Block S M, Goldstein L S, Schnapp B J
Rowland Institute for Science, Cambridge, Massachusetts 02142.
Nature. 1990 Nov 22;348(6299):348-52. doi: 10.1038/348348a0.
Kinesin, a mechanoenzyme that couples ATP hydrolysis to movement along microtubules, is thought to power vesicle transport and other forms of microtubule-based motility. Here, microscopic silica beads were precoated with carrier protein, exposed to low concentrations of kinesin, and individually manipulated with a single-beam gradient-force optical particle trap ('optical tweezers') directly onto microtubules. Optical tweezers greatly improved the efficiency of the bead assay, particularly at the lowest kinesin concentrations (corresponding to approximately 1 molecule per bead). Beads incubated with excess kinesin moved smoothly along a microtubule for many micrometres, but beads carrying from 0.17-3 kinesin molecules per bead, moved, on average, only about 1.4 microns and then spontaneously released from the microtubule. Application of the optical trap directly behind such moving beads often pulled them off the microtubule and back into the centre of the trap. This did not occur when a bead was bound by an AMP.PNP-induced rigor linkage, or when beads were propelled by several kinesin molecules. Our results are consistent with a model in which kinesin detaches briefly from the microtubule during a part of each mechanochemical cycle, rather than a model in which kinesin remains bound at all times.
驱动蛋白是一种将ATP水解与沿微管的运动相偶联的机械酶，被认为为囊泡运输和其他形式的基于微管的运动提供动力。在这里，微观二氧化硅珠预先用载体蛋白包被，暴露于低浓度的驱动蛋白，并使用单光束梯度力光学粒子阱（“光镊”）直接将其逐个操纵到微管上。光镊极大地提高了珠子检测的效率，特别是在最低驱动蛋白浓度下（相当于每个珠子约1个分子）。与过量驱动蛋白孵育的珠子沿着微管平稳移动许多微米，但每个珠子携带0.17 - 3个驱动蛋白分子的珠子平均仅移动约1.4微米，然后自发地从微管上释放。在这种移动的珠子后面直接应用光阱通常会将它们从微管上拉下来并回到阱的中心。当珠子通过AMP.PNP诱导的强直连接结合时，或者当珠子由几个驱动蛋白分子推动时，这种情况不会发生。我们的结果与一种模型一致，即驱动蛋白在每个机械化学循环的一部分期间会短暂地从微管上脱离，而不是一种驱动蛋白始终保持结合的模型。
