker and Myomerger–Minion are expressed in the do not directly regulate differentiation. Cultured myo- developing myotome beginning at embryonic (E) day 10 blasts from each genetically modified mouse line also but no longer detectable after postnatal (P) day 21, when displayed the ability to differentiate, but not fuse, con- muscle development has ceased. In healthy adult myofi- firming

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