Single Molecule Investigation of Kinesin-1 Motility Using Engineered Microtubule Defects.
Single Molecule Investigation of Kinesin-1 Motility Using Engineered Microtubule Defects.
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DOI：
10.1038/srep44290
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发表时间：
2017-03-13
影响因子：
4.6
通讯作者：
Ross JL
中科院分区：
文献类型：
--
作者：
Gramlich MW;Conway L;Liang WH;Labastide JA;King SJ;Xu J;Ross JL
微管的结构在细胞中通过许多微管相关的蛋白质和酶受到严格调节。微管在聚合过程中积累结构缺陷，并且缺陷尺寸在机械应力下可以进一步增加。有趣的是，微管缺陷已被证明是通过切断酶或自我修复去除的目标。细胞在缺陷去除中的控制表明缺陷可以影响基于微管的过程，包括基于分子马达的细胞内转运。我们以前证明，微管缺陷影响货物运输的多个驱动蛋白马达。然而，所观察到的效果的机制研究仍然具有挑战性，因为缺陷在聚合过程中随机发生，并且在当前的运动性测定中不能直接观察到。为了克服这一挑战，我们使用端到端退火来产生使用标准落射荧光显微镜直接观察到的缺陷。我们证明，退火网站概括了聚合衍生的缺陷对多电机运输的影响，因此代表了一个简单而适当的模型自然发生的缺陷。我们发现，单一驱动蛋白进行过早的解离，但没有优先暂停，在退火网站。我们的研究结果提供了第一个机制的洞察缺陷如何影响驱动蛋白为基础的运输。单分子水平上的优先解离有可能损害体内微管缺陷位点处的货物递送。
The structure of the microtubule is tightly regulated in cells via a number of microtubule associated proteins and enzymes. Microtubules accumulate structural defects during polymerization, and defect size can further increase under mechanical stresses. Intriguingly, microtubule defects have been shown to be targeted for removal via severing enzymes or self-repair. The cell’s control in defect removal suggests that defects can impact microtubule-based processes, including molecular motor-based intracellular transport. We previously demonstrated that microtubule defects influence cargo transport by multiple kinesin motors. However, mechanistic investigations of the observed effects remained challenging, since defects occur randomly during polymerization and are not directly observable in current motility assays. To overcome this challenge, we used end-to-end annealing to generate defects that are directly observable using standard epi-fluorescence microscopy. We demonstrate that the annealed sites recapitulate the effects of polymerization-derived defects on multiple-motor transport, and thus represent a simple and appropriate model for naturally-occurring defects. We found that single kinesins undergo premature dissociation, but not preferential pausing, at the annealed sites. Our findings provide the first mechanistic insight to how defects impact kinesin-based transport. Preferential dissociation on the single-molecule level has the potential to impair cargo delivery at locations of microtubule defect sites in vivo.