A few studies have investigated the regulatory mechanismsofmyomakerexpression. Myoblast fusion into multinucleated myotubes is a crucial step in skeletal muscle development and regeneration. Myoblast fusion in drosophila journal article osti. Live imaging of drosophila myoblast fusion europe pmc. Separating myoblast differentiation from muscle cell fusion using igfi and the p38 map kinase inhibitor sb202190 samantha gardner,1 sean m. Pctaire1cdk16 promotes skeletal myogenesis by inducing myoblast migration and fusion kouhei shimizua, atsushi uematsua, yuuki imaib, tatsuya sawasakia,c. Unfortunately, the molecular machinery, cell behaviors, and membrane and cytoskeletal remodeling events that govern fusion and myofiber formation remain poorly understood. We used conditional mutagenesis in mice to demonstrate that rac1 and cdc42 are essential for myoblast fusion in vivo and in vitro. Myoblast fusion is an essential step in skeletal muscle development and regeneration. Extracellular annexins and dynamin are important for.
Myoblast fusion requires a number of cellular behaviors, including cell migration, recognition and adhesion as well as a series of subcellular behaviors, such as cytoskeletal rearrangements, vesicle trafficking and membrane dynamics leading to two cells becoming one. Myoblast fusion requires several cell adhesion and transmembrane proteins, which accumulate at contact sites between two myogenic cells either in. Normal myoblast fusion requires myoferlin development. M during cell differentiation of the murine myoblast c2c12 cell line. Myoblast fusion, a cellcell fusion event, is not as extensively studied as. A two populations of myoblasts are specified for fusion, fcs orange and yellow cells and fcms green cells. Myogenesis in vitro involves myoblast cell cycle arrest, migration, and fusion to form multinucleated myotubes. Cellfusion is the leading business application vendor of endtoend lean enterprise solutions. After differentiation and at the myotube stage, dex treatment has an atrophic effect. Using timelapse imaging approaches on mouse c2c12 myoblasts, we identify discrete and specific. Myomaker, encoded by mymk tmem8c, is a wellconserved plasma membrane protein. In the same way, blocking nglycan synthesis impairs myoblast fusion and the in vivo invalidation of mgat1, a gene involved in the synthesis of complex nglycans, generates mouse embryo death in utero. Rac1 has been implicated in myoblast fusion in drosophila and in mouse c2c12 myoblasts 17,18. The fusion of myoblasts into multinucleate syncytia plays a fundamental role in muscle function, as it supports the formation of extended sarcomeric arrays, or myofibrils, within a large volume of cytoplasm.
Jun 02, 2009 rac1 and cdc42 are small gproteins that regulate actin dynamics and affect plasma membrane protrusion and vesicle traffic. Feb 15, 2012 cell culture models of myoblast fusion in mice. Cellcell fusion is a crucial and highly regulated event in the genesis of both form and function of many tissues. Cells exposed to the porphyrin compound showed cell survival, proliferation, and fusion forming polynucleated syncytia, devoid of mhc expression. Klimek,1 and peter rotwein1,2 1department of biochemistry and molecular biology, oregon health and. Lifting the block allowed us to explore a largely synchronized fusion. Structurefunction analysis of myomaker domains required for. In these cells, activation of rac1 distinctively peaks at day 2 in df medium, at the onset of myoblast fusion. A myocyte also known as a muscle cell is the type of cell found in some types of muscle tissue. Herein, we report that our microtopographical carbohydrate substrates composed of bioactive hexanacetyldglucosamine glcnac6 modulated the efficiency of myoblast fusion without requiring horse serum or any differentiation. Myoblasts fuse with each other as well as with multinucleated myotubes to enlarge the myofiber. On the other hand, skeletal muscles are formed by morphological units referred to as muscle fibers. Nadph oxidase 4 nox4 regulates cellular processes such as proliferation, differentiation, and survival by producing reactive oxygen species ros.
Pdf inhibition of myoblast migration by prostacyclin is. Myocytes develop from myoblasts to form muscles in a process known as myogenesis. Nadph oxidase 4 contributes to myoblast fusion and skeletal. Optical projections were created using the projection function of the zeiss lsm software. Herein, we report that our microtopographical carbohydrate substrates composed of bioactive hexanacetyldglucosamine glcnac6 modulated the efficiency of myoblast fusion without requiring horse serum or any. Myoblast fusion occurs both during development and throughout adulthood, as skeletal muscle growth and regeneration require the accumulation of additional nuclei within myofibers. Fusion of myoblasts is required for proper skeletal muscle formation during embryonic development and adult muscle regeneration. Mar 16, 2018 myoblast fusion, which is essential for muscle development, regeneration, and repair, can be assessed in vitro via the calculation of a fusion index.
Membrane fusion, hemifusion, myoblast fusion, lipid mixing. In short, live cell imaging data generated as described here provide a quantitative link between nuclear dynamics and the ability to form myotubes and can be used as input for a highthroughput quantitative characterization of nuclear dynamics and myoblast behavior during differentiation and fusion. Of note, cellcell fusion is a basic phenomenon for all species, from drosophila to humans, where such events are involved in numerous processes including, in addition to placentation in mammals, macrophage fusion for osteoclast formation, and most importantly myoblast fusion for myogenesis during development and for muscle regeneration after. May 01, 2010 cell cell fusion is a crucial and highly regulated event in the genesis of both form and function of many tissues.
Cell cell fusion is a crucial and highly regulated event in the genesis of both form and function of many tissues. To assess the role of pip3 in bubbling bleb dynamics and myoblast fusion, chemically. A mononucleate cell in drosophila that is committed to a musclespecific program of differentiation but not yet fused. Myoblast fusion, which is essential for muscle development, regeneration, and repair, can be assessed in vitro via the calculation of a fusion index. Dexamethasone treatment at the myoblast stage enhanced c2c12. Mesenchymal stem cells and myoblast differentiation under hgf. Traditionally, this requires use of either immunocytochemistry or fluorescentlylabeled cytoskeletal staining, followed by microscopy and laborious analysis.
One particular type of cell fusion, myoblast fusion, is a key cellular process that shapes both the formation and repair of muscle. Analysis and quantification of in vitro myoblast fusion using. Myomaker, encoded by mymk tmem8c, is a wellconserved plasma membrane protein required for myoblast. Novel myoblast differentiationfusion model defined by quantitative proteomics by amanda maxwell 12. Wipwaspbased actinpolymerization machinery is essential. We therefore measured the levels of active rac1 in the igfn1kd1 and control cell lines at. Myoblast differentiationfusion model defined by quantitative. Mrf4 function to execute the differentiation program for committed myoblasts and are. Dec 23, 2015 novel myoblast differentiationfusion model defined by quantitative proteomics by amanda maxwell 12. We therefore measured the levels of active rac1 in the igfn1kd1 and control cell lines at day 2 of differentiation.
Conversely, ncam1 oglycosylation promotes myoblast fusion 23, 24. Cell culture studies demonstrate that multinucleated myotubes form. Myoblast fusion is a critical process that contributes to the growth of muscle during development and to the regeneration of myofibers upon injury. Rac1 and cdc42 are small gproteins that regulate actin dynamics and affect plasma membrane protrusion and vesicle traffic. Myoblast fusion requires several cell adhesion and transmembrane proteins, which accumulate at contact sites between two myogenic cells either in an asymmetrical or symmetrical fashion 3, 42. We discovered a musclespecific protein named myomakertmem8c that is absolutely required for the fusion of myoblasts and are now investigating the biochemical and biophysical properties of this membrane protein. Fusion and cell death in the development of skeletal muscle. Clicking the images or links will redirect you to a website hosted by benchsci that provides thirdparty scientific content. To determine whether imagej analysis software could be. Glycogenome expression dynamics during mouse c2c12. Met promotes the proliferation and differentiation of.
Myoblast migration, best described in studies in vitro, enables formation of groups of elongated fusion competent cells that then align to achieve cell cell contact and recognition thus promoting myotube formation. Cd36 is required for myoblast fusion during myogenic. Differentiation is regulated by myogenic regulatory factors, including myod, myf5, myogenin, and mrf4. The fusion of postmitotic mononucleated myoblasts to form syncytial myofibers is a critical step in the formation of skeletal muscle. Myoblast fusion requires a number of cellular behaviors, including cell migration, recognition, and adhesion, as well as a series of subcellular behaviors, such as cytoskeletal rearrangements. Graf1 promotes ferlindependent myoblast fusion sciencedirect. Exploiting live imaging to track nuclei during myoblast. Founded in 1999 in the silicon valley, california usa the organization has been focusing on advanced lean manufacturing and value stream application suites and technology. C2c12 cells represent a cell culture model of muscle differentiation yaffe and saxel, 1977.
Autoinhibition of myoblast fusion by cyclic receptor signalling. Myoblast fusion into functionallydistinct myotubes to form in vitro skeletal muscle constructs under differentiation serumfree conditions still remains a challenge. The small gproteins rac1 and cdc42 are essential for. Using genetic and pharmacological approaches, we show that pgi2 is a negative regulator of myoblast migration that also enhances cell fusion. Myoblasts exit the cell cycle and undergo fusion to. Nov 20, 2017 myoblast fusion requires several cell adhesion and transmembrane proteins, which accumulate at contact sites between two myogenic cells either in an asymmetrical or symmetrical fashion 3, 42. Separating myoblast differentiation from muscle cell fusion.
While our data strongly support a model whereby graf1 facilitates membrane merging by promoting the recruitment of ferlincontaining vesicles, numerous cell surface receptors have been implicated in promoting the initial stages of myoblast fusion including mcadherin and ncam that mediate cell recognition and integrins that mediate cell cell. The microprotein minion controls cell fusion and muscle. The deficit in fusion of rac1 or cdc42 mutant myoblasts correlates with a deficit in the recruitment of actin fibers and vinculin to. In the differentiation step, myoblasts begin to produce the proteins that make the cells competent to fuse. Much of the work pertaining to myoblast fusion in mammals derives from in vitro studies using either primary muscle cells isolated from mouse, rat or human muscles, or established mouse muscle cell lines, such as c2c12 fig. Neither the content nor the benchsci technology and processes for selection have been evaluated by us. Nfatc2 controls myoblast fusion after the initial formation of a myotube and is necessary for further cell growth. Despite differences in the developmental program that specifies.
Extracellular matrix ecm integrity during these processes is maintained by the opposing actions of matrix metalloproteinase mmp proteases and their inhibitors, the tissue inhibitor of metalloproteinases timps. Analysis and quantification of in vitro myoblast fusion using the. Here, we accumulated murine myoblasts at the readytofuse stage by blocking formation of early fusion intermediates with lysophosphatidylcholine. This is a timelapse movie of a mm14 myoblast culture induced to differentiate by depletion of serum and bfgf in the culture medium. Images were captured every 15 minutes using volocity 5. A membrane activator of myoblast fusion and muscle formation.
Myoblast myogenic differentiation but not fusion process is. The primary structure of dysferlin and myoferlin suggest a role in membrane fusion processes, as these proteins include six c2 domains and a transmembrane domain. Volumetric muscle loss caused by trauma or after tumour surgery exceeds the natural regeneration capacity of skeletal muscle. Thus, pgi2 may act as a brake on migrating cells to. Myoblast differentiation and fusion is key for skeletal muscle development and for muscle repair in aging or diseased states. Initial studies demonstrated that myoblast fusion requires extracellular calcium and changes in cell membrane topography and cytoskeletal organization. The deficit in fusion of rac1 or cdc42 mutant myoblasts correlates with a deficit in the recruitment of actin fibers and vinculin to myoblast. Formation of syncytial muscle fibers involves repeated rounds of cell fusion between growing myotubes and neighboring myoblasts. Few if any infected but unfused cells are observed. Step 1 the initial cell adhesion between a founder cell and an fcm is mediated by ig domaincontaining cams. C2c12 cells represent a cellculture model of muscle differentiation yaffe and saxel, 1977. Ijms free fulltext spatial geometries of selfassembled.
Fcs express specific combinations of transcription factors identity genes that control the morphogenesis of the resultant muscle. One particular type of cell fusion, myoblast fusion, is a key cellular process. Nadph oxidase 4 contributes to myoblast fusion and. There are two specialized forms of myocytes with distinct properties. Myoblast fusion can be divided into a series of steps of differentiation, cellcell recognition, adhesion, alignment, and membrane fusion knudsen and horwitz, 1978. Multinucleate cellular syncytial formation is a hallmark of skeletal muscle differentiation. Dose and timedependent effect of dex on c2c12 myoblast cell line was analyzed at myoblast and myotube stage, respectively. Activated satellite cells myoblasts migrate to the site of muscle trauma. Since proliferation was reduced, we focused more on the entry into myogenic differentiation, rather than fusion index, which is affected by cell number. While it was initially considered that myoblast fusion is tightly coordinated with the muscle differentiation program and requires the expression of.
The mousederived myoblast cell line c2c12 obtained from atcc, was maintained under 5% co2 at 37 c in dmem gibco, usa supplemented with 10% fbs gibco, usa and with 1% penicillin and streptomycin gibco, usa. Lipid mixing assay for murine myoblast fusion and other slow cell. Apr 26, 20 fusion between cells, as in the formation of placental trophoblasts, osteoclasts, and myoblasts, share many of the genetic and biochemical pathways used for fusion processes occurring inside cells. Insulinlike growth factor 1 induces muscle hypertrophy via nox4, but its function in myoblast fusion remains elusive. Mechanisms of myoblast fusion during muscle development. Phospholipase d1 facilitates secondphase myoblast fusion and. Reducing pip3 by targeting pten to the cell surface improves myoblast fusion pten dephosphorylates pip3 to pip2 and thus antagonizes the action of pi3k to inhibit the akt signaling pathway. Fusion events were only considered valid if optical slices were available above and below the plane of fusion, to rule out mistaking a cell migration event for myoblast fusion.
Myoblasts exit the cell cycle and undergo fusion to form multi. Genetic evidence that captured retroviral envelope syncytins. Regulation of myotube formation by the actinbinding factor. Fusion between cells, as in the formation of placental trophoblasts, osteoclasts, and myoblasts, share many of the genetic and biochemical pathways used for. Gata4 and gata6 also play a role in myocyte differentiation. A myoblast is a type of embryonic progenitor cell that differentiates to give rise to muscle cells. Myomaker is the only identified musclespecific protein required for myoblast fusion. Our current focus is to understand the molecules and mechanisms that mediate myoblast fusion. Gene ontology analysis software indicated that the tgf signalling pathway was overrepresented within the group of molecules inhibiting the fusion of c2c12 cells fig. Glycolipids also play key roles in cell differentiation 25, 26. Analysis and quantification of in vitro myoblast fusion. Although these functional roles of pctaire1 thus far have been characterized mainly in nervous system, which seems to be coincidentally associated with the myogenesis because cell migration and formation of a large f. While our data strongly support a model whereby graf1 facilitates membrane merging by promoting the recruitment of ferlincontaining vesicles, numerous cellsurface receptors have been implicated in promoting the initial stages of myoblast fusion including mcadherin and ncam that mediate cell recognition and integrins that mediate cellcell. Myoblast fusion of the drosophila body wall muscles.
From an experimental perspective, the minimal twocomponent nature of this program offers. The independent and sequential membrane functions of different proteins myomaker and myomerger culminate in membrane coalescence. Numerous mechanisms are involved in controlling myoblast migration though nearly all of them are not muscle specific. Myd88 promotes myoblast fusion in a cellautonomous manner. A membrane activator of myoblast fusion and muscle. Here the authors identify minion as a microprotein required for myoblast fusion and skeletal muscle formation, and show that co. We also emphasize recent discoveries in vertebrate myoblast fusion in skeletal muscle, which is composed of numerous multinucleated myo. We have established that wsp, the drosophila homolog of the wasp family of microfilament nucleationpromoting factors, is an essential facilitator of myoblast fusion in drosophila embryos. Myoblast differentiation is a multistep process that involves withdrawal from the cell cycle, acquisition of a cell typespecific transcriptional program and morphological changes that include elongation, alignment and fusion of myoblasts to form myofibers 14. Cells free fulltext a lowtherapeutic dose of lithium.
Myoblast fusion, which is essential for muscle development. Jun 09, 2010 this is a timelapse movie of a mm14 myoblast culture induced to differentiate by depletion of serum and bfgf in the culture medium. Cellular fusion is essential for skeletal muscle development. The level of differentiation was determined by myh1, pax7, atrogin1, and myostatin mrna expression and fusion index. Myoblast fusion is essential for muscle development, regeneration, and growth. Molecular control of mammalian myoblast fusion springerlink. Dwip, a homolog of the conserved verprolinwasp interacting protein family of. Depdc1b is a key regulator of myoblast proliferation in mouse. Pctaire1cdk16 promotes skeletal myogenesis by inducing. Despite the central role for plasma membrane fusion, the molecules and mechanisms that govern this process have remained elusive. Glycogen synthase kinase 3 gsk3 slows myogenic differentiation and myoblast fusion partly by inhibiting the wnt. Of note, cell cell fusion is a basic phenomenon for all species, from drosophila to humans, where such events are involved in numerous processes including, in addition to placentation in mammals, macrophage fusion for osteoclast formation, and most importantly myoblast fusion for myogenesis during development and for muscle regeneration after. Results myoferlin is expressed in myoblasts undergoing fusion to myotubes.
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