Adhesion Proteins - An Impact on Skeletal Myoblast Differentiation

Przewoźniak, Marta; Czaplicka, Iwona; Czerwińska, Areta M.; Markowska-Zagrajek, Agnieszka; Moraczewski, Jerzy; Stremińska, Władysława; Jańczyk-Ilach, Katarzyna; Ciemerych, Maria A.; Brzóska, Edyta

doi:10.1371/journal.pone.0061760

Cited by 34 publications

(30 citation statements)

References 47 publications

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“…The relatively small number of amino acids that protrude from either the intracellular or extracellular space could provide a clue to the biochemical function of myomaker. For instance, it is unlikely that myomaker functions in the adhesion step of fusion, because it is not predicted to be glycosylated and contains only short extracellular segments (21). Muscle cell fusion has been shown to involve calcium and the formation of a hemifusion intermediate, in which the outer leaflets of the membranes fuse followed by fusion pore formation and cytoplasmic mixing (22)(23)(24).…”

Section: Discussionmentioning

confidence: 99%

Structure–function analysis of myomaker domains required for myoblast fusion

Millay

Gamage

Quinn

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Discussionmentioning

confidence: 99%

Structure–function analysis of myomaker domains required for myoblast fusion

Millay

Gamage

Quinn

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…CD9 and FRZB are positive regulators of ITGB1BP2 gene expression and ITGB1BP2 is a negative regulator of FRZB gene expression in myotubes As melusin and CD9 interact with integrin β1 ( Refs 12,14,15,16), and the expression of ITGB1BP2, CD9 and FRZB genes is upregulated in calpain 3-deficient patients, siRNA experiments were carried out in myotubes, to find out whether there was any coordinated regulation of these genes. At 48 h after treatment with CD9 siRNA, cultures showed markedly less CD9 mRNA (90%) than in those treated with scrambled LGMD2A CONTROL LGMD2A 25µM 25µM…”

Section: Frzb and Melusin Overexpressed In Lgmd2amentioning

confidence: 99%

FRZB and melusin, overexpressed in LGMD2A, regulate integrin β1D isoform replacement altering myoblast fusion and the integrin-signalling pathway

Jaka

Casas-Fraile

Azpitarte

et al. 2017

Expert Rev. Mol. Med.

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Limb-girdle muscular dystrophy type 2A (LGMD2A) is characterised by muscle wasting and progressive degeneration of proximal muscles because of mutations in the CAPN3 gene. However, the underlying pathophysiological mechanisms of muscle degeneration are still not well understood. The objective of this study was to assess the relevance of genes with differential expression in the muscle of LGMD2A patients. For this purpose, we analysed their in vitro expression in primary cultures of human myoblasts and myotubes. Abnormal fusion was observed in the myotubes of these patients, which may be explained by the lack of physiological replacement of integrin β1D. Owing to this observation, we focused on deregulated genes coding proteins that directly interact with integrin, ITGB1BP2 and CD9, as well as FRZB gene, because of its in vitro upregulation in myotubes. Silencing studies established that these genes are closely regulated, CD9 and FRZB being positive regulators of the expression of ITGB1BP2, and in turn, this gene being a negative regulator of the expression of FRZB. Interestingly, we observed that FRZB regulates integrin β1D expression, its silencing increasing integrin β1D expression to levels similar to those in controls. Finally, the administration of LiCl, an enhancer of the Wnt-signalling pathway showed similar experimentally beneficial effects, suggesting FRZB silencing or LiCl administration as potential therapeutic targets, though further studies are required.

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“…Although NELL1 is an intracellular signaling molecule, studies have shown that NELL1 can also act extracellularly through integrin α 3 β 1 to promote cell attachment and differentiation [Hasebe et al, 2012;Shen et al, 2012]. Both integrin α 3 and β 1 have been shown to play an important role in skeletal myoblast differentiation, with modifications in integrin expression significantly altering myoblast fusion in vitro [Przewozniak et al, 2013]. It is clear from the present study that extracellular delivery of NELL1 influences the constructive remodeling response in this skeletal muscle defect model.…”

Section: Discussionmentioning

confidence: 54%

Human NELL1 Protein Augments Constructive Tissue Remodeling with Biologic Scaffolds

Turner

Londoño²,

Dearth³

et al. 2013

Cells Tissues Organs

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Biologic scaffolds composed of extracellular matrix (ECM) derived from decellularized tissues effectively reprogram key stages of the mammalian response to injury, altering the wound microenvironment from one that promotes scar tissue formation to one that stimulates constructive and functional tissue remodeling. In contrast, engineered scaffolds, composed of purified ECM components such as collagen, lack the complex ultrastructure and composition of intact ECM and may promote wound healing but lack factors that facilitate constructive and functional tissue remodeling. The objective of the present study was to test the hypothesis that addition of NELL1, a signaling protein that controls cell growth and differentiation, enhances the constructive tissue remodeling of a purified collagen scaffold. An abdominal wall defect model in the rat of 1.5-cm² partial thickness was used to compare the constructive remodeling of a bovine type I collagen scaffold to a biologic scaffold derived from small intestinal submucosa (SIS)-ECM with and without augmentation with 17 μg NELL1 protein. Samples were evaluated histologically at 14 days and 4 months. The contractile response of the defect site was also evaluated at 4 months. Addition of NELL1 protein improved the constructive remodeling of collagen scaffolds but not SIS-ECM scaffolds. Results showed an increase in the contractile force of the remodeled skeletal muscle and a fast:slow muscle composition similar to native tissue in the collagen-treated group. The already robust remodeling response to SIS-ECM was not enhanced by NELL1 at the dose tested. These findings suggest that NELL1 protein does contribute to the enhanced constructive remodeling of skeletal muscle.

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Adhesion Proteins - An Impact on Skeletal Myoblast Differentiation

Cited by 34 publications

References 47 publications

Structure–function analysis of myomaker domains required for myoblast fusion

Structure–function analysis of myomaker domains required for myoblast fusion

FRZB and melusin, overexpressed in LGMD2A, regulate integrin β1D isoform replacement altering myoblast fusion and the integrin-signalling pathway

Human NELL1 Protein Augments Constructive Tissue Remodeling with Biologic Scaffolds

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