Myosin-1C differentially displaces tropomyosin isoforms altering their inhibition of motility

Figures

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  Figure 1. Tpm3.1 inhibition of Myo1C is not limited by motor availability.A, Myo1C actin gliding motility with increasing concentrations of free native- or MAS (acetyl-mimic)- Tpm3.1. Fits, dose-response inhibition model. B, effect of (native-) Tpm3.1 on gliding motility with variable concentrations of surface-attached Myo1C. Solid lines, connect the points. A and B, points, mean and SD from individual trials, n = 50 filaments (unless 0 or indicated next to the point). Open symbols, n ≤5 events. C, steady-state ATPases. (MAS-) Tpm3.1 and actin alone conditions were performed in parallel. Slopes of linear fits give second-order rates. Data are pooled from three trials employing N = 3 preparations of Myo1C (one experiment per point). D, effect of NEM-Sk-S1 (skeletal myosin) on 150 nM Myo1C gliding motility in the absence or presence of (MAS-) Tpm3.1. Solid lines connect the points. Left, total number (n) of motile filaments, right, speeds (mean and SD) from individual trials. E, NEM-Sk-S1 has no effect on (native-) Tpm3.1’s inhibition of 150 nM Myo1C gliding speed. Box and whiskers, median, quartiles, and range. Data are pooled from three days of trials with N = 2 preparations of Myo1C.…

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  Figure 2. Myo1C displaces Tpm3.1.A and B, cosedimentation assays with myosins (in rigor) and 2:1 actin-Tpm3.1. Left, example gel. A, Tpm3.1 sedimentation decreases with increasing Myo1C concentration (5 μM actin, 2.5 μM Tpm3.1). Data are from two trials and two preparations of Myo1C (left and Fig. S3A) Fit, dose-response inhibition model (IC50 = 1.0 ± 0.5 μM). Two additional trials in (Fig. S3D). B, class-II myosins binding in rigor have no effect on Tpm3.1 co-pelleting with actin (3 μM actin, 1.5 μM Tpm3.1). Data are from (left) smooth muscle myosin (SM)-S1 and (Fig. S3B) NEM-Sk-S1 (skeletal myosin). Linear fits have near-zero slopes. A and B, right, quantification of Tpm3.1 pelleting. (S) supernatant, (P) pellet, (M) molecular weight standards, (CaM) calmodulin light chains of Myo1C, (ELC) essential light chain MYL6. C, example kymographs from TIRF motility assays (see Experimental procedures) showing GFP-Tpm3.1, 0.8 μM in solution, associates with NEM-Sk myosin-bound filaments for many minutes but is gradually displaced from Myo1C-bound filaments undergoing motility (37 °C; see also Video S1). D, fluorescence transients, aligned at the start of decay (t = X0), from TIRF motility…

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  Figure 3. Kinetics of GFP-Tpm3.1 dissociation.A, TIRF GFP-Tpm3.1 displacement assays (25 °C) where reaction solutions were exchanged from 0.8 μM GFP-Tpm3.1 to 10 μM unlabeled Tpm3.1 with variable concentrations of free Myo1C (rigor) or NEM-Sk-S1 (C; green). B, averages of filaments (±SD) from each Myo1C concentration (color-coded to the left) fit to exponentials. C, apparent rate constants (log scale) with linear fits giving the second-order rate constant. Data are pooled from three days of trials with n = 10 filaments per condition, N = 2 preparations of Myo1C.

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  Figure 4. Tpm1.7 binds acto-Myo1C and inhibits gliding more than Tpm3.1.A, cosedimentation assays with Myo1C (in rigor) and 2:1 actin-Tpm1.7 show that displacement of Tpm1.7 is 75% less than Tpm3.1 (Fig. 2A). Left, example gel. Right, quantification of Tpm1.7 pelleting. Tpm3.1 fit (blue) taken from Figure 2A. (S) supernatant, (P) pellet, (M) molecular weight standards, (CaM) calmodulin. Data are from two trials and N = 2 preparations of Myo1C (left and Fig. S4C). Slope of linear fit: −3 ± 1 x 10−2 μM−1. An additional trial in Supplemental (Fig. S4D). B, inhibition of Myo1C gliding motility speed by Tpm1.7. Inset, Tpm1.7 inhibits speed over a range of Myo1C concentrations. Points, mean ± SD of each trial, n = 50 filaments, unless labeled. C, Tpm3.1 and Tpm1.7 reduce the number of filaments glided by Myo1C, with Tpm1.7 significantly inhibiting motility more than Tpm3.1. Median speeds: actin alone, 97 nm s−1; Tpm1.7, 15 nm s−1; and Tpm3.1, 27 nm s−1. Data are from N = 3 preparations of Myo1C, with conditions performed side-by-side. Statistics, Brown-Forsythe ANOVA. D, GFP-Tpm1.7 stays bound when pre-mixed 1:4 with actin in Myo1C TIRF gliding assays (final concentrations: 2.5 nM GFP-Tpm1.7, 10…

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