Drosophila class-I myosins that can impact left-right asymmetry have distinct ATPase kinetics

Figures

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  Figure 1. Drosophila myosin-I ATPase cycle.

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  Figure 2. Purified Drosophila myosin-Is and calmodulin light chain.A and B, a representative SDS-PAGE gel of (A) purified myo1C and (B) purified myo1D after anion exchange chromatography containing the heavy chain of 119 kDa and 117 kDa, respectively, and CaM light chain (16.7 kDa). myo1C, myosin-1C; myo1D, myosin-1D; CaM, calmodulin.

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  Figure 3. Actin-activated steady-state ATPase activity.A and B, observed rate of 0.5 μM (A and B) myo1C or (B) myo1D (blue) with increasing F-actin concentrations. The solid lines represent the best fits by Equation 1 with a Vmax of 0.44 ± 0.02 s−1 and 5.1 ± 0.3 s−1 and a KATPase of 48 ± 5 μM and 26 ± 4 μM, respectively. The error bars correspond to the standard deviation of three independent experiments using three different protein preparations per paralog. myo1C, myosin-1C; myo1D, myosin-1D.

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  Figure 4. MgATP-induced actomyosin-I dissociation.A and B, stopped-flow fluorescence transients of dissociation of (A) 0.5 μM myo1C and (B) 0.5 μM myo1D from 0.5 μM pyrene-actin-phalloidin by 20 μM MgATP. The solid lines represent single exponential fits to the fluorescence data with rates of (A) 30.3 ± 0.2 s−1 and (B) 28.6 ± 0.3 s−1. C, rate of MgATP-induced dissociation of myo1C (red) and myo1D (blue) from pyrene-actin-phalloidin as a function of MgATP concentration. The observed rates were obtained at each nucleotide concentration by fitting the stopped-flow data to a single exponential. The solid lines represent the best fits by Equation 2 with K1’= 73 ± 11 μM and 118 ± 15 μM, and k+2’= 174 ± 5 s−1 and 179 ± 5 s−1 for myo1C and myo1D, respectively. The error bars correspond to the standard deviation of three independent experiments using three different protein preparations per paralog. myo1C, myosin-1C; myo1D, myosin-1D.

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  Figure 5. Kinetic scheme for modeling MgATP binding.

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  Figure 6. Actin-activated phosphate (Pi) release is the rate-limiting step for myo1C.A and B, stopped-flow fluorescence transients of Pi release from (A) 0.75 μM myo1C in the presence of 60 μM F-actin and 60 μM MgATP and (B) 0.5 μM myo1D in the presence of 60 μM F-actin and 50 μM MgATP. The observed rates were obtained at each F-actin concentration by fitting the stopped-flow data to a single exponential and slope for (A) and a single exponential for (B). C and D, rate of Pi release from (C) myo1C and (D) myo1D as a function of F-actin concentration. Values of the actin-activated steady-state ATPase of myo1C (green points) are added for reference in panel (C). The solid lines in (C) and (D) represent the best fits by Equation 3 with k+4’ = 0.31 ± 0.06 s−1 and 27 ± 4 s−1 and K9’ = 31 ± 14 μM and 15 ± 12 μM, respectively. The error bars correspond to the standard deviation of 3 to 8 independent experiments using three different protein preparations for myo1D and eight different protein preparations for myo1C. myo1C, myosin-1C; myo1D, myosin-1D.

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  Figure 7. Kinetic scheme for modeling Pi release.

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  Figure 8. Kinetic scheme for modeling MgADP release.

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  Figure 9. Determination of the rate of MgADP release from myo1C and myo1D.A and B, stopped-flow fluorescence transients of MgATP (1 mM)-induced dissociation of (A) 0.5 μM pyrene-actomyo1C and (B) 0.5 μM pyrene-actomyo1D each preincubated with 2.5 μM MgADP. The solid lines represent single exponential fits to the fluorescence data with rates of (A) k5+’ = 1.0 ± 0.1 s−1 and (B) 8.7 ± 1.9 s−1. C, log scales of stopped-flow transients of 0.5 μM actomyo1C (red) and actomyo1D (blue) preincubated with 0.5 μM MgADP mixed with 1 mM MgATP. Transients are composed of a fast phase corresponding to MgATP-induced actomyosin-I dissociation, followed by a slow phase corresponding to MgADP release. D, MgADP affinity (K5’) of myo1C (red) and myo1D (blue) dependent on nucleotide concentration. The observed rate constants were obtained at each nucleotide concentration by fitting the stopped-flow data to a square fit equation. The solid lines represent the best fits by Equation 4 with K5’ = 89 ± 2 nM for myo1C and 44 ± 2 nM for myo1D (11). The error bars correspond to the standard deviation of three independent experiments using three different protein preparations per paralog. myo1C, myosin-1C; myo1D,…

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  Figure 10. Actin gliding and SUV motility assays.A, schematic of experimental settings for actin gliding assays with surface-immobilized myosin-I attached via biotin-neutravidin. B, biotinylated myosin-Is show average velocities decrease as a function of higher motor concentration. C, MgADP concentration-dependence of actin motility in the presence of 2 mM MgATP. The error bars for B and C correspond to the standard deviation of 3 to 6 independent experiments with one different protein preparation per paralog. D, schematic of SUV motility assay on immobilized actin filaments. Myo1D is attached to the SUV via the PtIns (4, 5)P2 – tail interaction. E, average speeds and run lengths for three independent experiments of (light blue) 10 nM or (dark blue) 50 nM myo1D bound to SUVs. myo1C, myosin-1C; myo1D, myosin-1D; SUVs, small unilamellar vesicles.