PubMed · 8528900
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P Sheterline, J C Sparrow. 1994. Actin.. https://pubmed.ncbi.nlm.nih.gov/8528900/
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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.
In the hindbrain, rhombomeres represent morphological units that develop characteristic, segment-specific structures. Similar segments, known as prosomeres, have been proposed to exist in the forebrain. The neuroepithelial cells of the sharp boundary regions that form the borders between many segments often exhibit distinct shapes, reflecting unique cytoskeletal organization. The present investigation examined the expression of one family of actin-binding, regulatory proteins, the tropomyosins (TM), in boundaries. We found that high molecular weight TMs selectively concentrate in boundary cells and other neuroepithelial zones that exhibit unique cell shapes and movements. Specific TM expression is found at hindbrain boundaries as early as embryonic day 10 in the rat, whereas rhombomeres themselves were TM-negative. Highly restricted TM localization also defined some prosomere boundaries in the early forebrain, particularly those exhibiting unique cell shapes. Furthermore, several regions of the neuroepithelium that evaginate are TM-immunoreactive, including tuberal and preoptic neuroepithelium. Most striking, a subpopulation of neuroepithelial cells in the medial telencephalic wall expresses TM, apparently marking the neuroepithelial region that gives rise to the choroid plexus at least 2 d before its formation. This suggests that the medial cerebral wall is not entirely dedicated to generating cells that comprise allocortex. TM expression in the choroid plexus is maintained through initial evagination and appearance in all ventricles. The spatially restricted expression of TMs implicates that this actin-binding protein is involved in the dynamic regulation of cell shape or motility associated with boundary formation and morphogenesis of the neuroepithelium during critical stages of brain development.
The ability of chicken gizzard smooth muscle caldesmon (CaD) to inhibit actomyosin ATPase activity is due mainly to an inhibitory domain that resides within the C-terminal 67 amino acid residues of the CaD molecule. In the present study, a series of C-terminal truncation and internal deletion mutants of chicken gizzard smooth muscle CaD were systematically designed using a site-directed mutagenesis approach, and these mutant proteins were overexpressed in a baculovirus expression system. Analysis of actin binding and inhibition of actomyosin ATPase activity using these mutants identified a strong actin-binding motif of 6 amino acid residues (from Lys718 to Glu723), which also form the core sequence for CaD-induced inhibition of actomyosin ATPase. However, maximal inhibition by CaD requires the presence of residues 728-731, which are not associated with actin binding. Our data provide direct evidence for the requirement of actin binding to a specific region in CaD for CaD-induced inhibition of actin activation of smooth muscle myosin ATPase. Furthermore, our findings also show that the region between residues 690 and 717 is responsible for the weak inhibition of actomyosin ATPase and reveal that the inhibitory determinants located in the regions between residues 690 and 717 and residues 718 and 756 can function independently.