PubMed · 15522968
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James A Spudich. 2004-11-02. Two important polymers cross paths.. https://doi.org/10.1073/pnas.0406932101
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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.
Rat neutrophils express the mRNA encoding for transient receptor potential (TRP) V1. However, capsaicin-stimulated [Ca2+]i elevation occurred only at high concentrations (> or = 100 microM). This response was substantially decreased in a Ca2+-free medium. Vanilloids displayed similar patterns of Ca2+ response with the rank order of potency as follows: scutigeral>resiniferatoxin>capsazepine>capsaicin=olvanil>isovelleral. Arachidonyl dopamine (AAD), an endogenous ligand for TRPV1, failed to desensitize the subsequent capsaicin challenge. Capsaicin-induced Ca2+ response was not affected by 8-bromo-cyclic ADP-ribose (8-Br-cADPR), the ryanodine receptor blocker, but was slightly attenuated by 1-[6-[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), the inhibitor of phospholipase C-coupled processes, 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365), the blocker of receptor-gated and store-operated Ca2+ (SOC) channels, 2-aminoethyldiphenyl borate (2-APB), the blocker of D-myo-inositol 1,4,5-trisphospahte (IP3) receptor and Ca2+ influx, and by ruthenium red, a blocker of TRPV channels, and enhanced by the Ca2+ channels blocker, cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine (MDL-12330A) and Na+-deprivation. In addition, capsaicin had no effect on the plasma membrane Ca2+-ATPase activity or the production of nitric oxide (NO) and reactive oxygen intermediates (ROI) or on the total thiols content. Capsaicin (> or = 100 microM) inhibited the cyclopiazonic acid (CPA)-induced store-operated Ca2+ entry (SOCE). In the absence of external Ca2+, the robust Ca2+ entry after subsequent addition of Ca2+ was decreased by capsaicin in CPA-activated cells. Capsaicin alone increased the actin cytoskeleton, and also increased the actin filament content in cell activation with CPA. These results indicate that capsaicin activates a TRPV1-independent non-SOCE pathway in neutrophils. The reorganization of the actin cytoskeleton is probably involved in the capsaicin inhibition of SOCE.
4-Hydroxy-2-nonenal (HNE), a major lipid peroxidation product, reacts with histidine, lysine or cysteine residues of proteins to form hemiacetal Michael adducts and thus interferes with the functions of the proteins. Here we undertook to identify HNE-modified proteins in the target organ of a ferric nitrilotriacetate (Fe-NTA)-induced renal carcinogenesis model with histidine-specific HNEJ-2 antibody. Immunoaffinity column separation and sequencing identified one of the major modified proteins as actin. To further explore the characteristics of actin as an HNE acceptor, we produced four novel monoclonal antibodies against HNE-modified keyhole limpet hemocyanin. All these antibodies (HNEJ-1, 3-5) recognized histidine adducts, but were different from HNEJ-2 in recognizing lysine and cysteine adducts to some extent. Actin, albumin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), metallothionein and superoxide dismutase were treated in vitro with HNE and evaluated with these antibodies. The results revealed that actin was most sensitive to HNE modification and metallothionein most resistant. Furthermore, the residue-specificity of GAPDH was in accord with that shown by our recent mass spectrometry data. Immunohistochemistry with the antibodies revealed cytoplasmic staining with or without nuclear staining in the renal proximal tubules after Fe-NTA administration. The results suggest that actin is a major target protein for HNE modification in vivo, and that our monoclonal antibodies are useful for evaluating the HNE adducts produced.