Lmod2[E34Q] assembles at thin filament pointed ends, does not significantly displace Tmod1 from the pointed ends, and reduces ability to elongate thin filaments compared with WT Lmod2. (A) Representative images showing Tmod1 assembly (red) in rat neonatal cardiomyocytes expressing (from top to bottom) GFP, GFP-Lmod2[WT], and GFP-Lmod2[E34Q] (green). (B) Percentage of cells with consistent Tmod1 assembly at pointed ends in cardiomyocytes expressing GFP (turquoise), GFP-Lmod2[WT] (WT, blue), and GFP-Lmod2[E34Q] (E34Q, red). Overexpression of WT Lmod2 caused significant displacement of Tmod1 from thin filament pointed ends. Similar levels of Lmod2[E34Q] (see Fig. S1 A) did not change Tmod1 distribution compared with GFP-expressing controls. (C) Samples extracted with a Triton-X–containing (cytoskeletal-extraction: CSK) buffer show a strong association of Tmod1 with pointed ends in the presence of GFP-Lmod2[E34Q], similar to that in GFP-expressing controls. (Note that GFP alone is substantially removed by CSK buffer treatment, as is the cytoplasmic and side binding for WT Lmod2 and Lmod2 E34Q). GFP-Lmod2 [E34Q] showed good association with pointed ends of thin filaments. Pointed ends are indicated by yellow triangles. Scale bars are 1 mm. (D) Thin filament lengths from rat neonatal cardiomyocytes expressing GFP, GFP-Lmod2[WT], and GFP-Lmod2[E34Q]. Representative images and the relationship between sarcomere length and filament lengths are available in Fig. S1, B and C. Each point represents the average measurement per single cell. Mean + SD, P values are shown on the graph.