PubMed · 8925001
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J Hasek, E Streiblová. 1996. Fluorescence microscopy methods.. https://doi.org/10.1385/0-89603-319-8%3A391
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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 examined the role that individual TGF-beta isoforms, and in particular TGF-beta3, play in control of epidermal homeostasis. Mice with a knockout mutation of the TGF-beta3 gene die a few hours after birth. A full-thickness skin grafting approach was used to investigate the postnatal development and homeostatic control of the skin of these mice. Grafted skin of mice with a disruption of the TGF-beta3 gene developed similarly to grafts of wild type and TGF-beta1 knockout animals. However, a strikingly different response was observed after acute treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). When exposed to TPA, the grafted skin of wild type and TGF-beta1 knockout mice underwent a hyperplastic response similar to that of normal mouse skin. In marked contrast, TPA treatment of TGF-beta3 knockout grafts induced widespread areas of keratinocyte cell death. Analysis of cultured keratinocytes treated with purified TGF-beta isoforms revealed that TGF-beta3 plays a direct and specific function in protecting keratinocytes against TPA-induced cell death. The protective function of TGF-beta3 on TPA-induced cell death was not because of general suppression of the signaling pathways triggered by this agent, as ERK1/2 activation occurred to a similar if not greater extent in TGF-beta3-treated versus control keratinocytes. Instead, TGF-beta3 treatment led to a significant reduction in TPA-induced c-Jun N-terminal kinase activity, which was associated and possibly explained by specific counteracting effects of TGF-beta3 on TPA-induced disruption of keratinocyte focal adhesions.
Participation of the actin cytoskeleton in the transduction of proliferative signals has been established through the use of compounds that disrupt the cytoskeleton. To address the possibility that actin also participates in the transduction of an apoptotic signal, we have studied the response of the murine interleukin 2 (IL-2)-dependent T cell line CTLL-20 to treatment with the actin-binding compound jasplakinolide upon IL-2 deprivation. Like phalloidin, jasplakinolide stabilizes F-actin and promotes actin polymerization. Treatment of CTLL-20 cells with jasplakinolide, in the presence or absence of recombinant human IL-2, altered actin morphology as assessed by confocal fluorescence microscopy. Jasplakinolide was not toxic to CTLL-20 cells, nor was apoptosis induced in the presence of exogenous recombinant human IL-2. However, actin stabilization at the time of IL-2 deprivation enhanced apoptosis by changing the time at which CTLL-20 cells committed to the apoptotic pathway. This effect of jasplakinolide correlated with its ability to stabilize polymerized actin, as treatment with a synthetic analog of jasplakinolide with a greatly reduced ability to bind actin, jasplakinolide B, did not enhance apoptosis. The enhancement occurred upstream of the induction of caspase-3-like activity and could be inhibited by the overexpression of the anti-apoptotic protein Bcl-xL. These data suggest that the actin cytoskeleton plays an active role in modulating lymphocyte apoptosis induced by cytokine deprivation.