The small G-proteins Rac1 and Cdc42 are essential for myoblast fusion in the mouse.
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ABSTRACT:
SUBMITTER: Vasyutina E
PROVIDER: S-EPMC2682539 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cadherins are transmembrane glycoproteins that mediate Ca(2+)-dependent homophilic cell-cell adhesion and play crucial role during skeletal myogenesis. M-cadherin is required for myoblast fusion into myotubes, but its mechanisms of action remain unknown. The goal of this study was to cast some light on the nature of the M-cadherin-mediated signals involved in myoblast fusion into myotubes. We found that the Rac1 GTPase activity is increased at the time of myoblast fusion and it is required for this process. Moreover, we showed that M-cadherin-dependent adhesion activates Rac1 and demonstrated the formation of a multiproteic complex containing M-cadherin, the Rho-GEF Trio, and Rac1 at the onset of myoblast fusion. Interestingly, Trio knockdown efficiently blocked both the increase in Rac1-GTP levels, observed after M-cadherin-dependent contact formation, and myoblast fusion. We conclude that M-cadherin-dependent adhesion can activate Rac1 via the Rho-GEF Trio at the time of myoblast fusion.
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Project description:Myoblast fusion is an intricate process that is initiated by cell recognition and adhesion, and culminates in cell membrane breakdown and formation of multinucleate syncytia. In the Drosophila embryo, this process occurs asymmetrically between founder cells that pattern the musculature and fusion-competent myoblasts (FCMs) that account for the bulk of the myoblasts. The present studies clarify and amplify current models of myoblast fusion in several important ways. We demonstrate that the non-conventional guanine nucleotide exchange factor (GEF) Mbc plays a fundamental role in the FCMs, where it functions to activate Rac1, but is not required in the founder cells for fusion. Mbc, active Rac1 and F-actin foci are highly enriched in the FCMs, where they localize to the Sns:Kirre junction. Furthermore, Mbc is crucial for the integrity of the F-actin foci and the FCM cytoskeleton, presumably via its activation of Rac1 in these cells. Finally, the local asymmetric distribution of these proteins at adhesion sites is reminiscent of invasive podosomes and, consistent with this model, they are enriched at sites of membrane deformation, where the FCM protrudes into the founder cell/myotube. These data are consistent with models promoting actin polymerization as the driving force for myoblast fusion.
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Project description:Myoblast fusion is crucial for the formation, growth, maintenance and regeneration of healthy skeletal muscle. Unfortunately, the molecular machinery, cell behaviors, and membrane and cytoskeletal remodeling events that govern fusion and myofiber formation remain poorly understood. Using time-lapse imaging approaches on mouse C2C12 myoblasts, we identify discrete and specific molecular events at myoblast membranes during fusion and myotube formation. These events include rearrangement of cell shape from fibroblast to spindle-like morphologies, changes in lamellipodial and filopodial extensions during different periods of differentiation, and changes in membrane alignment and organization during fusion. We find that actin-cytoskeleton remodeling is crucial for these events: pharmacological inhibition of F-actin polymerization leads to decreased lamellipodial and filopodial extensions and to reduced myoblast fusion. Additionally, shRNA-mediated inhibition of Nap1, a member of the WAVE actin-remodeling complex, results in accumulations of F-actin structures at the plasma membrane that are concomitant with a decrease in myoblast fusion. Our data highlight distinct and essential roles for actin cytoskeleton remodeling during mammalian myoblast fusion, provide a platform for cellular and molecular dissection of the fusion process, and suggest a functional conservation of Nap1-regulated actin-cytoskeleton remodeling during myoblast fusion between mammals and Drosophila.
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Project description:The Drosophila myoblast city (mbc) locus was previously identified on the basis of a defect in myoblast fusion (Rushton et al., 1995. Development [Camb.]. 121:1979-1988). We describe herein the isolation and characterization of the mbc gene. The mbc transcript and its encoded protein are expressed in a broad range of tissues, including somatic myoblasts, cardial cells, and visceral mesoderm. It is also expressed in the pole cells and in ectodermally derived tissues, including the epidermis. Consistent with this latter expression, mbc mutant embryos exhibit defects in dorsal closure and cytoskeletal organization in the migrating epidermis. Both the mesodermal and ectodermal defects are reminiscent of those induced by altered forms of Drac1 and suggest that mbc may function in the same pathway. MBC bears striking homology to human DOCK180, which interacts with the SH2-SH3 adapter protein Crk and may play a role in signal transduction from focal adhesions. Taken together, these results suggest the possibility that MBC is an intermediate in a signal transduction pathway from the rho/rac family of GTPases to events in the cytoskeleton and that this pathway may be used during myoblast fusion and dorsal closure.
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Project description:The Drosophila sticks-and-stones (sns) locus was identified on the basis of its mutant phenotype, the complete absence of body wall muscles and corresponding presence of unfused myoblasts. The genetic location of the mutation responsible for this apparent defect in myoblast fusion was determined by recombination and deficiency mapping, and the corresponding wild-type gene was isolated in a molecular walk. Identification of the SNS coding sequence revealed a putative member of the immunoglobulin superfamily (IgSF) of cell adhesion molecules. As anticipated from this homology, SNS is enriched at the membrane and clusters at discrete sites, coincident with the occurrence of myoblast fusion. Both the sns transcript and the encoded protein are expressed in precursors of the somatic and visceral musculature of the embryo. Within the presumptive somatic musculature, SNS expression is restricted to the putative fusion-competent cells and is not detected in unfused founder cells. Thus, SNS represents the first known marker for this subgroup of myoblasts, and provides an opportunity to identify pathways specifying this cell type as well as transcriptional regulators of fusion-specific genes. To these ends, we demonstrate that the presence of SNS-expressing cells is absolutely dependent on Notch, and that expression of SNS does not require the myogenic regulatory protein MEF2.
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Project description:Epsins are endocytic proteins with a structured epsin N-terminal homology (ENTH) domain that binds phosphoinositides and a poorly structured C-terminal region that interacts with ubiquitin and endocytic machinery, including clathrin and endocytic scaffolding proteins. Yeast has two redundant genes encoding epsins, ENT1 and ENT2; deleting both genes is lethal. We demonstrate that the ENTH domain is both necessary and sufficient for viability of ent1Deltaent2Delta cells. Mutational analysis of the ENTH domain revealed a surface patch that is essential for viability and that binds guanine nucleotide triphosphatase-activating proteins for Cdc42, a critical regulator of cell polarity in all eukaryotes. Furthermore, the epsins contribute to regulation of specific Cdc42 signaling pathways in yeast cells. These data support a model in which the epsins function as spatial and temporal coordinators of endocytosis and cell polarity.
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Project description:During cell migration, Rho GTPases spontaneously form spatial gradients that define the front and back of cells. At the front, active Cdc42 forms a steep gradient whereas active Rac1 forms a more extended pattern peaking a few microns away. What are the mechanisms shaping these gradients, and what is the functional role of the shape of these gradients? Here we report, using a combination of optogenetics and micropatterning, that Cdc42 and Rac1 gradients are set by spatial patterns of activators and deactivators and not directly by transport mechanisms. Cdc42 simply follows the distribution of Guanine nucleotide Exchange Factors, whereas Rac1 shaping requires the activity of a GTPase-Activating Protein, β2-chimaerin, which is sharply localized at the tip of the cell through feedbacks from Cdc42 and Rac1. Functionally, the spatial extent of Rho GTPases gradients governs cell migration, a sharp Cdc42 gradient maximizes directionality while an extended Rac1 gradient controls the speed.
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Project description:Satellite cells represent a heterogeneous population of stem and progenitor cells responsible for muscle growth, repair and regeneration. We investigated whether c-Myb could play a role in satellite cell biology because our previous results using satellite cell-derived mouse myoblast cell line C2C12 showed that c-Myb was expressed in growing cells and downregulated during differentiation. We detected c-Myb expression in activated satellite cells of regenerating muscle. c-Myb was also discovered in activated satellite cells associated with isolated viable myofiber and in descendants of activated satellite cells, proliferating myoblasts. However, no c-Myb expression was detected in multinucleated myotubes originated from fusing myoblasts. The constitutive expression of c-Myb lacking the 3' untranslated region (3' UTR) strongly inhibited the ability of myoblasts to fuse. The inhibition was dependent on intact c-Myb transactivation domain as myoblasts expressing mutated c-Myb in transactivation domain were able to fuse. The absence of 3' UTR of c-Myb was also important because the expression of c-Myb coding region with its 3' UTR did not inhibit myoblast fusion. The same results were repeated in C2C12 cells as well. Moreover, it was documented that 3' UTR of c-Myb was responsible for downregulation of c-Myb protein levels in differentiating C2C12 cells. DNA microarray analysis of C2C12 cells revealed that the expression of several muscle-specific genes was downregulated during differentiation of c-Myb-expressing cells, namely: ACTN2, MYH8, TNNC2, MYOG, CKM and LRRN1. A detailed qRT-PCR analysis of MYOG, TNNC2 and LRRN1 is presented. Our findings thus indicate that c-Myb is involved in regulating the differentiation program of myogenic progenitor cells as its expression blocks myoblast fusion.
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Project description:During peripheral nervous system (PNS) myelination, Schwann cells must interpret extracellular cues to sense their environment and regulate their intrinsic developmental program accordingly. The pathways and mechanisms involved in this process are only partially understood. We use tissue-specific conditional gene targeting to show that members of the Rho GTPases, cdc42 and rac1, have different and essential roles in axon sorting by Schwann cells. Our results indicate that although cdc42 is required for normal Schwann cell proliferation, rac1 regulates Schwann cell process extension and stabilization, allowing efficient radial sorting of axon bundles.
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Project description:Formation of the Drosophila larval body wall muscles requires the specification, coordinated cellular behaviors and fusion of two cell types: Founder Cells (FCs) that control the identity of the individual muscle and Fusion Competent Myoblasts (FCMs) that provide mass. These two cell types come together to control the final size, shape and attachment of individual muscles. However, the spatial arrangement of these cells over time, the sequence of fusion events and the contribution of these cellular relationships to the fusion process have not been addressed. We analyzed the three-dimensional arrangements of FCs and FCMs over the course of myoblast fusion and assayed whether these issues impact the process of myoblast fusion. We examined the timing of the fusion process by analyzing the fusion profile of individual muscles in wild type and fusion mutants. We showed that there are two temporal phases of myoblast fusion in wild type embryos. Limited fusion events occur during the first 3 h of fusion, while the majority of fusion events occur in the remaining 2.5 h. Altogether, our data have led us to propose a new model of myoblast fusion where the frequency of myoblast fusion events may be influenced by the spatial arrangements of FCs and FCMs.
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