Mechanochemical properties of human myosin-1C are modulated by isoform-specific differences in the N-terminal extension

Figures

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  Figure 1. Schematic representation of the domain structure of human myosin-1C isoforms and of the constructs used in this study. A, domain structure of myosin-1C consisting of a generic myosin motor domain, 3 IQ repeats, a post-IQ domain, and a TH1 domain corresponding to an extended Pleckstrin homology (PH) domain. IQ1 and IQ2 are generic calmodulin-binding motifs. IQ3 and the post-IQ domain together bind a third calmodulin in an unconventional manner, thereby creating a long rigid lever arm region connecting the converter region in the motor domain with the extended PH domain. B, sequence alignment showing isoform-specific differences in the N-terminal amino acid sequences of the human myosin-1C splice isoforms. C, schematic representation of the myosin-1C constructs used in this study.

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  Figure 2. Isoform-specific differences in actin-activated Mg2+-ATPase activity. A, steady-state actin-activated ATPase activity was measured at 37 °C with actin concentrations in the range from 0 to 50 μM. Error bars represent standard deviations from at least three measurements of each data point. B, global fitting simulations of the ATP turnover of Myo1C-ΔTH1 isoforms in the presence of 0 to 300 μM actin and at 20 °C. The parameters kcat, Kapp.actin, and kcat/Kapp.actin were obtained by fitting the data to the Michaelis–Menten equation. K0.5 defines Kapp.actin, plateau values define kcat, and kcat/Kapp.actin is defined by the initial slope of the fit curve at concentrations of actin much lower than Kapp.actin. Results are summarized in Table 1.

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  Figure 3. Minimal kinetic reaction scheme of the acto·myosin-1C ATPase cycle. The main pathway of myosin-1C is highlighted in blue and orange indicating Myo1C in the actin-bound and actin-unbound states, respectively. ‘A’ refers to actin, ‘M’ to myosin-1C, ‘T’ to ATP, and ‘D’ to ADP; subscript A refers to actin (KA); M' refers to closed state; rate constants are written as k+ for the forward and k− for the backward reaction.

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  Figure 4. ATP-induced dissociation of pyrene-labeled acto·Myo1C0-ΔTH1 and acto·Myo1C35-ΔTH1.A, pyrene fluorescence transients observed upon mixing 130 nM acto·Myo1C-ΔTH1 isoforms with 0.03 or 3 mM ATP. All concentrations are given as final concentrations after rapid mixing. The averaged fluorescence transients are best described by double exponentials. B, ratio of slow to fast phase amplitudes (Aslow/Afast) plotted against the ATP concentration. The data were fitted to a hyperbola in each case. The respective plateau values define the equilibrium constants for isomerization of the nucleotide-binding pocket Kα. C, the dependence of kobs,slow on ATP concentration was best fitted with a hyperbola in each case. The plateau values define k+α, the first-order rate constant for the closed-to-open isomerization of the nucleotide-binding pocket of Myo1C0-ΔTH1 and Myo1C35-ΔTH1. D, Similarly, the dependence of kobs,fast on ATP concentration is well described by hyperbolas for both isoforms. The best fits to kobs,fast = K1k+2[ATP]/(1 + K1[ATP]) are superimposed. The plateau values define near-identical values for k+2. The ATP concentration required for half-maximal saturation defines (1/K1). Here, we…

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  Figure 5. Interaction of Myo1C0-ΔTH1 and Myo1C35-ΔTH1 with the hydrolysis products ADP and Piin the presence of pyrene-labeled actin. A, inhibition of the ATP-induced dissociation of pyrene-acto·Myo1C by ADP. The observed increases in fluorescence are best described by double exponentials yielding values for kobs,slow, kobs,fast, Aslow, and Afast. B, the dependence of kobs,slow on ADP concentration was best fitted with a hyperbola. The apparent affinities for ADP correspond to 0.21 ± 0.06 μM and 0.10 ± 0.03 μM for acto·Myo1C0 and acto·Myo1C35. The minimum observed rates in the presence of saturating ADP concentrations define the rate constant for ADP dissociation (k+5). C, plot of the fraction of Aslow against [ADP]. The data were fitted to a hyperbola with Aslow/Atotal = [ADP]/(K5 + [ADP]). D, Pi release from acto·Myo1C was followed in a double mixing experiment with 1.8 μM of the phosphate sensor MDCC-PBP and 5 μM F-actin after mixing. The averaged fluorescence transients are best described by single exponentials yielding an apparent rate constant for phosphate release. Fitted parameters are summarized in Table 1. All concentrations are given as final concentrations after rapid mixing.…

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  Figure 6. Actin binding and release in the absence of nucleotides. Binding of the Myo1C-ΔTH1 constructs to pyrene-labeled F-actin quenches the pyrene fluorescence. The transients obtained upon mixing a Myo1C-ΔTH1 construct with pyrene-labeled actin in a stopped-flow spectrophotometer follow a mono-exponential. A, the observed rate constants show a linear dependence on the concentration of pyrene-labeled actin in the range from 0.25 to 3.0 μM. The second-order rate constants for binding to actin (k+A) is derived from the slopes. B, fluorescence transients observed after chasing pyrene-labeld actin from the pyrene–actomyosin complex with excess F-actin. The observed processes could be fit to single exponentials where kobs corresponds directly to the rates of actin dissociation (k-A). The fitted parameters are summarized in Table 1. All concentrations are given as final concentrations after rapid mixing. Error bars in panel A represent standard deviations from at least three determinations of each data point; the experimental curves in panel B correspond to the averaged signals from four independent measurements. Lines and symbols are shown in red and blue for Myo1C0-ΔTH1 and Myo1C35-ΔTH1,…

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  Figure 7. Isoform-specific changes of myosin-1C motor activity.A, schematic illustration of the flow cell coated with a supported lipid bilayer containing PtdIns(4,5)P2 (SLP). The SLP is spread on a glass coverslip, where it facilitates attachment of Myo1C-FL via the extended PH domain (blue). Motor domains (green) are optimally oriented for productive interactions with TRITC-phalloidin–labelled actin filaments (yellow). Following the addition of ATP, the actin filaments move in a unidirectional fashion. B, Box-and-whisker diagram of averaged filament sliding velocities for Myo1C0-FL, Myo1C35-FL, and Myo1C16-FL. C, Box-and-whisker diagram of the average filament sliding velocities of Myo1C0-ΔTH1 and Myo1C35-ΔTH1 are shown on the left side of the panel. Results obtained for Myo1C0-ΔTH1 in the presence of peptides NTE35, NTE16, and NTE35R21G are shown on the right side of the panel. Each data point in the box-and-whisker diagram represents the averaged filament sliding velocity determined using an independent flow cell and analyzing more than 100 unidirectional trajectories. D, Myosin-1C isoform-specific changes of actin filament sliding velocity measured at various myosin-1C surface…

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  Figure 8. Differences between Myo1C0-ΔTH1 and Myo1C35-ΔTH1 in force generation and power output. The mechanochemical behavior of Myo1C0 and Myo1C35 was compared using a frictional loading assay (29). A, the filament sliding velocity of F-actin driven by Myo1C-ΔTH1 is reduced by the addition of α-actinin, generating a resistive force that increases with increasing α-actinin concentrations. The fit curves shown were obtained using the equationy = A2+(A1−A2)/(1+e((x−x0)/dx)). The values obtained for the midpoint of the transition and the steepness of the transition correspond to 25 ± 8 nM and 18 ± 4 nM for Myo1C0-ΔTH1 and 24 ± 11 nM and 15 ± 7 nM for Myo1C35-ΔTH1. B, dependence of the observed filament sliding velocity on resistive force. To relate α-actinin concentration to resistive force per filament, we used Equation 1. The fit curves shown were obtained using Equation 2 (21, 28). The values for the force-independent rate (ki) and the force-dependent rate in the absence of external loads (kf0) shown in Table 1 were obtained by iterative fitting cycles. C, plot of power output against the resistive force. Based on the result shown in panel B and on the reported stall force for a single…

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  Figure 9. Model describing the biphasic ATP-induced detachment of acto•myosin-1C. The model defines the detachment of acto•myosin-1C as the result of a force-independent step and a force-dependent step, but the order of the steps cannot be distinguished by the model alone.

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  Figure 10. Comparison of kinetic and functional properties of myosin-1 isoforms. The graph shows the values obtained for myosin-1 C35 relative to those obtained for myosin-1 C0; n-fold larger values are positive, and n-fold smaller values are negative. The results are grouped in parameters determined using (i) steady-state ATPase measurements, (ii) stopped-flow measurements, and (iii) in vitro motility assays. All parameters were measured under zero-load conditions, with the exception of Pmax and FPmax. The dashed lines represent 30% deviation from 1.0.