Spontaneous Detachment of the Leading Head Contributes to Myosin VI Backward Steps

<div><p>Myosin VI is an ATP driven molecular motor that normally takes forward and processive steps on actin filaments, but also on occasion stochastic backward steps. While a number of models have attempted to explain the backwards steps, none offer an acceptable mechanism for their existence. We therefore performed single molecule imaging of myosin VI and calculated the stepping rates of forward and backward steps at the single molecule level. The forward stepping rate was proportional to the ATP concentration, whereas the backward stepping rate was independent. Using these data, we proposed that spontaneous detachment of the leading head is uncoupled from ATP binding and is responsible for the backward steps of myosin VI.</p> </div

Correlation between myosin VI step types.

<p>(A) Steps from the distant binding state. A large step occurs only from the distant binding state and results in the distant binding state; small steps 1 from the distant binding state result in the adjacent binding state; backward steps only occur from the distant binding state and result in the adjacent binding state. (B) Steps from the adjacent binding state. From the adjacent binding state, myosin VI can only take a small step. Here, we should note an apparent correlation between steps (e.g. a large step following a backward step and a large step or backward step following a small step 1), which is inconsistent with our models, appears in the example traces (Fig. 1B and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058912#pone.0058912.s001" target="_blank">Fig. S1</a>). The inconsistency is because which head is stepping from the adjacent binding state is unknown <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058912#pone.0058912-Ikezaki1" target="_blank">[11]</a>. We have previously shown that either head has an equal probability of taking the next step from the adjacent binding state <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058912#pone.0058912-Ikezaki1" target="_blank">[11]</a>. Our results here likely undercount the number of small steps by the labeled head, but at the same time equally overcount the number of steps following the adjacent binding state made by the other head because of our labeling method. Had we labeled both heads in our experiments, we would expect to see our model satisfied <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058912#pone.0058912-Nishikawa1" target="_blank">[2]</a>.</p

Actual stepping rate calculated from the apparent stepping rate and the step ratio.

<p>(A) Actual forward stepping rate. The distribution was well fitted to the function 1/k_f = 1/(k_ATP[ATP])+1/k_ADP with k_ATP = 0.035 µM·s⁻¹ and k_ADP = 8 s⁻¹. (B) Actual backward stepping rate. The distribution had a constant value of 0.62, meaning that ATP-binding does not couple with backward steps. Open circles indicate values estimated from the apparent large step rate; closed circles indicate those from the apparent backward step rate. The errors of the actual forward and backward stepping rates were calculated using the error for each apparent stepping rate, the law of the error propagation and the freeware Maxima.</p

The stepping rate and the probability of step type at various ATP concentrations.

<p>(A) Stepping rate of each step type at various ATP concentrations (red circles, large steps; blue circles, small steps; green circles, backward steps). The stepping rate for large and small steps were calculated by fitting the dwell time distribution using a convolution of two exponentials (tk² exp (-kt)) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058912#pone.0058912.s002" target="_blank">Fig. S2A</a>, B), while that of backward steps was calculated by fitting the dwell time distribution using a single exponential decay function (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058912#pone.0058912.s002" target="_blank">Fig. S2C</a>). (B) Probability of step types at various ATP concentrations (red circles, large steps; blue circles, small steps; green circles, backward step). The probabilities were calculated by fitting the stepping size distribution (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058912#pone.0058912.s002" target="_blank">Fig. S2D</a>) with a three-Gaussian function. All fits were performed using Origin 7.5 (OriginLab).</p

Calculation of small step frequency from the distant binding state.

<p>The distribution of small steps, <i>s</i>, from the distant binding state was constant (0.14) against ATP concentration. Errors for the frequency were calculated using errors for each apparent stepping rate, the law of error propagation and the freeware Maxima.</p

Experimental system for single molecule measurements of myosin VI.

<p>(A) A Qdot585-labeled myosin VI moving on an actin filament was illuminated using an evanescent field. Myosin VI was biotinylated via HaloTag at its N-terminus (motor domain) using biotin-Halo-ligand. A streptavidin conjugated Qdot585 (Life technologies) was attached to the motor domain of myosin VI using avidin-biotin interactions. (B) A typical stepping trace of myosin VI at 200 µM ATP. Examples of large, small and backward steps are indicated by the arrows.</p