Cargo-binding makes a wild-type single-headed myosin-VI move processively
Cargo-binding makes a wild-type single-headed myosin-VI move processively
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DOI：
10.1529/biophysj.105.075721
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发表时间：
2006-05-01
影响因子：
3.4
通讯作者：
Yanagida, T
中科院分区：
文献类型：
--
作者：
Iwaki, M;Tanaka, H;Yanagida, T
VI类肌球蛋白是一种沿肌动蛋白运动的细胞内囊泡和细胞器转运蛋白。哀叹的方向与大多数其他已知的肌球蛋白类别相反。肌球蛋白VI预计会形成一个二聚体，沿着肌动蛋白进行移动。哀叹与其他肌球蛋白细胞器转运体一样的手部机制。然而，最近，野生型肌球蛋白-VI被证明是单体和单头的，这使得人们对其加工性能产生了怀疑。利用单分子技术，我们证明了绿色荧光蛋白标记的单头野生型肌球蛋白VI不会连续运动。然而，当与200纳米聚苯乙烯微珠(大小相当于细胞内小泡)以每个微珠一个头的比率耦合时，单头肌球蛋白-VI以大(40纳米)的步长连续移动。这种单体驱动的运动与人工二聚体驱动的运动的特征不同：与人工二聚体相比，单体-微珠复合体具有更小的失速力(1pN比2pN)、平均行程长度缩短2.5倍(91 nm比220 nm)以及依赖负载的步长。此外，我们发现，在类似于细胞环境的高粘度溶液(比水高40倍)中，单体-微珠复合体的运动更连续。由于微珠的扩散常数比肌球蛋白VI在水中的扩散常数低60倍，我们提出了一个模型，即微珠作为肌球蛋白VI的扩散锚，增强其在脱离后的重新结合，并支持微珠-单体复合体的前进运动。虽然单头肌球蛋白-VI能够携带大量货物连续移动，但旅行距离相当短。细胞内可能存在多个分子参与远距离的货物运输。
Class VI myosin is an intracellular vesicle and organelle transporter that moves along actin. laments in a direction opposite to most other known myosin classes. The myosin-VI was expected to form a dimer to move processively along actin. laments with a hand-over-hand mechanism like other myosin organelle transporters. Recently, however, wild-type myosin-VI was demonstrated to be monomer and single-headed, casting a doubt on its processivity. By using single molecule techniques, we show that green-fluorescent-protein-tagged single-headed, wild-type myosin-VI does not move processively. However, when coupled to 200-nm polystyrene beads (comparable to intracellular vesicles in size) at a ratio of one head per bead, single-headed myosin-VI moves processively with large (40-nm) steps. The characteristics of this monomer-driven movement were different to that of artificial dimer-driven movement: Compared to the artificial dimer, the monomer-bead complex had a reduced stall force (1 pN compared to 2 pN), an average run length 2.5-fold shorter (91 nm compared to 220 nm) and load-dependent step size. Furthermore, we found that a monomer-bead complex moved more processively in a high viscous solution (40-fold higher than water) similar to cellular environment. Because the diffusion constant of the bead is 60-fold lower than myosin-VI heads alone in water, we propose a model in which the bead acts as a diffusional anchor for the myosin-VI, enhancing its rebinding following detachment and supporting processive movement of the bead-monomer complexes. Although a single-headed myosin-VI was able to move processively with a large cargo, the travel distance was rather short. Multiple molecules may be involved in the cargo transport for a long travel distance in cells.