PubMed · 9788166
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H Mukai, Y Ono. 1998. [Structure and function of PKN].. https://pubmed.ncbi.nlm.nih.gov/9788166/
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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.
According to the Lorenz et al. (Lorenz, M., Poole, K. J., Popp, D., Rosenbaum, G., and Holmes, K. C. (1995) J. Mol. Biol. 246, 108-119) atomic model of the actin-tropomyosin complex, actin residue Asp-311 (Glu-311 in yeast) is predicted to have a high binding energy contribution to actin-tropomyosin binding. Using the yeast actin mutant E311A/R312A in the in vitro motility assays, we have investigated the role of these residues in such interactions. Wild type (wt) yeast actin, like skeletal alpha-actin, is fully regulated when complexed with tropomyosin (Tm) and troponin (Tn). Structure-function comparisons of the wt and E311A/R312A actins show no significant differences between them, and the unregulated F-actins slide at similar speeds in the in vitro motility assay. However, in the presence of Tm and Tn, the mutation increases both the sliding speed and the number of moving filaments at high pCa values, shifting the speed-pCa curve nearly 0.5 pCa units to the left. Tm alone (no Tn) inhibits the motilities of both actins at low heavy meromyosin densities but potentiates only the motility of the mutant actin at high heavy meromyosin densities. Actin-Tm binding measurements indicate no significant difference between wt and E311A/R312A actin in Tm binding. These results implicate allosteric effects in the regulation of actomyosin function by tropomyosin.
The ubiquitously expressed heterotrimeric guanine nucleotide-binding proteins (G-proteins) G12 and G13 have been shown to activate the small GTPase Rho. Rho stimulation leads to a rapid remodeling of the actin cytoskeleton and subsequent stress fiber formation. We investigated the involvement of G12 or G13 in stress fiber formation induced through a variety of Gq/G11-coupled receptors. Using fibroblast cell lines derived from wild-type and Galphaq/Galpha11-deficient mice, we show that agonist-dependent activation of the endogenous receptors for thrombin or lysophosphatidic acid and of the heterologously expressed bradykinin B2, vasopressin V1A, endothelin ETA, and serotonin 5-HT2C receptors induced stress fiber formation in either the presence or absence of Galphaq/Galpha11. Stress fiber assembly induced through the muscarinic M1 and the metabotropic glutamate subtype 1alpha receptors was dependent on Gq/G11 proteins. The activation of the Gq/G11-coupled endothelin ETB and angiotensin AT1A receptors failed to induce stress fiber formation. Lysophosphatidic acid, B2, and 5-HT2C receptor-mediated stress fiber formation was dependent on Galpha13 and involved epidermal growth factor (EGF) receptors, whereas thrombin, ETA, and V1A receptors induced stress fiber accumulation via Galpha12 in an EGF receptor-independent manner. Our data demonstrate that many Gq/G11-coupled receptors induce stress fiber assembly in the absence of Galphaq and Galpha11 and that this involves either a Galpha12 or a Galpha13/EGF receptor-mediated pathway.