High-resolution tracking of microtubule motility driven by a single kinesin motor.
High-resolution tracking of microtubule motility driven by a single kinesin motor.
复制标题
由单个驱动蛋白马达驱动的微管运动的高分辨率跟踪。
DOI：
10.1073/pnas.91.10.4584
复制
发表时间：
1994
影响因子：
11.1
通讯作者：
Vale,RD
中科院分区：
文献类型：
--
作者：
Malik,F;Brillinger,D;Vale,RD
驱动蛋白是一种基于微管的运动蛋白，包含两个相同的产生力的亚基。沿微管的驱动蛋白结合位点相距 8 nm（微管蛋白二聚体的尺寸），这意味着每个水解循环驱动蛋白必须移位 8 nm 的最小距离。然而，对驱动蛋白微管刺激的 ATP 酶活性（约 20 ATP 每秒）和运输速度（约 0.6 微米/秒）的测量表明，在零负载条件下，每个 ATP 移动的净距离（约 30 nm）可能大于一个微管蛋白二聚体。为了探索驱动蛋白在其 ATP 酶循环过程中如何易位，我们构建了一个能够在 200 Hz 带宽下以 1 nm 分辨率跟踪运动的显微镜，并使用该设备来检查由单个驱动蛋白电机驱动的微管运动。尽管作用于驱动蛋白马达内弹性元件的布朗力排除了< 12 nm的步长的检测，但在移动微管的位移痕迹中没有观察到规则的逐步运动。虽然偶尔会观察到大约 16 nm 的单个步长，但它们很少出现，表明驱动蛋白在其 ATP 水解循环过程中很少突然移动两个或多个微管蛋白亚基的距离。相反，在零负载条件下，驱动蛋白更有可能向前移动仅单个微管蛋白亚基的距离。
Kinesin is a microtubule-based motor protein that contains two identical force-generating subunits. The kinesin binding sites along the microtubule lie 8 nm apart (the dimension of the tubulin dimer), which implies that kinesin must translocate a minimum distance of 8 nm per hydrolysis cycle. Measurements of kinesin's microtubule-stimulated ATPase activity (approximately 20 ATP per sec) and velocity of transport (approximately 0.6 micron/sec), however, suggest that the net distance moved per ATP (approximately 30 nm) may be greater than one tubulin dimer under zero load conditions. To explore how kinesin translocates during its ATPase cycle, we constructed a microscope capable of tracking movement with 1-nm resolution at a bandwidth of 200 Hz and used this device to examine microtubule movement driven by a single kinesin motor. Regular stepwise movements were not observed in displacement traces of moving microtubules, although Brownian forces acting on elastic elements within the kinesin motor precluded detection of steps that were < 12 nm. Though individual steps of approximately 16 nm were occasionally observed, their infrequent occurrence suggests that kinesin rarely moves abruptly by distances of two or more tubulin subunits during its ATP hydrolysis cycle. Instead it is more likely that kinesin moves forward by the distance of only a single tubulin subunit under zero load conditions.