PubMed · 8548558
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P Sheterline, J Clayton, J Sparrow. 1995. Actin.. https://pubmed.ncbi.nlm.nih.gov/8548558/
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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.
The role of myosins in Xenopus retinal ganglion cell growth cone motility in the optic tract was studied using two pharmacologic inhibitors with different specificities. 2,3-Butanedione monoxime (BDM) disrupts myosin-actin interactions of all myosins, and ML-7 specifically inhibits activation of myosin II. Both inhibitors caused growth cones to assume a collapsed morphology and decreased growth cone speed. Similar effects were observed in vitro. Interestingly, the effects of the two inhibitors, while similar, were clearly distinguishable, raising the possibility that different myosins may have different functional roles in growth cone motility. BDM caused growth cones to withdraw lamellipodia and some filopodia and eventually to freeze, whereas ML-7 caused total collapse and retraction. Concentrations of BDM and ML-7 that had no effect when applied independently stopped growth cones when applied simultaneously, suggesting that these inhibitors act synergistically on myosin function, thus providing evidence of specificity. These results imply that normal growth cone motility in the molecularly and spatially complex environment of the living brain requires myosin function.
We show that the requirement for Prp18 during the second step of actin pre-mRNA splicing in vitro is dictated by the distance between the branch point and the 3'splice site. Prp18 is dispensable for splicing of precursor RNAs in which the interval between the branch point and 3'splice site is <12 nt. This resembles the requirement for another second step factor, Slu7. Excess Slu7 protein can bypass the need for Prp18 in vitro , suggesting that Slu7 and Prp18 function in a concerted manner. Physical interaction between Slu7 and Prp18 was demonstrated by using the two-hybrid assay. Deletion mutants of SLU7 were tested for their ability to support growth of a slu7 null strain. Removal of 199 amino acids from the N-terminus of the 382 amino acid Slu7 protein did not affect cell viability at 25 degrees C. A more extensive N-terminal deletion of 221 amino acids was lethal, as was a C-terminal deletion of 47 amino acids. Deleted versions of Slu7 were also tested for interaction with Prp18 in the two-hybrid system. We define a segment of Slu7 from residue 200 to 224 that is necessary for interaction with Prp18.