PubMed · 5569013
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P Laugier, N Hunziker, V Ellena. 1971-07-24. [Reactive hyperplastic reticuloses].. https://pubmed.ncbi.nlm.nih.gov/5569013/
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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.
As transmembrane heterodimers, integrins bind to both extracellular ligands and intracellular proteins. We are currently investigating the interaction between integrins and the intracellular protein calreticulin. A prostatic carcinoma cell line (PC-3) was used to demonstrate that calreticulin can be found in the alpha3 immunoprecipitates of cells plated on collagen type IV, but not when plated on vitronectin. Conversely, alphav immunoprecipitates contained calreticulin only when cells were plated on vitronectin, i. e. not when plated on collagen IV. The interactions between these integrins and calreticulin were independent of actin cytoskeleton assembly and were transient, being maximal approx. 10-30 min after the cells came into contact with the substrates prior to complete cell spreading and formation of firm adhesive contacts. We demonstrate that okadaic acid, an inhibitor of intracellular serine/threonine protein phosphatases, inhibited the alpha3beta1-mediated adhesion of PC-3 cells to collagen IV and the alpha2beta1-mediated attachment of Jurkat cells to collagen I. This inhibition by okadaic acid was accompanied by inhibition of the ligand-specific interaction of calreticulin with the respective integrins in the two cell types. Additionally, we found that pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) resulted in prolongation of the calreticulin-integrin interaction, and enhancement of PC-3 cell attachment to collagen IV. We conclude that calreticulin interacts transiently with integrins during cell attachment and spreading. This interaction depends on receptor occupation, is ligand-specific, and can be modulated by protein phosphatase and MEK activity.
Dendritic spines are known to be extremely motile, providing a structural mechanism for synaptic plasticity. Actin filaments are thought to be responsible for the changes in the shape of spines. We tested our hypothesis that drebrin, an actin-binding protein, is a regulator of spine shape. In high-density long-term primary cultures of rat cerebral cortex neurons, drebrin was colocalized with actin filaments at spines. We introduced drebrin tagged with green fluorescent protein (GFP) into these neurons to test the ability of exogenous drebrin to localize at spines and the effect of overexpression of drebrin on spine shape. We observed that exogenous drebrin indeed accumulated in spines. But when the actin-binding domain of drebrin was deleted, the protein was distributed in both spines and dendritic shafts, indicating that accumulation of drebrin in the spines required its actin-binding activity. Statistical analysis of the lengths of spines as determined from confocal laser microscopic images revealed that the spines were significantly longer in GFP-drebrin-expressing neurons than in GFP-expressing neurons. The longer spines labeled with GFP-drebrin were demonstrated to be postsynaptic by double labeling of the presynaptic terminals with antibody against synaptophysin. These results directly indicate that drebrin binds to actin filaments at dendritic spines and alters spine shape.