PubMed · 10559874
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S H Zigmond. 1999. A method for movement.. https://doi.org/10.1038/8962
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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.
Messenger RNA (mRNA) targeting to specific subcellular domains has been studied extensively in many cell types, and there is increasing evidence suggesting that mRNA sorting also occurs in astrocytes. As a step toward developing strategies to evaluate the signals that govern mRNA sorting in astrocytes, the authors studied the subcellular distribution of several representative mRNAs, poly(A) RNA and ribosomal RNA, in process-bearing (type-2) astroglial cells in culture. Nonradioactive in situ hybridization analysis revealed a gradual increase in the expression of glial fibrillary acidic protein (GFAP) mRNA as type-2 astrocytes differentiated in culture. In mature cells, labeling was present in both cell bodies and processes. GFAP mRNA labeling was granular in nature and was particularly concentrated at branch points and at the tips of the processes. Unlike GFAP mRNA, vimentin, beta-tubulin, and beta- and gamma-actin mRNAs were mainly confined to the cell bodies, with only occasional labeling seen in the processes. Nonradioactive and radioactive in situ hybridization analysis of poly(A) and ribosomal RNA, respectively, revealed labeling in cell bodies and processes of immature and differentiated astrocytes. Treatment with nocodazole, a microtubule depolymerizing agent, resulted in a substantial reduction of GFAP mRNA labeling in the processes, whereas treatment with cytochalasin D, a microfilament-disrupting agent, did not alter GFAP mRNA distribution. The results indicate that cultured type-2 astrocytes have the capacity to sort mRNAs to different subcellular domains and that the localization of GFAP mRNA to astrocyte processes requires intact microtubules.
Marfan syndrome is associated with early death due to aortic aneurysm. The condition is caused by mutations in the gene (FBN1) encoding fibrillin-1, a major constituent of extracellular microfibrils. Prior observations suggested that a deficiency of microfibrils causes failure of elastic fiber assembly during late fetal development. Mice homozygous for a targeted hypomorphic allele (mgR) of Fbn1 revealed a predictable sequence of abnormalities in the vessel wall including elastic fiber calcification, excessive deposition of matrix elements, elastolysis, and intimal hyperplasia. Here we describe previously unrecognized concordant findings in elastic vessels from patients with Marfan syndrome. Furthermore, ultrastructural analysis of mgR mice revealed cellular events that initiate destructive changes. The first detectable abnormality was an unusually smooth surface of elastic laminae, manifesting the loss of cell attachments that are normally mediated by fibrillin-1. Adjacent cells adopted alteration in their expression profile accompanied by morphological changes but retained expression of vascular smooth muscle cell markers. The abnormal synthetic repertoire of these morphologically abnormal smooth muscle cells in early vascular lesions included elastin, among other matrix elements, and matrix metalloproteinase 9, a known mediator of elastolysis. Ultimately, cell processes associated with zones of elastic fiber thinning and fragmentation. These data suggest that the loss of cell attachments signals a nonproductive program to synthesize and remodel an elastic matrix. This refined understanding of the pathogenesis of vascular disease in Marfan syndrome will facilitate the development of therapeutic strategies.