Tropomyosin

Altogether, 103 mice (average age, 2 mo) were used in the study. Genotype was determined based on tailtip PCR. Nebulin expression was assessed with Western blot analysis using anti-nebulin N-terminal antibody (SI Appendix, Fig. S6). Protein levels of tropomyosin (Tm), troponin-C (TnC), troponin-T (TnT), and troponin-I (TnI) as well as MHC isoform expression were also determined. Routine picrosirius red, Gömöri-trichrome stains (SI Appendix, Fig. S8) and electron microscopy were performed on fixed soleus cross-sections. All animal experiments were approved by the University of Arizona and the Illinois Institute of Technology Institutional Animal Care and Use Committees and followed the Guide for the Care and Use of Laboratory Animals (39 ).
Intact soleus muscles were electrically stimulated at optimal muscle length in a custom-built test system (Fig. 1A). Blebbistatin was used to inhibit muscle contraction and lower tetanic force. Alternatively, the tetanic force was rapidly lowered by adjusting the muscle length (Fig. 2C). Sarcomere length was measured on fixed soleus fiber bundles using a CCD camera (SI Appendix, Fig. S7B). X-ray diffraction images were recorded using a high-flux 12 keV X-ray beam provided by Beamline 18 at the Advanced Photon Source (Argonne National Laboratory). Individual image frames were summed to be equivalent to at least 0.375 s exposure. The MUSICO computational platform (27 (link)) was used to simulate isometric force development and instantaneous muscle stiffness.
Descriptive statistical results are shown as mean ± SD unless stated otherwise. Differences between groups were considered to be statistically significant at a probability value of P < 0.05. Symbols used in statistical tests and on figures include ns, P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001. Detailed statistical evaluation is provided in SI Appendix, Table S1. Further details are in SI Appendix, Supplemental Methods.

To determine if the altered actin patch morphology observed in cap2Δ cells results from ectopic formin recruitment to the patches, we generated a strain that enabled temperature-sensitive inactivation of formin activity, cap2Δ bni1-1 bnr1Δ. This strain was generated by crossing BGY24 (cap2Δ) to BGY715 (bni1-1 bnr1Δ; Sagot et al., 2002 (link)). Two independent isolates of cap2Δ bni1-1 bnr1Δ were used in all experiments. We performed statistical tests (one-way ANOVA), which revealed that the observed changes in F-actin signal (at 25 and 37°C) are consistent between independent isolates and that any differences between the isolates are extremely small and not contributing to the statistical differences we observe between genotypes (as graphed in Fig. S5, A–C). The data presented are pooled from the two isolates. To visualize F-actin in wild-type and cap2Δ cells before and after formin inactivation, single colonies of wild-type, cap2Δ, bni1-1 bnr1Δ, and cap2Δ bni1-1 bnr1Δ strains were used to inoculate 5 ml yeast extract peptone dextrose cultures and grown at 25°C to OD₆₀₀ 0.3–0.6. Cultures were then split, such that half was shifted to the nonpermissive temperature of 37°C for 30 min before fixing in 3.7% formaldehyde for 40 min (at 37°C). The other half of the culture was maintained at 25°C and then fixed as above (at 25°C). Cells were stained with Alex488-phalloidin as above, mounted, and samples were imaged separately on a spinning-disk confocal and an Airyscan microscope, as described above for fixed cell imaging. As above, in all experiments, a wild-type control strain (marked with RFP^mScarlet) was grown at 25°C in parallel, then mixed with experimental cells during fixation and phalloidin stained to control for tube-to-tube variability in staining. The total F-actin signal per cell was measured as described above for fixed-cell imaging. The signal intensity of individual patches was measured in ImageJ from a single Airyscan Z-section by drawing a 5 × 5-pixel box around the central Z-plane of each patch. Signal background (outside of the cell) was subtracted from these measurements and values are reported relative to patch intensities of the internal control strain.
To determine whether formin activity is required for tropomyosin recruitment to patches, we generated a cap2Δ bni1-1 bnr1Δ strain expressing mNeon–Tpm1 and the patch marker Arc15-RFP^mScarlet. This strain was constructed by crossing a cap2Δ bni1-1 bnr1Δ strain to a strain expressing mNeon–Tpm1 and Arc15-RFP^mScarlet. These strains were then grown in synthetic complete media (2% glucose) and imaged on a spinning-disk confocal microscope as above for live cell imaging. To inactivate formins, cells were shifted to 37°C for 10 min, then imaged as above except at 37°C using a CherryTemp (Cherry Biotech) stage heater.

Fig. S1 compares the in vitro effects of yeast Cap1/2 and vertebrate CapZ on formin-mediated (Bni1, Bnr1, and mDia1) actin assembly in bulk assays, and the inhibitory effects of vertebrate CapZ on yeast formin (Bni1 and Bnr1) stimulated elongation of actin filaments in mf-TIRF assays. Fig. S2 shows the in vitro elongation rates of yeast actin filaments visualized with a SiR-actin probe in mf-TIRF assays, and the minimal effects of formins (Bni1 or Bnr1) on off rate of Cap1/2 from the barbed end (related to Fig. 2 in the main text). Also shown is quantification of the high-level (galactose-induced) expression of Cap1/2-GFP^Envy and GFP^Envy-CapZ in cells and evidence that it has no significant effect on Sac6-RFP^mScarlet marked actin patches (related to Fig. 3 in the main text). Fig. S3 shows the improved detection of formins Bni1 and Bnr1 in cells when tagged with a 3xmNeon tag and the quantification of formin (Bni1 and Bnr1) colocalization with Arc15-RFP^mScarlet labeled patches (related to Fig. 5 in the main text). Fig. S4 shows that even in the absence of formin activity in vivo, there is excessive accumulation of F-actin (indicated by phalloidin staining) at cortical actin patches in cap2∆ cells (related to Fig. 6 in the main text). Further, it shows that actin-binding proteins have altered dynamics at cortical patches in cap2∆ cells, and that aberrant decoration of older patches by tropomyosin (Tpm1) in cap2∆ cells occurs independent of formin activity. Fig. S5 shows that upon CK666 treatment the Arp2/3 complex signal at cortical patches is lost in both wild-type and cap2∆ cells, and provides additional examples of F-actin organization in phalloidin stained wild-type and cap2∆ cells (related to Fig. 7 in the main text). It also shows an alignment of the capping protein β-tentacle primary sequences from budding yeast (S. cerevisiae), fission yeast (S. pombe), human (Homo sapiens), and chicken (G. gallus). Video 1 shows dynamic puncta of 3xmNeon-tagged Cap2 visible at the cell cortex that is distinct from actin patches. Video 2 is an example time series of cells expressing Cap2–3xmNeon treated with control carrier, CK666 or LatB (analyzed in Fig. 5). Video 3 shows continuous imaging of example cells analyzed in Fig. S3 expressing formins (Bni1 or Bnr1) tagged with GFP or 3xmNeon, revealing that the 3xmNeon tag is brighter and more photostable. Videos 4 and 5 (related to Fig. 5) show that 3xmNeon-tagged Bni1 (Video 4) and Bnr1 (Video 5) colocalize with actin patches (marked by Arc15-RFP^mScarlet) in cap2Δ but not WT cells. Video 6 shows that Arc15-RFP^mScarlet marked actin patches in cap2∆ cells acquire cable-like characteristics as they age and inappropriately recruit tropomyosin (mNeon–Tpm1; related to Fig. 6). Table S1 contains a list of yeast strains used in this study. Table S2 contains a list of primers used and a description of each primer. Table S3 contains a list of plasmids used and a description of each plasmid.