PubMed · 6383637
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N Kamiya. 1984. Cytoplasmic streaming.. https://pubmed.ncbi.nlm.nih.gov/6383637/
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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 describe a new method that uses straightforward physics to apply force to substrate-attached cells. In this method, collagen-coated magnetic ferric oxide beads attach to the dorsal surface of cells via receptors of the integrin family, and a magnetic field gradient is applied to produce a force. In this paper we present a complete characterization of the method in a configuration that is easy to use, in which a permanent magnet provides a fairly uniform gradient over a relatively large area. This allows a fairly uniform average force that can be controlled in magnitude, direction, and duration to be applied to a large number of cells. We show how to determine the applied force per cell by measuring the force per unit volume of magnetic bead, the distribution of bead diameters, and the distribution of beads per cell. We also show how to calculate the force per unit volume of bead in a three-dimensional region near the permanent magnet on the basis of field measurements, and present results for three of the magnets. An upward force applied to fibroblasts by this method produces a measurable time-dependent increase in attachment of cytoskeletal actin filaments to the force application points, and an increase in actin cross-linking. This is accompanied by an actin-dependent retraction of the force-induced upward movement of the dorsal surface of the cells.
Platelet-derived growth factor-stimulated actin rearrangement and edge ruffle formation have previously been shown to be dependent on activation of phosphatidylinositol 3'-kinase, the activity of which also is important for directed migration of cells. This lipid kinase binds to phosphorylated tyrosine residues Y740 and Y751 in the kinase insert of the human platelet-derived growth factor ss-receptor. We examined the role of two other tyrosine residues in the kinase insert of this receptor, Y775 and Y778, for ligand-induced actin rearrangement. Both were shown to be phosphorylation sites; Y775 was only marginally phosphorylated in cells expressing the wild-type ss-receptor, whereas Y778 was phosphorylated at higher stoichiometry. Mutant receptors Y775F, Y778F and Y775/778F were active kinases and mediated proliferative responses when expressed in porcine aortic endothelial cells. Fluorescence staining of actin in platelet-derived growth factor-stimulated PAE cells revealed that Y778 is involved in regulation of the actin cytoskeleton since the cells contained, apart from edge ruffles and circular ruffles, a novel type of giant ruffle on the dorsal side of the cell, which consisted of irregular multilayered actin structures. Mutation at Y778 had no effect on activation of phosphatidylinositol 3'-kinase, nor on the GTPase activating protein of Ras and phospholipase C(gamma), and the extent of directed migration towards platelet-derived growth factor of these cells was not changed. We conclude that actin rearrangement is regulated in part by Y778 in the platelet-derived growth factor ss-receptor, potentially through binding of a novel signaling molecule to this site.