DEV022723 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. MS spectrum of the arginylated actin peptide identified in an alpha cardiac actin spot. The spot was excised from a 2D gel similar to those shown in Fig. 1. The identified peptide had the sequence (R) GIVLDSGDGVTHNVPIYEGY, with the added R identified in brackets. Dominant b and y ions are indicated on the spectrum. The tabulated data are b and y ion masses observed in the spectrum (right columns, in red and gray, respectively) and their deviation from the theoretically calculated masses (left columns).

• Supplemental Figure S2 -

  Fig. S2. Myofibril preparations from wild-type and Ate1 knockout E12.5 mouse hearts and relative amounts of actin in the different fractions. One-dimensional gel comparison of myofibril preparations from wild type (WT) and Ate1 knockout (KO) E12.5 mouse hearts (left) and relative amounts of actin in different fractions (bottom right) show no consistent differences in the myofibril protein composition or the distribution of actin between the soluble pool and the myofibril fraction. The most similar preparations are shown in the figure. Myosin to actin ratios were averaged from three wild-type and four knockout hearts using gel densitometry. Numbers represent average and s.d. Buffers and actin purification steps are designated as described in Materials and methods.

• Supplemental Figure S3 -

  Fig. S3. Ate1 knockout results in delayed myofibril development. (Top panels) Comparison of the ‘worst’ (less developed, top) and ‘best’ (more developed, bottom) myofibrils in wild-type (WT) and Ate1 knockout (KO) E9.5 hearts. KO myofibrils are thinner than WT and contain less prominent Z-bands, suggesting a delay in myofibril development in KO hearts. (Bottom) Measurement of Z-band lengths (a parameter corresponding to the myofibril thickness and reflecting the overall size of the myofibrils) show that both at E9.5 and, less prominently, at E14.5, the mean thickness of the myofibrils is higher in the wild type, suggesting that myofibril development is delayed overall and that this delay can be prominently observed at earlier embryonic stages.

• Supplemental Figure S4 -

  Fig. S4. Beating frequency curves for wild-type cultured myocytes derived from E12.5 embryonic hearts. As used for the calculations shown in Fig. 5.

• Supplemental Figure S5 -

  Fig. S5. Beating frequency curves for Ate1 knockout cultured myocytes derived from E12.5 embryonic hearts. As used for the calculations shown in Fig. 5.

• Supplemental Figure S6 -

  Fig. S6. Beating frequency curves for wild-type and Ate1 knockout cultured myocytes derived from E12.5 embryonic hearts imaged by phase contrast and Fluo-4 calcium indicator fluorescence. Wild type (WT, top); Ate1 knockout (KO, bottom); phase, phase contrast; calcium, Fluo-4 calcium indicator. As used for the calculations and the correlation plot shown in Fig. 5.