FIGURE 2.

Both leucine 27 and isoleucine 131 residues in Tmod1 are required for thin filament pointed end localization. Neonatal rat cardiomyocytes expressing GFP-Tmod1 (WT or mutants) or GFP alone were stained for GFP (a, e, i, m, and q), F-actin using fluorescently conjugated phalloidin (b, f, j, n, and r), and sarcomeric α-actinin to stain the Z-discs (c, g, k, o, and s). d, h, l, p, and t show merged images of the triple staining (GFP, green; F-actin, red; α-actinin, blue). Clear and consistent striated localization of WT GFP-Tmod1 was observed at thin filament pointed ends (e). GFP-Tmod1(L27E) assembled like WT GFP-Tmod1 (m), but the percentage of the cells displaying striations was lower, and the soluble/unbound GFP molecule in cytoplasm was higher compared with those expressing WT GFP-Tmod1 (Fig. 3). In contrast, GFP-Tmod1(I131D) was rarely observed at the pointed ends (q, arrowheads), and GFP-Tmod1(L27E/I131D) was never observed in a striated pattern (i). GFP-Tmod1(I131D), GFP-Tmod1(L27E/I131D), and GFP alone were observed to be localized in the nucleus (N), all of which do not assemble at thin filament pointed ends. Scale bar = 10 μm.