Valentine Megan T, Fordyce Polly M, Krzysiak Troy C, Gilbert Susan P, Block Steven M
Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA.
Nat Cell Biol. 2006 May;8(5):470-6. doi: 10.1038/ncb1394. Epub 2006 Apr 2.
Eg5, a member of the kinesin superfamily of microtubule-based motors, is essential for bipolar spindle assembly and maintenance during mitosis, yet little is known about the mechanisms by which it accomplishes these tasks. Here, we used an automated optical trapping apparatus in conjunction with a novel motility assay that employed chemically modified surfaces to probe the mechanochemistry of Eg5. Individual dimers, formed by a recombinant human construct Eg5-513-5His, stepped processively along microtubules in 8-nm increments, with short run lengths averaging approximately eight steps. By varying the applied load (with a force clamp) and the ATP concentration, we found that the velocity of Eg5 was slower and less sensitive to external load than that of conventional kinesin, possibly reflecting the distinct demands of spindle assembly as compared with vesicle transport. The Eg5-513-5His velocity data were described by a minimal, three-state model where a force-dependent transition follows nucleotide binding.
驱动蛋白超家族中基于微管的马达蛋白Eg5，对于有丝分裂期间双极纺锤体的组装和维持至关重要，但对于它完成这些任务的机制却知之甚少。在此，我们使用了一种自动光镊装置，并结合一种新型的运动分析方法，该方法采用化学修饰表面来探究Eg5的机械化学性质。由重组人构建体Eg5-513-5His形成的单个二聚体，以8纳米的增量沿着微管连续步进，短程平均约为八步。通过改变施加的负载（采用力钳）和ATP浓度，我们发现，与传统驱动蛋白相比，Eg5的速度较慢，对外部负载的敏感性较低，这可能反映出与囊泡运输相比，纺锤体组装有不同的需求。Eg5-513-5His的速度数据由一个最小的三态模型描述，其中力依赖性转变发生在核苷酸结合之后。
