Figure 4

Expression of the IRM-linked H40Y- and V163L-actin mutants. EGFP-tagged H40Y- (A-C) and V163L- (D, E) actin mutantswere expressed in C2C12 myoblasts (A) and differentiatedmyotubes.(B-E). EGFP fluorescence showed intranuclear (A; long arrows) and cytoplasmic aggregates (A; short arrow) formed by the H40Y-mutant in undifferentiated cells, with some co-staining with phalloidin (A"; arrows), but not with alpha-actinin (A'; arrows). A similar phenotype was observed for the V163L-isoform (not shown). In myotubes, some integration of EGFP-actin into stress fibres or sarcomeric structures was observed for both, H40Y- actin (B-B"; long arrows) and V163L-actin mutants (E, inset). However, in many differentiated cells, aberrant stress fibres were produced with a thickened and wavy appearance (C, C" long arrows; E, E", short arrows). The H40Y mutant might also induce fragmentation of stress fibres (B, B"; short arrow), but this could also be a short cytoplasmic rod. The V163L-mutant also formed nuclear aggregates in some myotubes (D; long arrow) that co-stained with phalloidin (D"; long arrow) and also very faintly with alpha-actinin (D'; long arrow). Nuclear aggregates were never found with the H40Y-mutant after differentiation. (Scale bars: 5 μm)