Autoregulation and dual stepping mode of MYA2, an Arabidopsis myosin XI responsible for cytoplasmic streaming

Figures

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  Figure 1. Schematic diagrams of the MYA2 constructs used in this work. (a) Full: Full-length construct comprising MD, six IQ motifs to which six calmodulins bind, coiled-coil, and GTD domains. (b) HMM: HMM construct comprising of MD, six IQ motifs to which six calmodulins bind, and a coiled-coil domain. 6IQ: 6IQ construct comprising of MD and six IQ motifs to which six calmodulins bind. MD: MD construct comprising only MD. GTD: GTD construct comprising only GTD.

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  Figure 2. Actin-activated Mg2+-ATPase activities and inhibitory effects on Mg2+-ATPase activities of Full, HMM, 6IQ, and MD. (a) Actin-activated Mg2+-ATPase activities of Full, HMM, 6IQ, and MD. Mg2+-ATPase activities in the presence of various actin concentrations. The data were fitted using Michaelis–Menten dynamics, and the Vmax and Kapp values were measured. The concentrations of Full, HMM, 6IQ, and MD used 0.005–0.019, 0.001–0.009, 0.002–0.018, and 0.001–0.008 µM, respectively. (b) Inhibitory effects of GTD on Mg2+-ATPase activities of HMM, MD, and 6IQ. The actin-activated ATPase activities of HMM and 6IQ in the presence of various concentrations of GTD and 23.8 μM actin were fit to a hyperbola, and the Ki values of GTD for HMM and 6IQ were calculated to be 0.5 μM and 0.8 μM, respectively. (c) Inhibitory effects of Mg2+-ATPase activities of 6IQ by 1 µM GTD in the presence of various actin concentrations.

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  Figure 3. Inhibitory effects of actin sliding velocities of 6IQ and the dissociation rates of mant-ADP from 6IQ. (a) Inhibitory effects of actin sliding velocities of 6IQ by 1 µM GTD and 2 µM GTD in the presence of 3 mM ATP. (b) Mant-ADP dissociation from acto-6IQ in the presence of 1 µM GTD and absence of GTD. The dissociation rates of mant-ADP from 6IQ were measured using fluorescence energy transfer between the tryptophans of MD of 6IQ and mant-ADP. The transient shown is the average of six separate recordings. The red line is a single exponential fit yielding a rate constant of 403 s−1 in the absence of GTD (left) and 24 s−1 in the presence of 1 µM GTD (right). The average of three independent assays was 22 ± 3.4 s−1 in the presence of 1 µM GTD and 430 ± 40 s−1 in the absence of GTD. (c) A model of the mechanism by which GTD inhibits the movement of MYA2 in vitro and in vivo. In vitro, in the absence of GTD, the ADP dissociation rate of acto-6IQ is 430 s−1 and the actin sliding velocity of 6IQ is 5.2 μm s−1. In the presence of 1 μM GTD, the ADP dissociation rate of acto-6IQ is 22 s−1 and the actin sliding velocity of 6IQ is 2.4 μm s−1. In vivo, when GTD is not bound to the organelle,…

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  Figure 4. Stepping dynamics and force measurement of MYA2. (a) QD attached to a motor domain in a MYA2 dimer was observed using TIRFM at 2 nm and 33 ms spatiotemporal resolution. Steps were analyzed by an automated step-finding algorithm43 (gray line). ATP concentration, 1 µM. (b) Step size histogram of the motor domain. The histogram fit to three Gaussian functions with peaks at 60.5 ± 14.8 nm (red line), 29.3 ± 6.9 nm (blue line), and − 28.1 ± 9.9 nm (green line), respectively. Black line indicates the convolution of three Gaussian functions. (c) Typical trace of optical trapping nanometry at 1 µM ATP. Gray line: raw trace; Black line: data passed through a 200 Hz low pass filter. (d) Step size histogram of an optically trapped bead attached to the MYA2 C-terminal tail. The histogram was fit to two Gaussian functions with peaks at 31.6 ± 15.2 nm (orange line), and − 31.9 ± 13.6 nm (purple line), respectively. Black line indicates the convolution of the two Gaussian functions. (e) Histogram of MYA2 stall force. The histogram was fitted to single Gaussian function with peaks at 0.85 ± 0.16 pN (black line).

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  Figure 5. Walking model of MYA2. Left (a–d), large hand-over-hand steps; right (e–h), small inchworm-like steps. Colors denote the following: N-terminal motor domain (red or blue), lever arm (six calmodulins, green), coiled-coil domain (black), and the globular C-terminal tail domain (gray). Red, blue, and green arrows indicate the step of the MD for large, small, and backward steps, respectively, observed using FIONA. Orange and purple arrows indicate the step of the C-terminal tail for forward and backward steps, respectively, observed by optical trapping assay.