PubMed · 11156599
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E Fuchs, I Karakesisoglou. 2001-01-01. Bridging cytoskeletal intersections.. https://doi.org/10.1101/gad.861501
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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.
An immunohistochemical examination of cultured corpus cavernosum cells of the rabbit showed that they were composed of many smooth muscle cells retaining -smooth muscle actin and a few of endothelial cells. Application of noradrenaline (NA, 0.5-100 microM) to the culture dose-dependently increased cytosolic concentrations of Ca2+ ([Ca2+]i) in smooth muscle cells. The NA (10 microM)-induced [Ca2+]i increase was dose-dependently inhibited by phenoxybenzamine (1 nM-1 microM). The inhibition was 71% at 0.1 microM and was completely made at 1 microM. An alpha(1)-adrenoceptor antagonist, prazosin (0.1 microM), inhibited the [Ca2+]i response by 42%, and propranolol (1 microM) enhanced the response by 118%. These adrenoceptor antagonists showed almost the same effects on a contractile response of corporal tissue strips to NA (10 microM) as the effects on the [Ca2+]i response. Our results suggest that NA-induced [Ca2+]i increase was mixed with a large increase through 1-adrenoceptors and a small decrease through beta-adrenoceptors. The increased [Ca2+]i could play a role of intracellular messenger for leading to contraction of cavernosal smooth muscle cells.
EXPERIMENTAL OBJECTIVES: Stimulation of low-affinity CCK-1 receptors on pancreatic acini leads to inhibition of enzyme secretion. We studied signal transduction mechanisms to identify potential causes for the reduced secretion. RESULTS: Co-stimulation experiments with CCK, CCK-JMV-180, and bombesin revealed an inhibition of bombesin-stimulated enzyme secretion by low-affinity CCK-1 receptors. Binding of 125I-gastrin-releasing peptide (the mammalian analogue of bombesin) to acini after CCK preincubation was not altered. After a short preincubation of acini with high concentrations of CCK, intracellular calcium remained responsive to bombesin. In contrast to bombesin or CCK at concentrations of 10(-10) M or lower, high concentrations of CCK caused a strong activation of p125 focal adhesion kinase (p125(FAK)) and a marked reorganisation of the actin cytoskeleton. CONCLUSIONS: Inhibitory mechanisms triggered by low-affinity CCK-1 receptors interrupt enzyme secretion from pancreatic acini at late stages in the signal transduction cascades since bombesin receptor binding and early signalling events remained intact after CCK preincubation. A reorganisation of the actin cytoskeleton is suggested to be the mechanism by which low-affinity CCK-1 receptors actively interrupt enzyme secretion stimulated by other receptors.