PubMed · 8646850
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S J Winder, M P Walsh. 1996. Calponin.. https://doi.org/10.1016/s0070-2137(96)80002-1
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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 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.
Cortactin, a prominent substrate for pp60(c-src), is a filamentous actin (F-actin) binding protein. We show here that cortactin can promote sedimentation of F-actin at centrifugation forces under which F-actin is otherwise not able to be precipitated. Electron microscopic analysis after negative staining further revealed that actin filaments in the presence of cortactin are cross-linked into bundles of various degrees of thickness. Hence, cortactin is also an F-actin cross-linking protein. We also demonstrate that the optimal F-actin cross-linking activity of cortactin requires a physiological pH in a range of 7.3-7.5. Furthermore, pp60(c-src) phosphorylates cortactin in vitro, resulting in a dramatic reduction of its F-actin cross-linking activity in a manner depending on levels of tyrosine phosphorylation. In addition, pp60(c-src) moderately inhibits the F-actin binding activity of cortactin. This study presents the first evidence that pp60(c-src) can directly regulate the activity of its substrate toward the cytoskeleton and implies a role of cortactin as an F-actin modulator in tyrosine kinase-regulated cytoskeleton reorganization.