Comparative analysis of widely used methods to remove nonfunctional myosin heads for the in vitro motility assay

Figures

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  Fig. 1. Schematic illustration of the ‘affinity purification’ (left) and ‘blocking actin’ (right) approaches in the IVMA

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  Fig. 2. Effect of affinity purification on actin filament motility in the in vitro motility assay. Fraction of motile filaments (%) on a trimethylchlorosilane (TMCS) and b nitrocellulose surface before and after affinity purification of HMM. Actin sliding speed on c TMCS and d nitrocellulose surface before and after affinity purification of HMM. The insets in a and c show the % mean reduction in velocity upon affinity purification as function of the fraction of motile filaments (data in main Fig. 2a) and sliding velocity (data in main Fig. 2c) before the affinity purification. Negative values represent increase in velocity. Regression lines shown with 95% confidence band. Spearman correlation by rank (due to non-Gaussian residual around line) gives statistically significant positive correlation in both a and c (r = 0.85; p = 0.025). In c 12–30 actin filaments were analyzed for each flow cell in the 7 pairs, before and after affinity purification. More details in Fig. S2 and text. In d 30 actin filaments were analyzed in pairs as in c. The data from 7 different experimental occasions in a and c and 4 different occasions in b and d, using 2 different HMM preparations corresponding to 2…

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  Fig. 3. Effect of blocking actin (1 µM) on actin filament motility in the in vitro motility assay. a Fraction of motile filaments (%) on TMCS and b nitrocellulose surfaces studied in the absence and presence of 1 µM blocking actin. c Actin sliding speed on TMCS and d nitrocellulose surfaces studied in the absence and presence of 1 µM blocking actin. In c 12–30 actin filaments were analyzed in 5 pairs of flow cells either in the absence or presence of blocking actin. In d 30 actin filaments were analyzed for each flow cell. The experiments on 5 different occasions were performed using 2 different HMM preparations (each from a different myosin preparation). The myosin preparations are the same as those in Fig. 2 as indicated by color coding. Temperature was 27.5–28.7 °C on TMCS and 28.5–29.0 °C on nitrocellulose surfaces

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  Fig. 4. Comparison between effects of blocking actin and affinity purification using both procedures in series on TMCS surfaces. a Fraction of motile filaments and b sliding velocity before affinity purification, after affinity purification and after affinity purification and addition of blocking actin (BA). In b 12–30 actin filaments were analyzed for each flow cell using 3 different HMM tubes from 1 HMM preparation. Experiments before and after affinity purification also included in Fig. 2 where it can be seen that the effects of affinity purification are representative. Same HMM tubes also used for analysis of affinity purification procedure by SDS–PAGE (cf. Figs. S1, S2). Temperature, 27.8–28.2 °C

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  Fig. 5. Effect of the presence of MgATP (1 mM) in the HMM (120 µg/ml) incubation solution on actin sliding speed in the in vitro motility assay. a Actin sliding speed on TMCS and b nitrocellulose surface. c Actin sliding speed for individual filaments plotted against actin filament length on TMCS surface and d nitrocellulose surface. The data are for experiments in a and b with large reduction in velocity following HMM incubation in the presence of MgATP. In a 7–30 actin filaments were analyzed for each flow cell out of 6 pairs. In d 30 actin filaments were analyzed for each flow cell. In these experiments, on different occasions, we used 3 different HMM preparations (each from a different myosin preparation). These preparations are those used above as indicated by color coding and one further preparation (blue). Note very similar overall effects of the presence of MgATP during HMM incubation for different HMM preparations despite differences in sliding velocity. The latter difference is attributed to different fraction of dead heads (cf. Fig. 3a, c), ∼ 1 °C lower temperature in experiments with “blue” and “red” preparations (∼ 28 °C) compared to the “black preparation” (∼ 29 °C) and…

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  Fig. 6. Effect of MgATP (1 mM) incubation of surface prior to incubation with HMM for an in vitro motility assay. a Actin sliding speed on TMCS and b nitrocellulose surfaces. In a 10–30 actin filaments were analyzed for each flow cell in a pair. Two different HMM preparations (2 Myosin preparations) were used. Color coding as in Fig. 5. In b 30 actin filaments were analyzed for each flow cell using 2 different HMM tubes from 1 HMM preparation. Temperature was 27.8–29.2 °C on TMCS and 28.6–29.0 °C on nitrocellulose surfaces. (Color figure online)

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  Fig. 7. Effect of the presence of MgATP (1 mM) in the S1 (120 µg/ml) incubation solution on fraction of motile filaments and actin sliding speed in the in vitro motility assay on TMCS surface. a Fraction of motile filaments and b Sliding velocity. In b 15–30 actin filaments were analyzed for each flow cell using 2 different S1 preparations (2 different myosin preparations). Temperature was 27.9–28.4 °C

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  Fig. 8. Model for adsorption of heavy meromyosin (HMM) and subfragment 1 (S1) to surfaces. a Schematic illustration of HMM with two motor domains (S1; heads) to the right and the coiled–coiled subfragment 2 (S2) domain to the left. Green circles indicate most likely sites for adsorption to a TMCS surface. The experimental and theoretical basis for the model with high probability of adsorption via the C-terminal tail (chymotrypsin cleavage site; large circle) and lower probability via the actin-binding region (small circle) is given in detail previously (Albet-Torres et al. 2007; Månsson et al. 2008; Persson et al. 2010; Månsson 2012; Lindberg et al. 2018). Question marks indicate the possibility of further interaction sites between surfaces and HMM. b Schematic illustration of S1 domain assuming greatest probability of adsorption via the papain cleavage site at the C-terminal end of S1. c At high HMM incubation concentration, the HMM molecules are most likely to approach the surface either via the C-terminal tail domain or the head domain (Månsson et al. 2008). If the surface approach leads to adsorption or not depends on the binding probability at the approaching end of the molecule.…