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Cell fusion is a process in which cells unite their membranes and cytoplasm. It is fundamental for sexual reproduction and embryonic development. Among the best-known cell fusion processes during animal development are fertilization, myoblast fusion, osteoclast generation, and vulva formation in Caenorhabditis elegans. Although it is involved in many other functions in unicellular and multicellular organisms, little is known about the mechanisms of cell fusion and the genes that code for the proteins participating in this process. Benjamin Podbilewicz has dedicated many years to understanding the processes and mechanisms of cell fusion. In this interview, he spoke to us about how he began his studies of this process, his contributions to this exciting field, his scientific ties with Ibero-America and his strategies for a well-balanced scientific/personal life.

Purpose Among the members of the DOCK family, DOCK1–5 function as guanine-nucleotide exchange factors for small GTPase Rac1, which regulates the actin cytoskeleton. It has been reported that in model organisms the Dock-Rac axis is required for myoblast fusion. We examined the role of DOCK1–5 in trophoblast fusion herein. Methods We used a quantitative polymerase chain reaction (qPCR) to examine the mRNA expressions of DOCK1–5 and differentiation-related genes, i.e., fusogenic genes, in human trophoblastic cell lines, BeWo and JEG-3. We treated BeWo cells with TBOPP and C21 to inhibit DOCK1 and DOCK5. Cell dynamics and cell fusion were assessed by live imaging and immunostaining. The signaling pathways induced by DOCK1/5 inhibition were examined by western blotting. Results DOCK1 and DOCK5 were expressed in BeWo cells. The inhibition of DOCK1 or DOCK5 did not prevent the cell fusion induced by forskolin (a common reagent for cell fusion); it induced cell fusion. DOCK1 inhibition induced cell death, as did forskolin. DOCK1 and DOCK5 inhibition for 24 and 48 h increased the expression of the genes ASCT2 and SYNCYTIN2, which code responsive proteins of trophoblast cell fusion, respectively. Conclusion DOCK1 and DOCK5 inhibition participates in BeWo cell fusion, probably via pathways independent from forskolin-mediated pathways.

The fusion of skeletal myoblasts is a critical step in myotube formation and muscle maturation. Previously, we demonstrated that nanogrooves improved myoblast fusion via end-to-end fusion, which in turn generated parallel myotubes. However, the effect of serum components on the end-to-end fusion mechanism is unclear. In the current study, the synergistic effect of nanogrooves and human platelet lysate (hPL) on the growth and fusion of skeletal myoblasts was studied. Four types of nanogrooves (400 and 800 nm width; 100 and 400 nm depth) were used. Cell spreading, growth, and differentiation were screened on these nanogrooves in media formulated with hPL or fetal bovine serum (FBS), along with the flat substrate as a control group. The results showed that the deeper nanogrooves induced better alignment of myoblasts. hPL-adapted skeletal myoblasts (i.e., myoblasts@hPL) showed a smaller cell size with a more elongated morphology than myoblasts@FBS. During cell growth, the expression of myogenic genes (Myf5, MyoD, and MyoG) in myoblasts@hPL was lower than in myoblasts@FBS. During cell differentiation, myoblasts@hPL also expressed a lower level of myogenic and myosin heavy chain (MHC) genes. MHC-positive myoblasts@hPL without myotubes were found on all surfaces. Myomaker, an essential myoblast fusion gene, was upregulated during growth but downregulated during differentiation in myoblasts@hPL. Fibronectin-coated surfaces facilitate cell spreading and growth but still cannot support myoblast@hPL fusion. The results implied that hPL either lacks promotor factors or contains inhibitors on mouse skeletal myoblast fusion. This study reveals the effect of biophysical and biochemical cues on myoblast fusion and their potential for muscle tissue engineering.

Myoblast fusion is essential for the formation of multinucleated muscle fibers and is promoted by transient changes in the plasma membrane lipid distribution. However, little is known about the lipid transporters regulating these dynamic changes. Here, we show that proliferating myoblasts exhibit an aminophospholipid flippase activity that is downregulated during differentiation. Deletion of the P4-ATPase flippase subunit CDC50A (also known as TMEM30A) results in loss of the aminophospholipid flippase activity and compromises actin remodeling, RAC1 GTPase membrane targeting and cell fusion. In contrast, deletion of the P4-ATPase ATP11A affects aminophospholipid uptake without having a strong impact on cell fusion. Our results demonstrate that myoblast fusion depends on CDC50A and may involve multiple CDC50A-dependent P4-ATPases that help to regulate actin remodeling. This article has an associated First Person interview with the first author of the paper.

Fusion of nascent myoblasts to pre-existing myofibres is critical for skeletal muscle growth and repair. The vast majority of molecules known to regulate myoblast fusion are necessary in this process. Here, we uncover, through high-throughput in vitro assays and in vivo studies in the chicken embryo, that TGFβ (SMAD2/3-dependent) signalling acts specifically and uniquely as a molecular brake on muscle fusion. While constitutive activation of the pathway arrests fusion, its inhibition leads to a striking over-fusion phenotype. This dynamic control of TGFβ signalling in the embryonic muscle relies on a receptor complementation mechanism, prompted by the merging of myoblasts with myofibres, each carrying one component of the heterodimer receptor complex. The competence of myofibres to fuse is likely restored through endocytic degradation of activated receptors. Altogether, this study shows that muscle fusion relies on TGFβ signalling to regulate its pace., Fusion of myoblasts is essential for muscle development and repair, but the molecular mechanism underlying this process remains unclear. Here, the authors show, using chicken embryos as a model, that TGFβ signalling inhibits fusion via a receptor complementation mechanism, and indicate the involvement of endocytic degradation of activated receptors in modulation of this process.

Skeletal muscle plays essential roles in motor function, energy, and glucose metabolism. Skeletal muscle formation occurs through a process called myogenesis, in which a crucial step is the fusion of mononucleated myoblasts to form multinucleated myofibers. The myoblast/myocyte fusion is triggered and coordinated in a muscle-specific way that is essential for muscle development and post-natal muscle regeneration. Many molecules and proteins have been found and demonstrated to have the capacity to regulate the fusion of myoblast/myocytes. Interestingly, two newly discovered muscle-specific membrane proteins, Myomaker and Myomixer (also called Myomerger and Minion), have been identified as fusogenic regulators in vertebrates. Both Myomaker and Myomixer-Myomerger-Minion have the capacity to directly control the myogenic fusion process. Here, we review and discuss the latest studies related to these two proteins, including the discovery, structure, expression pattern, functions, and regulation of Myomaker and Myomixer-Myomerger-Minion. We also emphasize and discuss the interaction between Myomaker and Myomixer-Myomerger-Minion, as well as their cooperative regulatory roles in cell-cell fusion. Moreover, we highlight the areas for exploration of Myomaker and Myomixer-Myomerger-Minion in future studies and consider their potential application to control cell fusion for cell-therapy purposes.

Casein kinase 2 (CK2) is a tetrameric protein kinase composed of 2 catalytic (α and α′) and 2 regulatory β subunits. Our study provides the first molecular and cellular characterization of the different CK2 subunits, highlighting their individual roles in skeletal muscle specification and differentiation. Analysis of C2C12 cell knockout for each CK2 subunit reveals that: 1) CK2β is mandatory for the expression of the muscle master regulator myogenic differentiation 1 in proliferating myoblasts, thus controlling both myogenic commitment and subsequent muscle-specific gene expression and myotube formation; 2) CK2α is involved in the activation of the muscle-specific gene program; and 3) CK2α′ activity regulates myoblast fusion by mediating plasma membrane translocation of fusogenic proteins essential for membrane coalescence, like myomixer. Accordingly, CK2α′ overexpression in C2C12 cells and in mouse regenerating muscle is sufficient to increase myofiber size and myonuclei content via enhanced satellite cell fusion. Consistent with these results, pharmacological inhibition of CK2 activity substantially blocks the expression of myogenic markers and muscle cell fusion both in vitro in C2C12 and primary myoblasts and in vivo in mouse regenerating muscle and zebrafish development. Overall, our work describes the specific and coordinated functions of CK2 subunits in orchestrating muscle differentiation and fusogenic activity, highlighting CK2 relevance in the physiopathology of skeletal muscle tissue.—Salizzato, V., Zanin, S., Borgo, C., Lidron, E., Salvi, M., Rizzuto, R., Pallafacchina, G., Donella-Deana, A. Protein kinase CK2 subunits exert specific and coordinated functions in skeletal muscle differentiation and fusogenic activity.

The aim of this study was to determine the effect of thermal stimulation at 39°C on the fusion and elongation of skeletal muscle cells. During a 5 day differentiation process of C2C12 cells, nine groups subjected to varying lengths of thermal stimulation at 39°C were established. Afterward, all groups were immunostained using anti-muscle heavy-chain antibody to test for myotube formation. Quantification of the myotube area demonstrated a significant increase in the group subjected to thermal stimulation at 39°C during the latter half of the differentiation compared with the control group, but the fusion index was significantly higher in the group that received hyperthermic treatment during the first half of the differentiation period. Moreover, the longitudinal length of myotubes was significantly increased in the groups that were subjected to thermal stimulation at 39°C during the latter half of the differentiation period. The distance between the center of myotubes and the nucleus farthest away from the center was substantially extended in the group receiving thermal stimulation at 39°C only on the fourth day of the differentiation. Together, these results demonstrate that thermal stimulation at 39°C facilitates myoblast fusion and elongation.

Skeletal muscle regeneration is triggered by local inflammation and is accompanied by phagocytosis of dead cells at the injury site. Efferocytosis regulates the inflammatory program in macrophages by initiating the conversion of their inflammatory phenotype into the healing one. While pro-inflammatory cytokines induce satellite cell proliferation and differentiation into myoblasts, growth factors, such as GDF3, released by healing macrophages drive myoblast fusion and myotube growth. Therefore, improper efferocytosis may lead to impaired muscle regeneration. Transglutaminase 2 (TG2) is a versatile enzyme participating in efferocytosis. Here, we show that TG2 ablation did not alter the skeletal muscle weights or sizes but led to the generation of small size myofibers and to decreased grip force in TG2 null mice. Following cardiotoxin-induced injury, the size of regenerating fibers was smaller, and the myoblast fusion was delayed in the tibialis anterior muscle of TG2 null mice. Loss of TG2 did not affect the efferocytic capacity of muscle macrophages but delayed their conversion to Ly6C−CD206+, GDF3 expressing cells. Finally, TG2 promoted myoblast fusion in differentiating C2C12 myoblasts. These results indicate that TG2 expressed by both macrophages and myoblasts contributes to proper myoblast fusion, and its ablation leads to impaired muscle development and regeneration in mice.

Skeletal muscle fibers are multinucleated cellular giants formed by the fusion of mononuclear myoblasts. Several molecules involved in myoblast fusion have been discovered, and finger-like projections coincident with myoblast fusion have also been implicated in the fusion process. The role of these cellular projections in muscle cell fusion was investigated herein. We demonstrate that these projections are filopodia generated by class X myosin (Myo10), an unconventional myosin motor protein specialized for filopodia. We further show that Myo10 is highly expressed by differentiating myoblasts, and Myo10 ablation inhibits both filopodia formation and myoblast fusion in vitro. In vivo, Myo10 labels regenerating muscle fibers associated with Duchenne muscular dystrophy and acute muscle injury. In mice, conditional loss of Myo10 from muscle-resident stem cells, known as satellite cells, severely impairs postnatal muscle regeneration. Furthermore, the muscle fusion proteins Myomaker and Myomixer are detected in myoblast filopodia. These data demonstrate that Myo10-driven filopodia facilitate multinucleated mammalian muscle formation.

Homozygosity for the R51Q mutation in sorting nexin 10 (SNX10) inactivates osteoclasts (OCLs) and induces autosomal recessive osteopetrosis in humans and in mice. We show here that the fusion of wild-type murine monocytes to form OCLs is highly regulated, and that its extent is limited by blocking fusion between mature OCLs. In contrast, monocytes from homozygous R51Q SNX10 mice fuse uncontrollably, forming giant dysfunctional OCLs that can become 10- to 100-fold larger than their wild-type counterparts. Furthermore, mutant OCLs display reduced endocytotic activity, suggesting that their deregulated fusion is due to alterations in membrane homeostasis caused by loss of SNX10 function. This is supported by the finding that the R51Q SNX10 protein is unstable and exhibits altered lipid-binding properties, and is consistent with a key role for SNX10 in vesicular trafficking. We propose that OCL size and functionality are regulated by a cell-autonomous SNX10-dependent mechanism that downregulates fusion between mature OCLs. The R51Q mutation abolishes this regulatory activity, leading to excessive fusion, loss of bone resorption capacity and, consequently, to an osteopetrotic phenotype in vivo. This article has an associated First Person interview with the joint first authors of the paper., Summary: Fusion of monocytes to become bone-resorbing osteoclasts is limited by an SNX10-dependent cell-autonomous mechanism. Loss of SNX10 function deregulates fusion and generates giant inactive osteoclasts.

Muscle cell fusion is a multistep process involving cell migration, adhesion, membrane remodeling and actin-nucleation pathways to generate multinucleated myotubes. However, molecular brakes restraining cell–cell fusion events have remained elusive. Here we show that transforming growth factor beta (TGFβ) pathway is active in adult muscle cells throughout fusion. We find TGFβ signaling reduces cell fusion, regardless of the cells’ ability to move and establish cell-cell contacts. In contrast, inhibition of TGFβ signaling enhances cell fusion and promotes branching between myotubes in mouse and human. Exogenous addition of TGFβ protein in vivo during muscle regeneration results in a loss of muscle function while inhibition of TGFβR2 induces the formation of giant myofibers. Transcriptome analyses and functional assays reveal that TGFβ controls the expression of actin-related genes to reduce cell spreading. TGFβ signaling is therefore requisite to limit mammalian myoblast fusion, determining myonuclei numbers and myofiber size., The fusion of muscle progenitor cells to form syncytial myofibers is required for skeletal muscle development and regeneration. Here, the authors describe a novel and specific molecular regulation of muscle cell fusion driven by transforming growth factor beta (TGFβ) signaling.

Myomerger is a muscle-specific membrane protein involved in formation of multinucleated muscle cells by mediating the transition from the early hemifusion stage to complete fusion. Here, we considered the physical mechanism of the Myomerger action based on the hypothesis that Myomerger shifts the spontaneous curvature of the outer membrane leaflets to more positive values. We predicted, theoretically, that Myomerger generates the outer leaflet elastic stresses, which propagate into the hemifusion diaphragm and accelerate the fusion pore formation. We showed that Myomerger ectodomain indeed generates positive spontaneous curvature of lipid monolayers. We substantiated the mechanism by experiments on myoblast fusion and influenza hemagglutinin-mediated cell fusion. In both processes, the effects of Myomerger ectodomain were strikingly similar to those of lysophosphatidylcholine known to generate a positive spontaneous curvature of lipid monolayers. The control of post-hemifusion stages by shifting the spontaneous curvature of proximal membrane monolayers may be utilized in diverse fusion processes., Myomerger mediates the transition from the early hemifusion stage to complete fusion during the formation of multinucleated muscle cells. Here, authors use theoretical modeling and cell fusion experiments to show that Myomerger promotes a fusion pore by coupling the elastic stresses of the proximal and distal leaflets of myoblast membrane.

The actin cytoskeleton drives formation of membrane protrusions that promote cell–cell fusion. Chuang et al. now find that the invadosome scaffold protein Tks5 is required for myoblast fusion. Tks5 regulates the assembly of dynamin-2 around actin bundles, thus strengthening the stiffness of the invadosome to propel cell–cell fusion., Skeletal muscle development requires the cell–cell fusion of differentiated myoblasts to form muscle fibers. The actin cytoskeleton is known to be the main driving force for myoblast fusion; however, how actin is organized to direct intercellular fusion remains unclear. Here we show that an actin- and dynamin-2–enriched protrusive structure, the invadosome, is required for the fusion process of myogenesis. Upon differentiation, myoblasts acquire the ability to form invadosomes through isoform switching of a critical invadosome scaffold protein, Tks5. Tks5 directly interacts with and recruits dynamin-2 to the invadosome and regulates its assembly around actin filaments to strengthen the stiffness of dynamin-actin bundles and invadosomes. These findings provide a mechanistic framework for the acquisition of myogenic fusion machinery during myogenesis and reveal a novel structural function for Tks5 and dynamin-2 in organizing actin filaments in the invadosome to drive membrane fusion.

Myoblast fusion is tightly regulated during development and regeneration of muscle fibers. BAI3 is a receptor that orchestrates myoblast fusion via Elmo/Dock1 signaling, but the mechanisms regulating its activity remain elusive. Here we report that mice lacking BAI3 display small muscle fibers and inefficient muscle regeneration after cardiotoxin-induced injury. We describe two proteins that repress or activate BAI3 in muscle progenitors. We find that the secreted C1q-like1–4 proteins repress fusion by specifically interacting with BAI3. Using a proteomic approach, we identify Stabilin-2 as a protein that interacts with BAI3 and stimulates its fusion promoting activity. We demonstrate that Stabilin-2 activates the GPCR activity of BAI3. The resulting activated heterotrimeric G-proteins contribute to the initial recruitment of Elmo proteins to the membrane, which are then stabilized on BAI3 through a direct interaction. Collectively, our results demonstrate that the activity of BAI3 is spatiotemporally regulated by C1qL4 and Stabilin-2 during myoblast fusion., Myoblast fusion is an essential step in muscle growth and regeneration, and is regulated by the G-protein coupled receptor (GPCR) BAI3. Here Hamoud et al. show that the GPCR activity of BAI3 is spatiotemporally regulated during myoblast fusion, and identify C1qL4 and Stabilin-2 as, respectively, negative and positive regulators of its activity.

Adult skeletal muscles are comprised of multinuclear muscle cells called myofibers. During skeletal muscle development and regeneration, mononuclear progenitor cells (myoblasts) fuse to form multinuclear myotubes, which mature and become myofibers. The molecular events mediating myoblast fusion are not fully understood. Here we report that Shisa2, an endoplasmic reticulum (ER) localized protein, regulates the fusion of muscle satellite cell-derived primary myoblasts. Shisa2 expression is repressed by Notch signaling, elevated in activated compared to quiescent satellite cells, and further upregulated during myogenic differentiation. Knockdown of Shisa2 inhibits the fusion of myoblasts without affecting proliferation. Conversely, Shisa2 overexpression in proliferating myoblasts inhibits their proliferation but promotes premature fusion. Interestingly, Shisa2-overexpressing nascent myotubes actively recruit myoblasts to fuse with. At the molecular level, Rac1/Cdc42-mediated cytoskeletal F-actin remodeling is required for Shisa2 to promote myoblast fusion. These results provide a novel mechanism through which an ER protein regulates myogenesis.

Myoblast fusion is critical for muscle growth, regeneration, and repair. We previously reported that the enzyme peptidyl-prolyl cis-trans isomerase NIMA interacting 1 (Pin1) is involved in osteoclast fusion. The objective of this study was to investigate the possibility that Pin1 also inhibits myoblast fusion. Here, we show the increased number of nuclei in the Pin1+/- mice muscle fiber compared to that in wild-type mice. Moreover, we show that low dose of the Pin1 inhibitor dipentamethylene thiuram monosulfide treatment caused enhanced fusion in C2C12 cells. The R-Smads are well-known mediators of muscle hypertrophy and hyperplasia as well as being substrates of Pin1. We found that Pin1 is crucial for maintaining the stability of Smad3 (homologues of the Drosophila protein, mothers against decapentaplegic (Mad) and the Caenorhabditis elegans protein Sma). Our results show that serine 204 within Smad3 is the key Pin1-binding site during inhibition of myoblast fusion and that both the transforming growth factor-β receptor and extracellular signal-regulated kinase (ERK)-mediated phosphorylation are required for the interaction of Pin1 with Smad3. These findings suggest that a precise level of Pin1 activity is essential for regulating myoblast fusion during myogenesis and muscle regeneration.

Spectrin is a membrane skeletal protein best known for its structural role in maintaining cell shape and protecting cells from mechanical damage. Here, we report that α/βH-spectrin (βH is also called karst) dynamically accumulates and dissolves at the fusogenic synapse between fusing Drosophila muscle cells, where an attacking fusion partner invades its receiving partner with actin-propelled protrusions to promote cell fusion. Using genetics, cell biology, biophysics and mathematical modelling, we demonstrate that spectrin exhibits a mechanosensitive accumulation in response to shear deformation, which is highly elevated at the fusogenic synapse. The transiently accumulated spectrin network functions as a cellular fence to restrict the diffusion of cell-adhesion molecules and a cellular sieve to constrict the invasive protrusions, thereby increasing the mechanical tension of the fusogenic synapse to promote cell membrane fusion. Our study reveals a function of spectrin as a mechanoresponsive protein and has general implications for understanding spectrin function in dynamic cellular processes.

Regeneration of skeletal muscle in response to injury occurs through fusion of a population of stem cells, known as satellite cells, with injured myofibers. Myomixer, a muscle-specific membrane micropeptide, cooperates with the transmembrane protein Myomaker to regulate embryonic myoblast fusion and muscle formation. To investigate the role of Myomixer in muscle regeneration, we used CRISPR/Cas9-mediated genome editing to generate conditional knockout Myomixer alleles in mice. We show that genetic deletion of Myomixer in satellite cells using a tamoxifen-regulated Cre recombinase transgene under control of the Pax7 promoter abolishes satellite cell fusion and prevents muscle regeneration, resulting in severe muscle degeneration after injury. Satellite cells devoid of Myomixer maintain expression of Myomaker, demonstrating that Myomaker alone is insufficient to drive myoblast fusion. These findings, together with prior studies demonstrating the essentiality of Myomaker for muscle regeneration, highlight the obligatory partnership of Myomixer and Myomaker for myofiber formation throughout embryogenesis and adulthood.

The AT-rich interaction domain (ARID) family of proteins regulates gene expression, development, and differentiation. Although Arid5b has important functions in adipogenesis and chondrogenesis, the role of Arid5b in skeletal muscle myogenesis has not been investigated. Therefore, we isolated primary skeletal muscle cells from Arid5b+/+ and Arid5b-/- mice and characterized differentiation in these cells. We found that Arid5b-/- primary skeletal muscle cells showed differentiation defects and impaired sarcomeric assembly. Microarray analysis revealed down-regulation of the prostanoid biosynthesis pathway in Arid5b-/- myoblasts, including the genes encoding cyclooxygenase (COX)-1 ( Ptgs1) and prostaglandin (PG)I synthase ( Ptgis). Down-regulation of COX-1 and PGI synthase was confirmed by real-time PCR and Western blot analyses. Correspondingly, the production of PGI2, as measured by ELISA, was reduced in Arid5b-/- cells relative to Arid5b+/+ cells. Boyden chamber assays showed that migration was increased but chemotaxis was impaired in Arid5b-/- cells. Myoblast fusion was also inhibited in Arid5b-/- cells compared with Arid5b+/+ cells. Treatment with the PGI2 analog iloprost rescued the defects in myotube formation, migration, and fusion. These results demonstrate that Arid5b has a novel and essential role in skeletal muscle differentiation by regulating PGI2 production.-Murray, J., Whitson, R. H., Itakura, K. Reduced prostaglandin I2 signaling in Arid5b-/- primary skeletal muscle cells attenuates myogenesis.

Summary Myoblast fusion is essential for muscle development and regeneration. Yet, it remains poorly understood how mononucleated myoblasts fuse with preexisting fibers. We demonstrate that ERK1/2 inhibition (ERKi) induces robust differentiation and fusion of primary mouse myoblasts through a linear pathway involving RXR, ryanodine receptors, and calcium-dependent activation of CaMKII in nascent myotubes. CaMKII activation results in myotube growth via fusion with mononucleated myoblasts at a fusogenic synapse. Mechanistically, CaMKII interacts with and regulates MYMK and Rac1, and CaMKIIδ/γ knockout mice exhibit smaller regenerated myofibers following injury. In addition, the expression of a dominant negative CaMKII inhibits the formation of large multinucleated myotubes. Finally, we demonstrate the evolutionary conservation of the pathway in chicken myoblasts. We conclude that ERK1/2 represses a signaling cascade leading to CaMKII-mediated fusion of myoblasts to myotubes, providing an attractive target for the cultivated meat industry and regenerative medicine., Graphical abstract, Highlights • ERK inhibition induces robust mouse and chicken myoblast differentiation and fusion • Myotubes fuse with mononucleated myoblasts at an asymmetric fusogenic synapse • ERKi-driven signaling cascade leads to Ca2+-CaMKII-dependent myoblasts to myotube fusion • CaMKII is required for efficient muscle regeneration following injury, Eigler et al. show that an evolutionarily conserved signaling cascade initiated by ERK inhibition in myoblasts leads to CaMKII-dependent fusion of mononucleated myoblasts with early myotubes at a fusogenic synapse. Moreover, CaMKII is required for efficient muscle regeneration following injury.

The study of Drosophila muscle development dates back to the middle of the last century. Since that time, Drosophila has proved to be an ideal system for studying muscle development, differentiation, function, and disease. As in humans, Drosophila muscle forms via a series of conserved steps, starting with muscle specification, myoblast fusion, attachment to tendon cells, interactions with motorneurons, and sarcomere and myofibril formation. The genes and mechanisms required for these processes share striking similarities to those found in humans. The highly tractable genetic system and imaging approaches available in Drosophila allow for an efficient interrogation of muscle biology and for application of what we learn to other systems. In this article, we review our current understanding of muscle development in Drosophila, with a focus on myoblast fusion, the process responsible for the generation of syncytial muscle cells. We also compare and contrast those genes required for fusion in Drosophila and vertebrates.

Myoblast fusion is an indispensable step for skeletal muscle development, postnatal growth, and regeneration. Myeloid differentiation primary response gene 88 (MyD88) is an adaptor protein that mediates Toll-like receptors and interleukin-1 receptor signaling. Here we report a cell-autonomous role of MyD88 in the regulation of myoblast fusion. MyD88 protein levels are increased during in vitro myogenesis and in conditions that promote skeletal muscle growth in vivo. Deletion of MyD88 impairs fusion of myoblasts without affecting their survival, proliferation, or differentiation. MyD88 regulates non-canonical NF-κB and canonical Wnt signaling during myogenesis and promotes skeletal muscle growth and overload-induced myofiber hypertrophy in mice. Ablation of MyD88 reduces myofiber size during muscle regeneration, whereas its overexpression promotes fusion of exogenous myoblasts to injured myofibers. Our study shows that MyD88 modulates myoblast fusion and suggests that augmenting its levels may be a therapeutic approach to improve skeletal muscle formation in degenerative muscle disorders., Myoblast fusion is essential for skeletal muscle development and regeneration. Here the authors show that MyD88 is upregulated during myogenesis and during muscle growth, signals via the NF-κB and Wnt pathways, and that its expression modulates myoblast fusion and myofiber size in mice.

Skeletal muscle formation requires fusion of mononucleated myoblasts to form multinucleated myofibers. The muscle-specific membrane proteins myomaker and myomixer cooperate to drive mammalian myoblast fusion. Whereas myomaker is highly conserved across diverse vertebrate species, myomixer is a micropeptide that shows relatively weak cross-species conservation. To explore the functional conservation of myomixer, we investigated the expression and function of the zebrafish myomixer ortholog. Here we show that myomixer expression during zebrafish embryogenesis coincides with myoblast fusion, and genetic deletion of myomixer using CRISPR/Cas9 mutagenesis abolishes myoblast fusion in vivo. We also identify myomixer orthologs in other species of fish and reptiles, which can cooperate with myomaker and substitute for the fusogenic activity of mammalian myomixer. Sequence comparison of these diverse myomixer orthologs reveals key amino acid residues and a minimal fusogenic peptide motif that is necessary for promoting cell-cell fusion with myomaker. Our findings highlight the evolutionary conservation of the myomaker-myomixer partnership and provide insights into the molecular basis of myoblast fusion.

Micromanaging muscle cell fusion Adult skeletal muscles are characterized by long, multinucleated cells called myofibers. Myofibers form when muscle precursor cells, or myoblasts, differentiate and fuse together during embryogenesis. The fusion process is not fully understood. Studying cell culture and mouse models, Bi et al. identified an 84–amino acid peptide that promotes myoblast fusion. This small peptide, called Myomixer, physically interacts with and stimulates the activity of a fusogenic membrane protein called Myomaker. Notably, the Myomaker-Myomixer pair can also promote the fusion of nonmuscle cells, such as fibroblasts. Science , this issue p. 323

Myoblast fusion is required for myotube formation during myogenesis, and defects in myoblast differentiation and fusion have been implicated in a number of diseases, including human rhabdomyosarcoma. While the transcriptional regulation of the myogenic program has been studied extensively, the mechanisms controlling myoblast fusion remain largely unknown. This study identified and characterized the dynamics of a distinct class of blebs, termed bubbling blebs, which are smaller than those that participate in migration. The formation of these bubbling blebs occurred during differentiation and decreased alongside a decline in phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) at the plasma membrane before myoblast fusion. In a human rhabdomyosarcoma-derived (RD) cell line that exhibits strong blebbing dynamics and myoblast fusion defects, PIP3 was constitutively abundant on the membrane during myogenesis. Targeting phosphatase and tensin homolog (PTEN) to the plasma membrane reduced PIP3 levels, inhibited bubbling blebs, and rescued myoblast fusion defects in RD cells. These findings highlight the differential distribution and crucial role of PIP3 during myoblast fusion and reveal a novel mechanism underlying myogenesis defects in human rhabdomyosarcoma.Summary StatementThis study reveals a novel mechanism underlying myogenesis defects in human rhabdomyosarcoma cells involving PIP3, whose depletion with PTEN rescues myoblast fusion defects.

In response to injury, skeletal muscle stem cells (MuSCs) undergo myogenesis where they become activated, proliferate rapidly, differentiate and undergo fusion to form multinucleated myotubes. Dramatic changes in cell size, shape, metabolism and motility occur during myogenesis, which cause cellular stress and alter proteostasis. The molecular chaperone heat shock protein 70 (HSP70) maintains proteostasis by regulating protein biosynthesis and folding, facilitating transport of polypeptides across intracellular membranes and preventing stress-induced protein unfolding/aggregation. Although HSP70 overexpression can exert beneficial effects in skeletal muscle diseases and enhance skeletal muscle repair after injury, its effect on myogenesis has not been investigated. Plasmid-mediated overexpression of HSP70 did not affect the rate of C2C12 proliferation or differentiation, but the median number of myonuclei per myotube and median myotube width in differentiated C2C12 myotubes were increased with HSP70 overexpression. These findings reveal that increased HSP70 expression can promote myoblast fusion, identifying a mechanism for its therapeutic potential to enhance muscle repair after injury. This article has an associated First Person interview with the first author of the paper., Summary: In C2C12 myoblasts, HSP70 overexpression does not alter proliferation or differentiation but enhances fusion to increase myotube size.

Leucine-rich repeat containing family 8 (LRRC8) proteins form the volume-regulated anion channel (VRAC). Recently, they were shown to be required for normal differentiation and fusion of C2C12 myoblasts, by promoting membrane hyperpolarization and intracellular Ca2+ signals. However, the mechanism by which they are involved remained obscure. Here, using a FRET-based sensor for VRAC activity, we show temporary activation of VRAC within the first 2 h of myogenic differentiation. During this period, we also observed a significant decrease in the intracellular Cl− concentration that was abolished by the VRAC inhibitor carbenoxolone. However, lowering the intracellular Cl− concentration by extracellular Cl− depletion did not promote differentiation as judged by the percentage of myogenin-positive nuclei or total myogenin levels in C2C12 cells. Instead, it inhibited myosin expression and myotube formation. Together, these data suggest that VRAC is activated and mediates Cl− efflux early on during myogenic differentiation, and a moderate intracellular Cl− concentration is necessary for myoblast fusion.

Muscle-forming myotubes are formed from the cell-cell fusion of myoblasts. In this issue of Developmental Cell, Leikina et al. (2018) provide compelling evidence that myoblast fusion is dependent on a two-component membrane fusion complex of Myomaker and Myomerger, which function in hemifusion and pore formation activity, respectively.

Glycogen synthase kinase 3 (GSK3) slows myogenic differentiation and myoblast fusion partly by inhibiting the Wnt/&beta, catenin signaling pathway. Lithium, a common medication for bipolar disorder, inhibits GSK3 via Mg+ competition and increased Ser21 (GSK3&alpha, ) or Ser9 (GSK3&beta, ) phosphorylation, leading to enhanced myoblast fusion and myogenic differentiation. However, previous studies demonstrating the effect of lithium on GSK3 have used concentrations up to 10 mM, which greatly exceeds concentrations measured in the serum of patients being treated for bipolar disorder (0.5&ndash, 1.2 mM). Here, we determined whether a low-therapeutic (0.5 mM) dose of lithium could promote myoblast fusion and myogenic differentiation in C2C12 cells. C2C12 myotubes differentiated for three days in media containing 0.5 mM lithium chloride (LiCl) had significantly higher GSK3&beta, (ser9) and GSK3&alpha, (ser21) phosphorylation compared with control myotubes differentiated in the same media without LiCl (+2&ndash, 2.5 fold, p &lt, 0.05), a result associated with an increase in total &beta, catenin. To further demonstrate that 0.5 mM LiCl inhibited GSK3 activity, we also developed a novel GSK3-specific activity assay. Using this enzyme-linked spectrophotometric assay, we showed that 0.5 mM LiCl-treated myotubes had significantly reduced GSK3 activity (&minus, 86%, p &lt, 0.001). Correspondingly, 0.5 mM LiCl treated myotubes had a higher myoblast fusion index compared with control (p &lt, 0.001) and significantly higher levels of markers of myogenesis (myogenin, +3-fold, p &lt, 0.001) and myogenic differentiation (myosin heavy chain, +10-fold, p &lt, 0.001). These results indicate that a low-therapeutic dose of LiCl is sufficient to promote myoblast fusion and myogenic differentiation in muscle cells, which has implications for the treatment of several myopathic conditions.

Cell–cell fusion is a fundamental process underlying fertilization, development, regeneration and physiology of metazoans. It is a multi-step process involving cell recognition and adhesion, actin cytoskeletal rearrangements, fusogen engagement, lipid mixing and fusion pore formation, ultimately resulting in the integration of two fusion partners. Here, we focus on the asymmetric actin cytoskeletal rearrangements at the site of fusion, known as the fusogenic synapse, which was first discovered during myoblast fusion in Drosophila embryos and later also found in mammalian muscle and non-muscle cells. At the asymmetric fusogenic synapse, actin-propelled invasive membrane protrusions from an attacking fusion partner trigger actomyosin-based mechanosensory responses in the receiving cell. The interplay between the invasive and resisting forces generated by the two fusion partners puts the fusogenic synapse under high mechanical tension and brings the two cell membranes into close proximity, promoting the engagement of fusogens to initiate fusion pore formation. In this Cell Science at a Glance article and the accompanying poster, we highlight the molecular, cellular and biophysical events at the asymmetric fusogenic synapse using Drosophila myoblast fusion as a model.