PubMed · 14237861
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M E CARSTEN, A M KATZ. 1964-09-04. ACTIN: A COMPARATIVE STUDY.. https://doi.org/10.1016/0304-4165(64)90232-6
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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.
We have developed and tested successfully a general method based on Cre-mediated recombination that can be used for ubiquitous or tissue-specific expression of protein products, including tumor-inducing oncoproteins. Depending on the specificity of a chosen promoter driving cre expression, tumors develop by design in bitransgenic mouse progeny derived by crossing Cre-producing mice with partners carrying a dormant oncogenic transgene (targeted into the 3' noncoding region of the cytoplasmic beta-actin locus) that becomes functional after excision of a 'floxed' DNA segment. To provide proof-of-principle, we have used as models transgenes encoding the polyomavirus middle T antigen (PVMT) and the T antigens of the SV40 early region (SVER). Cre-dependent activation of widespread SVER expression resulted in hyperplasias or invasive tumors affecting particular visceral smooth muscles, whereas Cre-dependent, mammary gland-specific expression of PVMT-induced adenocarcinomas, according to plan. Unexpectedly, we also encountered spontaneous (Cre-independent) oncogene expression occurring as a rare event, which simulates the initiation of sporadic tumors and leads to PVMT-induced hemangiomas and mammary carcinomas or SVER-induced disseminated sarcomas, thus, revealing particular tissue susceptibilities to the actions of these oncoproteins.
One important step in bone formation on hard tissue implants is adhesion of osteoblast precursors to the implant surface. In this study, we used function-blocking antibodies against integrin subunits to characterize the mechanisms used by human marrow stromal cells and Saos-2 osteosarcoma cells to adhere to protein-coated hydroxylapatite (HA). We found that Saos-2 use both alpha5- and alphav-containing integrins, whereas stromal cells use alphav-containing integrins but not alpha5-containing integrins, despite the presence of alpha5-containing integrins on cell surfaces. On the basis of this difference, we examined binding of these cell types to HA coated with fibronectin (FN) or vitronectin (VN), to determine whether these ligands for alpha5 and alphav integrins could enhance the numbers or morphology of cells adhered to them. We also examined the adhesion of cells to HA coated with RGD peptides designed to bind to FN or VN receptors. Morphology and number of adherent stromal cells were markedly enhanced on serum-coated surfaces compared with FN or VN alone, whereas, surprisingly, Saos-2 cells failed to spread on serum-coated HA and displayed superior spreading and stress fiber formation on FN-coated [corrected] HA. Collectively, these results have important implications for the design of protein coatings to enhance the performance of HA implants.