Domain structure of human FHOD3.

GBD=GTPase Binding Domian, DID=Diaphanous Inhibitory Domain, CC=Coiled-coil (putative), FH=Formin Homology, DAD=Diaphanous Autoregulatory Domain. Mutations tested in this study are indicated with black arrows. Numbers correspond to FHOD3L sequence (Uniprot Isoform 4). The FHOD3L-CT construct spans residues 963–1622, including the FH1 domain, FH2 domain, and tail. The FHOD3S-CT construct spans residues 771-1422 (Uniprot Isoform 1 numbering). FHOD3L-specific exons are in gray. The 8-residue exon (T(D/E)5XE) that distinguishes FHOD3L-CT from FHOD3S-CT is circled in red.

Coomassie-stained polyacrylamide gel of purified FHOD3 constructs.

Lower molecular weight contaminants were difficult to remove, were relatively consistent for each construct, and had no detectable impact on FHOD activity.

Biochemical characterization of human FHOD3L.

(A) Assembly of 4 µM actin (5% pyrene-labeled) and the indicated concentrations of FHOD3L-CT from a 2-fold dilution series. (Higher concentrations of FHOD3L-CT are darker shades of blue. Symbols reflect the highest, middle and lowest concentrations tested. In this case, circle = 60 nM, square = 15 nM, and triangle = 3.75 nM FHOD3LT-CT.) The inset shows the first 200 seconds of data normalized to the plateau of actin alone. (B) Relative nucleation activities of FHOD3L-CT (n=5), FHOD3S-CT (n=4), FHOD3L-CT K1309A (n=3) and I1163A (n=3). Nucleation strength is described by the rate of assembly (i.e., slopes from traces like those in (A)), at an early timepoint, when nucleation dominates (t1/8), as a function of formin added. Data points are means and error bars are standard deviations. Slopes reported are the average slopes of independent experiments. The asterisks indicate significance of difference from FHOD3L-CT (see below for details). (C) Barbed-end elongation assay. Final conditions were 0.25 µM F-actin seeds (∼0.1 nM barbed ends), 0.5 µM actin (10% pyrene-labeled), and indicated concentrations FHOD3L-CT. (D) Quantification of barbed-end affinity for FHOD3L-CT (from C), FHOD3S-CT (from Figure 2—figure supplement 2B) and FHOD3L-CT I1163A (see Figure 2, supplement 1C for extended axes). Raw data are shown and lines are fist to all data points. The Kds reported are the averages of three independent trials. (n=3, each; mean ± SD.) (E) Barbed-end elongation assay with profilin. Final conditions as in (C) plus 1.5 µM S. pombe profilin. (F) Kymograph of a growing filament from a TIRF assay with FHOD3L-CT. Conditions: 1 µM actin (10%-Alexa Fluor 488-labeled), 5 µM Hs profilin-1, and 0.1 nM FHOD3L-CT. The green lines indicate bright regions of growing filament. The yellow lines represent dim regions. The orange line is a pause. Rates calculated for each region are reported as subunits/s. (G) Kymograph of a growing filament from a TIRF assay without added FHOD3L-CT. Conditions: 1 µM actin (10%-Alexa Fluor 488-labeled), 5 µM Hs profilin-1. The green lines indicate bright regions of growing filament. The red lines represent dim regions. Rates calculated for each region are reported as subunits/s. (H) Elongation rates from TIRF assays. Average elongation rates (10-100s of seconds) and elongation rates from brief (1-10 seconds) dim regions are shown separately. (n=21, profilin-actin (avg); n=27 profilin-actin (dim); n=20, FHOD3L-CT (avg); n=112, FHOD3L-CT (dim); 3 channels for all samples; mean ± SD.) (I) Coomassie-stained polyacrylamide gel of pellet fractions from low-speed bundling assays with 5 µM actin and 0-60 nM FHOD3L-CT. (J) Quantification of bundling from (I) via densitometry (n=3 each group; mean ± SD.) * p < 0.05, ** p < 0.001, *** p < 0.0001. p-values were determined by one-way ANOVA with post-hoc Tukey test.

(A) Actin polymerized in the absence or presence of 12 nM FHOD3L-CT, stabilized with fluorescent phalloidin diluted to 5 nM. (n=3, each; mean ± SD) Scale bars, 10 µm. (B) Pyrene fluorescence readings of 4 µM actin filaments (5% pyrene-labeled) incubated with different concentrations of FHOD3L-CT I1163A for 5 minutes. (n=2; mean ± SD.) (C) Quantification of barbed-end affinity (from Figure 2C,D) with axes extended. (D) Time-lapse images from TIRF microscopy. Conditions: 1 µM actin (10%-Alexa Fluor 488-labeled), 5 µM Hs profilin-1 ± 0.1 nM FHOD3L-CT. Yellow arrows indicate beginning and end of dim portion of filament elongated by FHOD3L-CT. 2.5 s / frame. Scale bar, 10 µm. (E) Quantification of (dim) run lengths for FHOD3L-CT TIRF assays. (F) Duration of pauses observed before bursts in TIRF assays. (For (E-F) n=112, FHOD3L-CT (dim); 3 flow channels for all samples; mean ± SD.) (G) Comparison of dim event frequencies as a function of filament length. (H) Epifluorescence micrographs of phalloidin-stabilized filaments from low-speed bundling assays. Conditions: 5 µM actin without or with 15 nM FHOD3L-CT, diluted to 5 nM actin after centrifugation. Scale bars, 10 µm.

Comparison of FHOD3S to FHOD3L.

(A) Assembly of 4 µM actin (5% pyrene-labeled) and the indicated concentrations of FHOD3S from a 2-fold dilution series. (Higher concentrations of FHOD3L-CT are darker shades of blue. Symbol reflect the highest, middle and lowest concentration tested. In this case, circle = 48 nM, square = 12 nM, and triangle = 3 nM FHOD3LT-CT.) (B) Barbed-end elongation assay. Final conditions: 0.25 µM F-actin seeds (∼0.1 nM barbed ends), 0.5 µM G-actin (10% pyrene-labeled), and 312.5 pM-5 nM FHOD3S-CT. (C) Kymograph of a growing filament from a TIRF assay with FHOD3S-CT. Conditions: 1 µM actin (10%-Alexa Fluor 488-labeled), 5 µM Hs profilin-1, and 1 nM FHOD3S-CT. The green lines indicate bright regions of growing filament. The yellow lines represent dim regions. The orange lines are pauses. Rates calculated for each region are reported as subunits/s. (D) Elongation rates from TIRF assays. Average elongation rates (over 10s of seconds) and formin-mediated elongation rates (dim) are shown separately. Conditions: 1 µM actin (10%-Alexa Fluor 488-labeled), 5 µM Hs profilin-1 ± 0.1 nM FHOD3L-CT or 1 nM FHOD3S-CT. (n=21, profilin-actin; n=20, 3L (avg); n=24, 3S (avg), n=112, 3L (dim), n=78, 3S (dim); 4 flow channels for 3S, 3 flow channels for all others; mean ± SD, p-values by one-way ANOVA with post-hoc Tukey test.) (E) Comparison of dim event frequencies as a function of filament length. (F) Quantification of (dim) run lengths for FHOD3S/L-CT TIRF assays. (n=97, FHOD3L-CT; n=73, FHOD3S-CT; 3 flow channels for 3L, 4 flow channels for 3S; mean ± SD, p-values by Mann-Whitney U test.) (G) Duration of capping events, i.e. pauses before bursts, by FHOD3L-CT vs FHOD3S-CT observed by TIRF microscopy. (n=97, FHOD3L-CT; n=73, FHOD3S-CT; 3 flow channels for 3L, 4 flow channels for 3S; mean ± SD, p>0.05 by Mann-Whitney U test.) (H) Coomassie-stained polyacrylamide gel of pellet fractions from low-speed bundling assays with 5 µM actin and 0-60 nM FHOD3S-CT. (I) Comparison of bundling for FHOD3S/L-CT via densitometry (n=3 each group; mean ± SD.) * p < 0.05, *** p < 0.0001

Summary of biochemical measurements for FHOD3L-CT and mutants.

All data shown are means ± standard deviation each from at least 3 independent experiments. n.d.= no data. Light grey columns are data acquired with bulk pyrene-actin based assays without profilin added. Medium grey columns provide data from TIRF analysis of individual filaments (profilin was present). The dark grey column report data from cosedimentation assay.

* Statistically different from FHOD3L-CT. Analysis by ANOVA and Tukey post hoc tests, p < 0.05. More details are in the figure legends.

Biochemical validation of function-separating mutants.

(A) Relative nucleation activity for FHOD3L-CT (n=5) and FHOD3L-CT K1193L (n=5). Data points are means and error bars are standard deviations. Slopes reported are the average slopes of independent experiments. (B) Barbed-end affinity measurements for FHOD3L-CT, FHOD3L-CT GS-FH1, and FHOD3L-CT K1193L. Raw data are shown and the line is a fit to all data points. The Kds reported are the average of three independent trials. (n=3, each; mean ± SD.) (C) Elongation rates from TIRF assays. Conditions: 1 µM actin (10%-Alexa Fluor 488-labeled), 5 µM Hs profilin-1 ± 0.1 nM FHOD3L-CT or 1 nM FHOD3L-CT K1193L. Average elongation rates (over 10-100s of seconds) and formin-mediated elongation rates (dim) are shown separately. (n=19, FHOD3L-CT K1193L (avg); n=67, FHOD3L-CT K1193L (dim); 3 flow channels; mean ± SD, p-values by one-way ANOVA with post-hoc Tukey test.) (D) Quantification of bundling by FHOD3L-CT GS-FH1 and K1193L (n=3, each; mean ± SD.) (E) Nucleation test. Quantification of number of filaments per field of view (FOV) for FHOD3L-CT and FHOD3L-CT GS-FH1. Conditions: 4 µM actin with indicated construct. Reaction was diluted in Alexa488-phalloidin to 5 nM actin for visualization. 5 images were taken per independent experiment. (n=15 images, each; 3 biological replicates, each; mean ± SD, p-values by one-way ANOVA with post-hoc Tukey test.) Representative images are shown in Figure 3—figure supplement 2B. (F) Barbed-end elongation assay for FHOD3L-CT GS-FH1 in the presence of profilin. (Higher concentrations of FHOD3L-CT are darker shades of blue. Symbols highlight the highest, middle and lowest concentrations tested. In this case, circle = 20 nM, square = 5 nM, and triangle = 1.25 nM FHOD3LT-CT.) Final conditions: 0.25 µM F-actin seeds (∼0.1 nM barbed ends), 0.5 µM actin (10% pyrene-labeled), 1.5 µM S. pombe profilin, and 1.25 nM-20 nM FHOD3L-CT GS-FH1. (n=2; mean ± SD.) ** p < 0.001, *** p < 0.0001

(A) Kymograph of a growing filament from a TIRF assay with FHOD3L-CT K1193L. Conditions: 1 µM actin (10%-Alexa Fluor 488-labeled), 5 µM Hs profilin-1, and 1 nM FHOD3L-CT K1193L. The green lines indicate bright regions of growing filament. The yellow lines represent dim regions. The orange line is a pause. Rates calculated for each region are given as subunits/s. (B) Quantification of run lengths for FHOD3L-CT and FHOD3L-CT K1193L. (p>0.05 by Mann-Whitney U test.) (C) Duration of capping events by FHOD3L-CT and FHOD3L-CT K1193L observed by TIRF microscopy. (p>0.05 by Mann-Whitney U test.)

(A) TIRF micrographs from nucleation assays using 4 µM actin diluted to 5 nM actin in the presence or absence of 46 nM FHOD3L-CT GS-FH1. Aggregates are apparent in the presence of FHOD3L CT GS-FH1. (B) TIRF micrographs from nucleation assays using 4 µM actin and indicated concentration of FHOD3 construct. Samples were diluted to 5 nM actin in Alexa488 phalloidin for visualization. The number of aggregates was negligible at lower concentrations of FHOD3L-CT GS-FH1. Quantification shown in Figure 3E.

FHOD3L rescues sarcomere organization in neonatal rat ventricular myocytes.

(A) Overview of the rescue-experiment protocol. Reverse transfection of siRNA upon plating NRVMs followed by infection with adenovirus to drive exogenous expression. (B) Western blot showing depletion of endogenous FHOD3 after knockdown, and exogenous FHOD3L expression levels after rescue. GAPDH used as a loading control. (C) Quantification of Western blot in (B) normalized to GAPDH for each lane and then normalized to endogenous levels in the mock knockdown. (n=3, each; mean ± SD.) (D) Sarcomere integrity indicated by immunofluorescent staining of α-actinin (green). Localization of exogenous HA-FHOD3L is shown in magenta. DAPI (blue) is included in the merged images. WGA is not shown for clarity. (E) Quantification of sarcomere number per NRVM. (n=160 cells, mock KD; n=334 cells, mock rescue; n=110 cells, FHOD3L rescue.) (F) Average sarcomere lengths per NRVM. (n=100 cells, mock KD; n=71 cells, mock rescue; n=92 cells, FHOD3L rescue). (G) Average sarcomere widths (Z-line lengths) per NRVM. (n=100 cells, mock KD; n=71 cells, mock rescue; n=92 cells, FHOD3L rescue) (H) Epifluorescent micrographs showing mock knockdown and FHOD3L-rescued NRVMs stained with phalloidin (green) to visualize thin filaments and anti-HA (magenta) to show expression of exogenous FHOD3L. (I) Quantification of thin filament lengths for mock knockdown and FHOD3L-rescued NRVMs. (n=94 cells, mock KD; n=84 cells, WT rescue) For (E,F,G,I) Data from three biological replicates for each condition are represented by different shades. Mean ± SD is shown. p-values were calculated with Mann-Whitney U tests, * p < 0.05; ** p < 0.001.).

Method establishment for FHOD3L rescues in neonatal rat ventricular myocytes.

(A) Relative mRNA expression of FHOD3 in NRVMs after knockdown with two different siRNA oligos compared to GAPDH mRNA expression (n=2 wells, 1 biological replicate; mean). (B) Typical image of CellPose segmentation of wild-type FHOD3L-rescued NRVMs, used for per cell quantification of sarcomeres, overlaid with the α-actinin channel. Regions of interest (ROIs) are numbered. Scale bar, 20 µm (C) Pairwise correlation of normalized DsRed vs. HA fluorescence intensity from FHOD3L rescue, plotted per NRVM area. Correlation is too weak to use DsRed as an expression level reporter (n=347 cells; 1 biological replicate). (D) Normalized and background corrected 3xHA-FHOD3L fluorescence intensity per NRVM area from rescue experiments. Magenta line indicates the upper expression level cutoff (2700 a.u./µm2) used to select cells for further analysis (n=913 cells; 3 biological replicates; mean ± SD.) (E) Normalized and background corrected 3xHA-FHOD3L fluorescence intensity per NRVM area from the same three replicate as in (D). These cells were randomly selected for further analysis. (F) Pairwise correlation of HA fluorescence intensity vs. sarcomere number, sarcomere length, or sarcomere width from FHOD3L rescue, plotted per NRVM area. No correlation is detected. (Dashed line shows trend when cells lacking sarcomeres (red) are excluded. Solid line shows trend for all cells analyzed.)

Summary of FHOD3L and mutant rescue experiments in NRVMs.

All data shown are means ± standard deviation each from 3 independent experiments (except for GS-FH1 rescue: maximal contraction and relaxation velocity measurements and average % rhythmic contractions from 2 independent experiments due to one biological replicate not contracting.) n.d.= no data. In the lightest columns, “-” indicates treatment with negative control siRNA or empty virus, “+” indicates treatment with FHOD3 siRNA or corresponding FHOD3L adenovirus. Data in the medium-colored columns were acquired from fixed and stained cells. Data in the darkest columns were acquired from live cells. Statistical analyses are described in figure legends.

a-statistically different from mock KD (mock rescue and Fhod3L rescue)

b-statistically different from mock rescue (FHOD3L and GS-FH1)

c- statistically different from FHOD3L rescue (all mutant rescues and overexpression)

FHOD3L rescues contractility in neonatal rat ventricular myocytes.

(A-C) Motion analysis by digital image correlation. Mock knockdown, mock rescue, and FHOD3L rescue beating patterns are shown. The first (•) and the second (▴) peak of each duplex represent the contraction and the relaxation, respectively. (D) Maximal contraction velocities quantified for mock knockdown, mock rescue, and FHOD3L wild-type rescue (n=82 ROIs, mock KD, n=81 ROIs, mock rescue, n=72, ROIs, FHOD3L rescue; 3 biological replicates, each; mean ± SD, p-values by Student’s two-sample, unpaired t-test.) (E) Quantification of the percentage of analyzed ROI’s from the videos that contained rhythmic contractions for mock knockdown, mock rescue, and FHOD3L wild-type rescue. (n=3, each; mean ± SD.) ** p < 0.001.

(A) Maximal relaxation velocities quantified for mock knockdown, mock rescue, and FHOD3L rescue (n=82 ROIs, mock KD; n=81 ROIs, mock rescue; n=72, ROIs, FHOD3L rescue; 3 biological replicates, each; mean ± SD, p-values by Student’s two-sample, unpaired t-test.) (B) Estimate of the percentage of NRVMs contracting in each video for mock knockdown, mock rescue, and FHOD3L rescue. (n=3, each; mean ± SD.) * p < 0.05, ** p < 0.001, *** p< 0.0001

(A) Comparison of 3xHA-FHOD3L fluorescence intensity per NRVM area from rescue experiments and overexpression experiments. Data are normalized and background corrected as in Fig. 4-supplemental figure 1D. (B) Maximal contraction velocities quantified for mock knockdown and FHOD3L overexpression NRVMs (n=82 ROIs, mock KD; n=79 ROIs, FHOD3L overexpression; 3 biological replicates, each; mean ± SD, p-value by Student’s two-sample, unpaired t-test.) (C) Maximal relaxation velocities quantified for mock knockdown and FHOD3L overexpression NRVMs (n=82 ROIs, mock KD, n=79 ROIs; FHOD3L overexpression; 3 biological replicates, each; mean ± SD, p-value by Student’s two-sample, unpaired t-test.) (D) Quantification of rhythmic contractions as in Fig. 5E for mock knockdown and overexpression NRVMs (n=3, each; mean ± SD.) (E) Estimate of the percentage of NRVMs contracting in each video for mock knockdown and overexpression NRVMs (n=3, each; mean ± SD.) * p < 0.05, ** p < 0.001, *** p< 0.0001

Loss of nucleation but not elongation is tolerated for sarcomere formation and function.

(A) Images of NRVMs rescued with K1193L or GS-FH1. Sarcomere integrity indicated by immunofluorescent staining of α-actinin (green). Localization of exogenous HA-FHOD3L is shown in magenta. DAPI (blue) is included in the merged images. WGA is not shown, for clarity. (B) Quantification of sarcomere number per NRVM in the FHOD3L, K1193L, and GS-FH1 rescues. (n=148 cells, K1193L; n=259 cells GS-FH1; 3 biological replicates; mean ± SD, p-values by Mann-Whitney-U test.) (C) Average sarcomere lengths per NRVM in the FHOD3L, K1193L, and GS-FH1-rescues. (n=95 cells, K1193L; n=73 cells, GS-FH1; 3 biological replicates; mean ± SD, p-value for FHOD3L comparison to GS-FH1 by Student’s two-sample, unpaired t-test, all other p-values by Mann-Whitney-U test.) (D) Average sarcomere widths (Z-line lengths) per NRVM in the FHOD3L, K1193L, and GS-FH1-rescues. (n=95 cells, K1193L; n=73 cells, GS-FH1; 3 biological replicates; mean ± SD, p-values by Mann-Whitney-U test.) (E) Quantification of thin filament lengths for FHOD3L and K1193L-rescued NRVMs (n=99 cells, K1193L; 3 biological replicates, each; mean ± SD, p-value by Mann-Whitney-U test.) (G) Quantification of contracting NRVMs, as in Fig. 5D, for FHOD3L, K1193L, and GS-FH1-rescued NRVMs. (n=3, each; mean ± SD.) (H) Quantification of rhythmic contractions, as in Fig. 5E, for FHOD3L, K1193L, and GS-FH1-rescued NRVMs. (n=3, FHOD3L and K1193L; n=2, GS-FH1; mean ± SD.) ** p < 0.001

(A) Normalized and background-corrected 3xHA-FHOD3L fluorescence intensity per NRVM area from the FHOD3L, K1193L, and GS-FH1 rescues. (n=897 cells, K1193L, n=536 cells, GS-FH1; 3 biological replicates, each; mean ± SD.) (B) Fluorescence distributions of the cells randomly selected for further analysis. (C-E) Analysis of correlation between HA intensity per cell and sarcomere number, length, and width for indicated rescue constructs.

(A) Epifluorescent micrographs showing K1193L-rescued NRVMs stained with phalloidin (green) to visualize thin filaments and anti-HA (magenta) to show expression of exogenous FHOD3L. (A’) 3x zoom of indicated region in phalloidin image. (B) Phalloidin staining (green) reveals that cardiomyocytes expression GS-FH1 have actin puncta that may be aligned. Stress fibers from a fibroblast are visible in the upper right-hand corner of the image. HA-FHOD3L (magenta) shows diffuse GS-FH1 protein. (B’) 3x zoom of indicated region in phalloidin image. (C) Striated HA-FHOD3L demonstrates that the GS-FH1 mutant can localize correctly if sarcomeres are present. Sarcomere integrity indicated by immunofluorescent staining of α-actinin (green). Localization of exogenous HA-FHOD3L is shown in magenta. DAPI (blue) is included in the merged images. WGA is not shown for clarity. (D) Maximal relaxation velocities for FHOD3L, K1193L, and GS-FH1-rescued NRVMs. (n=81 ROIs, K1193L; n=31 ROIs, GS-FH1; 3 biological replicates, WT and K1193L; 2 for GS-FH1; mean ± SD, p-values from Mann-Whitney U test.) (E) Estimate of the percentage of NRVMs contracting in each video for FHOD3L, K1193L, and GS-FH1-rescued NRVMs. (n=81 ROIs, K1193L; n=31 ROIs, GS-FH1; 3 biological replicates, WT and K1193L; 2 for GS-FH1; mean ± SD; p-values determined with Mann-Whitney-U test.) ** p < 0.001

(A) HA intensity distributions from full complement of FHOD3L (WT) rescue experiments compared to those with HA intensity < 720 a.u./μm2, those from the first biological replicate only, and with the GS-FH1 rescue data set. (B) Comparison of sarcomere metrics for the four indicated distributions. astatistically different from FHOD3L rescue (C) Analysis of correlation between HA intensity per cell and sarcomere number, length, and width for the three indicated low intensity populations.