JCS046805 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Subcellular localizations of actin-GFP fusion proteins. (A) After 48 hours of expression, GFP fusions of β-actin and α-cardiac actin have similar sarcomeric localization to F-actin (visualized by Rhodamine-phalloidin) in neonatal rat cardiomyocytes. Scale bar: 10 µm. (B) Higher magnification images of the boxed areas in A. Scale bar: 5 µm.

• Supplemental Figure S2 -

  Fig. S2. Localization and dynamics of GFP and mCherry in cultured cardiomyocytes. (A) Isolated GFP has a mainly diffuse localization in cardiomyocytes. FRAP analysis revealed that in a contracting cell (upper panel) and in a cell where contractility was inhibited by BDM (lower panel) GFP shows very rapid diffusion dynamics. White boxes indicate bleached areas and arrowheads the selected region over the time. (B) Isolated mCherry has a mainly diffuse localization in cardiomyocytes. Similarly to GFP, mCherry shows rapid diffusion dynamics in contracting (upper panel) and blebbistatin-treated (lower panel) cells. White boxes indicate bleached areas and arrowheads the selected region over the time. Scale bar: 5 µm.

• Supplemental Figure S3 -

  Fig. S3. α-cardiac actin dynamics in cardiomyocyte sarcomeres is dependent on the contractility. Recovery profile of bleached zone over time course of FRAP experiment for a cell expressing GFP−α-cardiac-actin. BDM treatment halts actin turnover.

• Supplemental Figure S4 -

  Fig. S4. The rates of recovery of the dynamic actin pool in cardiomyocytes of different maturation stages. The half-time of GFP−β-actin and GFP−cardiac-α-actin fluorescence recovery was quantified from cardiomyocytes after 2, 3 and 4 days in culture. For each column, the t₁/₂ for >20 cells was measured. Error bars indicate s.e.m. values.

• Supplemental Figure S5 -

  Fig. S5. Subcellular localization of tropomodulin in cardiomyocytes of different maturation stages. In pre-mature cardiomyocytes (cultured for 1 day) tropomodulin has a mainly diffuse localization (upper panel), whereas in mature cardiomyocytes (cultured for 3 days) tropomodulin typically concentrates at filament pointed ends/M-band region (lower panel). Scale bar: 10 µm.

• Supplemental Figure S6 -

  Fig. S6. Cofilin 1/2 knockdown. (A) Western blot analysis demonstrating the cofilin 1/2 protein level in wild type (wt) and cofilin 1 and cofilin 2 siRNA transfected cells (cof1/cof2 siRNA). Equal amounts of the cell lysates (8 µg) were run on polyacrylamide gels and cofilin 1/2 and tubulin were visualized by western blotting. (B) GFP−β-actin-expressing cell co-transfected with cofilin 1 and cofilin 2 siRNA oligonucleotides reveals a disorganized sarcomeric structure. (C) Actin dynamics are halted in a cofilin knockdown cell, as revealed by FRAP. Scale bar: 5 µm.

• Movie 1 -

  Movie 1. Selected cells before latrunculin B treatment. The majority of the cells are beating synchronously.

• Movie 2 -

  Movie 2. The same region as shown in Movie 1, after 20 minutes of 20 µM latrunculin B treatment. The presence of latrunculin B does not change the number of beating cells, but the beating is less synchronous than before the addition of latrunculin B.