Tpm modulates the Myo1C motility

Table 10 Mechanical parameters for Myo1C on acto•Tpm cofilaments Signal and measured µm²; ; ^a derived from the equation described in chapter 2.2.22; ^b derived from the equation described in chapter 2.2.22 for a single motor and extended by a force term. ^c calculated by using the rate constants obtained from the transient kinetic analysis at 20°C

The kinetic analyses of Tpm isoforms to acto•Myo1C complexes reveal changes in various kinetic parameters as summarised in Table 9. The duty ratio defines the fraction of time that myosin is strongly attached to F-actin during the ATPase cycle and was calculated as previously described in chapter 3.1.3.1. Thus, the calculated duty ratio of Myo1C equals to 0.044 ± 0.002 in the absence of Tpm, 0.043 ± 0.003 in the presence of Tpm1.7 and 0.045 ± 0.003 in the presence of Tpm3.1. The duty ratios show minor changes by Tpm isoforms in the fraction of time that Myo1C spends in actin bound states during the ATPase cycle (Table 10).

I performed in vitro motility assays to elevate the Tpm mediated changes in the rate constants of kcat as well as k_+α, k+2, k+4 and k+5 in the motile activity of Myo1C motor function. The tail-truncated Myo1C construct attaches at the surface via immobilised antibodies as described in chapter 2.2.20. In the presence of ATP, Myo1C displayed continuous motility with actin filaments and acto•Tpm cofilaments. The histogram of a single in vitro motility measurement showed in the presence of Tpm decreasing filament sliding velocities of the complete filament population (Figure 26A). To determine the dependency of the myosin motor density on the filament sliding velocity, I observed the sliding velocity at myosin densities in the range from 180 to 5400 motors per µm² and fitted the data with the equation as described in chapter 2.2.20 (Figure 26B). I found differences for the dependence of the sliding velocity on the myosin surface density in the presence of Tpm.

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Figure 26 Tpm isoform-specific changes of Myo1C motility. (A) Representative histogram of a single in vitro motility measurement shows the Gaussian distribution of the filament sliding velocity with >100 trajectories for Myo1C with acto•Tpm cofilaments at 37°C (B) Sliding velocities of acto•Tpm cofilaments measured with various Myo1C loading concentrations in the range from 0.1 to 3 µM corresponding to myosin densities from 180 to 5400 motors µm^-2. The density of Myo1C for maximal motility was determined in the absence of Tpm (~900 motors µm^-2) and in the presence of Tpm1.7 and Tpm3.1 (~3,600 motors µm^-2). The value for duty ratio was obtained by fitting the data to the equation described in chapter 2.2.20. (C) Box-and-whisker diagram of the average filament sliding velocities of Myo1C with actin filaments (52.1 ± 4.9 nm s^-1) decorated with Tpm1.6 (11.5 ± 2.1 nm s^-1), Tpm1.7 (10.5 ± 1.3 nm s^-1), Tpm1.12 (11.1 ± 2.9 nm s^-1), Tpm2.1 (11.8 ± 2.3 nm s^-1), Tpm3.1 (11.5 ± 1.7 nm s^-1), and Tpm4.2 (10.4 ± 2.1 nm s^-1). Each data point in the box-and-whisker diagram represents the averaged filament sliding velocity of a single in vitro motility measurement with >100 trajectories. (D) Filament sliding velocity against [tropomyosin] (0 to 70 µM), which decorates F-actin, were measured in an in vitro motility assay using Myo1C at 37°C. The parameter Kapp.Tpm was obtained by fitting the data to Boltzmann sigmoidal equation. Kapp.Tpm corresponds to the concentration at the 50 % threshold of the in vitro motility assay K50 %. In the case of acto•Tpm cofilaments, the final concentrations of 10 µM Tpm provide the complete decoration of F-actin with Tpm. Error bars represent standard deviations from at least three determinations of each data point.

Statistical significance was assessed by Student’s paired t test (2-tailed) and is assigned as follows: ns (p ≥ 0.05); * (p < 0.05); ** (p < 0.01); *** (p < 0.001). Lines and symbols are shown in red, orange and blue for No Tpm, Tpm1.7 and Tpm3.1 as well as dark violet, green, purple and cyan for Tpm1.6, Tpm1.12, Tpm2.1 and Tpm4.2, respectively.

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In the absence of Tpm, Myo1C supports the continuous movement of actin filaments with a velocity >52 nm s^-1 above 900 motors per µm². Addition of Myo1C <900 motors per µm² increased inconsistent, non-directional movement and fast detachment of the actin filaments due to the reduced probability of a myosin motor molecule to perform productive interaction with actin filaments. Myo1C in the presence of Tpm supports the continuous movement of acto•Tpm cofilaments with a plateau value of ~11 nm s^-1 only when motor densities of 3,600 motors per µm² or greater are used. The relationship between motor density and sliding velocity was used to yield the duty ratio from the fit of the data. Here, I observed for Myo1C in the absence of Tpm a duty ratio of 0.043 ± 0.005 similar to the calculated duty ratio of the kinetic parameters (Table 10). In contrast, the duty ratios of Myo1C in the presence of acto•Tpm cofilaments compared to actin filaments showed a 4-fold decreased duty ratio to 0.008 ± 0.004 and 0.009 ± 0.004 in the presence of Tpm1.7 and Tpm3.1, respectively, which was not observed for the calculated duty ratio (Table 10). This could be due to differences in the temperature conditions as the transient kinetic analyses were performed at 20°C, whereas in vitro motility assays were performed at 37°C. To test the effect of the temperature, I measured the rate constant of ADP release with acto•Myo1C in the presence of saturating [ADP] at 37°C (Figure 25A). I observed only negligible changes in the absence of Tpm with 7.8 ± 0.1 s^-1 compared to 7.3 ± 0.3 s^-1 and 7.1 ± 0.2 s^-1 in the presence of Tpm1.7 and Tpm3.1, respectively (Table 9), which can partially contribute to the change in the motor duty ratio observed in the in vitro motility measurements. It appears that differences of Myo1C motor activity between actin filaments and acto•Tpm cofilaments increases at higher temperatures.

To analyse the Tpm isoform-specific modulation of motile activity of Myo1C motor function as previously described (Hundt et al., 2016; Pathan-Chhatbar et al., 2018), the physiological relevant cytoplasmic Tpm isoforms Tpm1.6, Tpm1.7, Tpm1.12, Tpm2.1, Tpm3.1 and Tpm4.2 were used to measure the filament sliding velocities by similar myosin motor surface densities (Figure 26C). In the presence of acto•Tpm cofilaments, I measured 4.5- to 5-fold slower averaged filament sliding velocities in the range of 10.4 to 11.5 nm s^-1 compared to 52.1 ± 4.9 nm s^-1 with Tpm-free filaments. Analyses of the Tpm-induced transition of the filament sliding velocity at a Myo1C surface density of 3,600 molecules per µm² were performed by a Tpm titration from 0 to 70 µM. The data of the decrease in filament sliding velocity in dependence of [tropomyosin] were fitted to the Boltzmann sigmoidal equation (Figure 26D). Kapp.Tpm is defined as the Tpm concentration of the 50 % threshold of the filament sliding velocity K50 %. I observed similar values of 1.2 ± 0.2 µM for Tpm1.6, 1.0 ± 0.1 µM for Tpm1.7, 1.3 ± 0.9 µM for Tpm3.1, and 1.5 ± 0.1 µM for Tpm4.2, but was

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fold increased to 4.2 ± 0.4 µM for Tpm2.1 and 15-fold increased to 19.3 ± 1.1 µM for Tpm1.12. The results show Tpm isoform-specific differences in the reduction of Myo1C motor activity in dependence of [tropomyosin]. The observed Tpm isoform-specific changes in the concentration of Tpm to decrease the sliding velocity corresponds to the previously reported affinity differences of Tpm to actin filaments (Gateva et al., 2017; Pathan-Chhatbar et al., 2018). Any used Tpm isoform at fully decorated acto•Tpm cofilaments supports the continuous motility of the filaments by Myo1C in contrast to previous reports of a reduced interaction of Myo1C and other class 1 myosins with acto•Tpm cofilaments accompanied with diffusive non-directional movement in the in vitro motility measurements (Lieto-Trivedi et al., 2007; Kee et al., 2015). It appears that high myosin motor surface densities are required to enables productive interaction between myosin motor and acto•Tpm cofilaments.