The Antiparallel Coiled-Coil Domain Allows Multiple Forward Step Sizes of Myosin X

Figures

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  Figure 1. Motility of full-length myosin X. (A) Schematic of full-length myosin X structure. Full-length myosin X contains mApple-fused motor, 3 IQs for calmodulin-binding, single alpha helical (SAH), proline, glutamate, serine and threonine enriched domain (PEST), pleckstrin homology (PH), myosin tail homology 4 (MyTH4), Band 4.1 protein, Ezrin, Radixin, Moesin (FERM) domains. (B) Representative traces of full-length myosin X moving on actin filaments (B1) and actin bundles (B2) at 2 μM ATP. (C) Step size histograms of full-length myosin X moving on actin filaments (C1) and actin bundles (C2) at 2 μM ATP. The step size histogram of full-length myosin X moving on actin filaments was fitted with the sum of 6 Gaussian components centered at −36.4 ± 10.7 (± SD), 26.6 ± 5.9, 38.1 ± 6.3, 46.7 ± 4.9, 61.4 ± 4.4, and 68.8 ± 15.0 nm (n = 239 steps). The histogram of full-length myosin X moving on actin bundles was fitted with the sum of 6 Gaussian components entered at −46.5 ± 14.0, 26.5 ± 4.2, 49.5 ± 10.0, 62.4 ± 6.9, 78.2 ± 8.4, and 102.7 ± 6.4 (n = 196 steps). These fittings indicate that full-length myosin X has multiple forward step sizes. (D) Cumulative run-length of full-length myosin X on…

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  Figure 2. Motility of chimera with the motor domain from myosin V and the lever arm and antiparallel coiled-coil domain from myosin X. (A) Schematic of the chimera which contains the motor and the first IQ domain from myosin V (blue) and the second and third IQs and antiparallel coiled-coil domain from myosin X (green). (B) Representative traces of the chimera with the antiparallel coiled-coil domain moving on actin filaments (B1) and actin bundles (B2) at 2 μM ATP. (C) Step size histograms of the chimera with the antiparallel coiled-coil domain moving on actin filaments (C1) and actin bundles (C2) at 2 μM ATP. The step size histogram of the chimera on actin filaments was fitted with the sum of 7 Gaussian components centered at −40.1 ± 11.9, 26.1 ± 0.5, 42.9 ± 9.8, 59.4 ± 2.1, 71.7 ± 4.8, 90.7 ± 5.4, and 115.8 ± 4.3 nm (n = 130 steps). The step size histogram of the chimera on actin bundles was fitted with the sum of 7 Gaussian components centered at −46.9 ± 9.2, 33.7 ± 1.2, 45.0 ± 10.5, 56.8 ± 1.7, 73.6 ± 9.3, 94.3 ± 2.2, and 115.9 ± 11.4 nm (n = 138 steps). These fittings indicate that the chimera with the antiparallel coiled-coil domain has multiple step sizes. (D) Run-length of chimera…

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  Figure 3. Motility of chimera with the motor and lever arm domain from myosin X and the parallel coiled-coil domain from myosin V on actin filaments and actin bundles. (A) Schematic of chimera which contains the motor and lever arm domain from myosin X (green) and the parallel coiled-coil domain from myosin V (blue). (B) Representative traces of the chimera with the parallel coiled-coil domain moving on actin filaments (B1) and actin bundles (B2) at 2 μM ATP. (C) Step size histogram of the chimera with the parallel coiled-coil domain moving on actin filaments (C1) and actin bundles (C2) at 2 μM ATP. The step size histogram with the parallel coiled-coil domain on actin filaments was fitted the sum of 2 Gaussian components centered at −30.9 ± 15.5 and 40.3 ± 17.5 nm (n = 208). The step size histogram on actin bundles was fitted with the sum of 3 Gaussian components centered at −28.0 ± 18.8, 40.6 ± 11.6, and 72.7 ± 5.9 nm (n = 202). (D) Run-length of the chimera with the parallel coiled-coil domain at different ATP concentrations. Chimera with the parallel coiled-coil domain was nonprocessive at high ATP concentrations. (E) Velocity of the chimera at different ATP concentrations.

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  Figure 4. Mutations in the antiparallel coiled-coil domain disrupt the dimerization of myosin X. (A) Schematic of mutant myosin X which contains four mutations in the antiparallel coiled-coil domain. (B) Representative fluorescent intensity trace of mutant myosin X showing one-step photobleaching. (C) Prevalence of one-step and two-step photobleaching of mutant myosin X (N = 40 molecules).