Fig. 4: The p.R189W variant weakens TMOD1’s ability to cap the pointed ends of actin filaments.

a Representative curves of pyrene-actin fluorescence (A.U.: arbitrary units) from pointed ends of gelsolin-capped actin filaments (1:10 gelsolin-to-actin molar ratio) over time (min) in the presence of 0.8 μM TMOD1^wt or TMOD1^R189W (control: spontaneous actin pointed end polymerization) and in the absence of TPM1 (−TPM1). b Concentration-dependent inhibition of actin polymerization in the presence of TMOD1^wt or TMOD1^R189W (p-value = 0.0299, 0.0480, 0.0146, 0.8498, respectively). c Representative curves of pyrene-actin fluorescence (A.U.) from pointed ends of gelsolin-capped actin filaments (1:100 gelsolin-to-actin molar ratio) over time (min) in the presence of TPM1 (+TPM1), with 25 nM TMOD1^wt or TMOD1^R189W. d Concentration-dependent inhibition of actin polymerization in the presence of TPM1 and TMOD1^wt or TMOD1^R189W (p-value = 0.0153, 0.0474, 0.0485, 0.0161, 0.9125, respectively). e Representative curves of decrease in pyrene-actin fluorescence (A.U.) resulting from depolymerization from the pointed ends of gelsolin-capped actin filaments (1:10 gelsolin-to-actin molar ratio) over time (min) in the presence of 0.4 μM TMOD1^wt or TMOD1^R189W (control: spontaneous actin pointed end depolymerization), in the absence of TPM1 (−TPM1). f Concentration-dependent inhibition of actin depolymerization in the presence of TMOD1^wt or TMOD1^R189W (p-value = 0.0329, 0.0165, 0.0279, 0.0125, respectively). g Representative curves of decrease in pyrene-actin fluorescence (A.U.) resulting from depolymerization from the pointed ends of gelsolin-capped actin filaments (1:100 gelsolin-to-actin molar ratio) over time (min) in the presence of TPM1 (+TPM1), with 100 nM TMOD1^wt or TMOD1^R189W. h Concentration-dependent inhibition of actin depolymerization in the presence of TPM1 (+TPM1) and TMOD1^wt or TMOD1^R189W (p-value = 0.0411, 0.0104, 0.0237, 0.0056, 0.4570, respectively). Actin polymerization or depolymerization rates relative to the control (R_exp/R_control) were calculated as the first derivatives at time zero after exponential fit. Data shown are mean ± standard deviation (n = 3 independent experiments, *p < 0.05, **p < 0.01, Student’s t-test). Control, TMOD1^wt and TMOD1^R189W are shown in white, black and red circles, respectively. i Immunofluorescence images of neonatal rat cardiomyocytes fixed and stained with Texas Red-conjugated phalloidin to mark F-actin and an anti-α-actinin antibody to indicate the Z-disc (“Z”) after being transfected with GFP (control), GFP-TMOD1^wt or GFP-TMOD1^R189W (Scale bar = 2 µm). Magenta bars show representative thin filament arrays (two bundles of thin filaments extending into opposite half-sarcomeres toward the M line [“M”]) that length measurements were taken from. j Thin filament length measurements collected from F-actin images of transfected cells (GFP = 0.770 ± 0.003 µm; GFP-TMOD1^wt = 0.729 ± 0.003 µm; GFP-TMOD1^R189W = 0.748 ± 0.003 µm). Data shown are mean ± standard error of the mean (n = 3 independent cultures, 11 cells per culture, 5 measurements per cell, 3 sarcomeres per measurement for a total of 141, 162 or 133 independent measurements, ****p < 0.0001, One-way ANOVA). GFP, GFP-TMOD1^wt and GFP-TMOD1^R189W are shown in white, light gray and dark gray bars, respectively.