PubMed · 11390229
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G H Thomas. 2001. Keeping bugs in shape.. https://doi.org/10.1016/s0966-842x(01)02050-9
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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 have found that MIM-B, a putative metastasis suppressor protein, is implicated in actin cytoskeletal control and interaction with a protein tyrosine phosphatase (PTP). MIM was originally described as a protein whose mRNA was Missing in Metastasis, as it was found not to be present in metastatic bladder carcinoma cell lines [Lee, Y. G., Macoska, J. A., Korenchuk, S. and Pienta, K. J. (2002) Neoplasia 4, 291-294]. We further characterized a variant of MIM, which we call MIM-B, and which we believe may be a link between tyrosine kinase signalling and the actin cytoskeleton. We have shown, using purified proteins and cell extracts, that MIM-B is an actin-binding protein, probably via a WASP (Wiskott-Aldrich syndrome protein)-homology 2 domain at its C-terminus. We have also found that MIM-B binds to the cytoplasmic domain of receptor PTPdelta. Expression of full-length MIM-B induces actin-rich protrusions resembling microspikes and lamellipodia at the plasma membrane and promotes disassembly of actin stress fibres. The C-terminal portion of MIM-B is localized in the cytoplasm and does not affect the actin cytoskeleton when expressed, while the N-terminal portion localizes to internal vesicles and probably targets the protein to membranes. We postulate that MIM-B may be a regulator of actin assembly downstream of tyrosine kinase signalling and that this activity may explain the involvement of MIM in the metastasis of cancer cells.
The tendency for tetramerization is the main disadvantage in the green fluorescent protein homologues from Anthozoa species. We report a universal method called hetero-oligomeric tagging, which diminishes troublesome consequences of tetramerization of Anthozoa-derived fluorescent proteins (FP) in intracellular protein labelling. This approach is based on the co-expression of the FP-tagged protein of interest together with an excess of free non-fluorescent FP mutant. The resulting FP heterotetramers contain only a single target polypeptide and, therefore, can be considered pseudo-monomeric. Feasibility of the method has been demonstrated with a red FP fused with cytoplasmic beta-actin or tubulin-binding protein Tau34. In addition, heterotetramers appeared to be a unique model for biophysical characterization of Anthozoa FPs in pseudo-monomeric state.