PubMed · 10023778
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F C Luft. 1999. Differentiating one smooth operator from another.. https://doi.org/10.1007/s001090050347
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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.
Neutrophil granulocytes are known to rapidly adhere and undergo frustrated phagocytosis upon contact with immunoglobulin and/or complement protein opsonized artificial surfaces. In this study, we examined the relation between serum protein deposition and human neutrophil activation on hydrophobic glass and silicon model surfaces that were coated with immunoglobulin G or M (IgG/IgM), both initiators of the classical complement pathway. Protein adsorption from normal human serum (NHS) was quantified with null-ellipsometry combined with antibody techniques. The neutrophil oxygen radical production was registered by luminol-amplified chemiluminescence (CL) and the morphology, as well as changes in the content of filamentous actin (F-actin), were documented by fluorescence microscopy. Complement factor 3 (C3) bound to both IgG- and IgM-coated surfaces, but surprisingly C1q was found only on IgG-coated surfaces. Both immunoglobulins triggered complement dependent neutrophil activation. However, CL and F-actin accumulation were found sensitive to the presence of C1q in the serum only at the IgG-coated surface. We suggest that spontaneously adsorbed IgM activates the complement system and interacts with neutrophils by C1q-independent mechanisms.
Gicerin is a cell adhesion molecule belonging to the immunoglobulin superfamily. To study the functional differences between l- and s-gicerin, we first examined the distribution of endogenous gicerin in B16 cells and found that l-gicerin was densely localized in microvilli. To clarify the relationship between gicerin and the microvilli, we established independent stable cell lines expressing l- and s-gicerin in L cells and found that l-gicerin localized to the microvilli. Scanning electron microscopic analysis revealed that the microvilli of l-gicerin-transfected cells were longer than those of s-gicerin and control transfectants. This suggested that l-gicerin might participate in the elongation of the microvilli. When cells were double-stained with antibodies to gicerin and moesin, a microvilli-specific protein, the staining of l-gicerin corresponded to that of moesin in the elongated microvilli. Moesin was coprecipitated with glutathione S-transferase-fusion proteins of the l-gicerin cytoplasmic domain but not with the s-gicerin cytoplasmic domain. To determine the region involved in the extension of microvilli, we generated transfectants of two truncated forms of l-gicerin cytoplasmic domain, and we found that only the transfectants of the longer mutant had the longer microvilli, while the shorter mutant exhibited short microvilli. These results suggested that l-gicerin-specific amino acid residues, especially amino acids 16-39, within the cytoplasmic domain of l-gicerin might be involved in the extension of microvilli.