Studies Of Myosin V

最近的研究提供了强有力的证据，表明单个肌凝蛋白V类分子沿着f -肌动蛋白运输囊泡和细胞器，每次扩散相遇都要经过几个36纳米的步骤，“手牵手”。调控肌凝蛋白- v的机制尚不清楚。在这里，我们使用基于光学镊子的传感器来测量负载对兔骨骼f -肌动蛋白和单个小鼠脑肌球蛋白- v之间的机械相互作用的影响，后者分两个阶段产生其工作行程。我们发现，在+/- 1.5 pN的推力和拉力范围内，工作冲程第一阶段的寿命呈指数变化，约为10倍。刚度测量表明，当两个头部都与f -肌动蛋白结合时，分子内力可能接近3.6 pN，在这种情况下，外推可以预测前头的脱离动力学将减慢50倍，后头的动力学将分别加速。这种对头部化学-机械循环的同步效应增加了尾头首先分离的可能性，并导致在没有应变依赖的系统中每次扩散遇到的前进步骤的数量大幅增加。

Recent studies provide strong evidence that single myosin class V molecules transport vesicles and organelles processively along F-actin, taking several 36-nm steps, 'hand over hand', for each diffusional encounter. The mechanisms regulating myosin-V's processivity remain unknown. Here, we have used an optical-tweezers-based transducer to measure the effect of load on the mechanical interactions between rabbit skeletal F-actin and a single head of mouse brain myosin-V, which produces its working stroke in two phases. We found that the lifetimes of the first phase of the working stroke changed exponentially and about 10-fold over a range of pushing and pulling forces of +/- 1.5 pN. Stiffness measurements suggest that intramolecular forces could approach 3.6 pN when both heads are bound to F-actin, in which case, extrapolation would predict the detachment kinetics of the front head to slow down 50-fold and the kinetics of the rear head to accelerate, respectively. This synchronizing effect on the chemo-mechanical cycles of the heads increases the probability of the trail head detaching first and causes a strong increase in the number of forward steps per diffusional encounter over a system with no strain dependence. Myosin V has an extremely long neck containing six calmodulin-binding IQ motifs that allows it to take multiple 36 nm steps corresponding to the pseudo-repeat of actin. To further investigate how myosin V moves processively on actin filaments, we altered the length of the neck by adding or deleting IQ motifs in myosin constructs lacking the globular tail domain. These myosin V IQ mutants were fluorescently labeled by exchange of a single Cy3-labeled calmodulin into the neck region of one head. We measured the step-size of these individual IQ mutants with nanometer precision and sub-second resolution using FIONA. The step-size was proportional to neck length for constructs containing 2, 4, 6 and 8 IQ motifs, providing strong support for the swinging lever arm model of myosin motility. In addition, the kinetics of stepping provided additional support for the hand-over-hand model whereby the two heads alternately assume the leading position. Interestingly, the 8 IQ myosin V mutants gave a broad distribution of step-sizes with multiple peaks, suggesting that this mutant has many choices of binding sites on an actin filament. These data demonstrate that the step-size of myosin V is affected by the length of its neck and is not solely determined by the pseudo-repeat of the actin filament.