Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo, 060-0810, Japan.
Faculty of Science, Hokkaido University, Sapporo, 060-0810, Japan.
Sci Rep. 2017 Jul 21;7(1):6166. doi: 10.1038/s41598-017-06249-1.
In vitro gliding assay of microtubules (MTs) on kinesins has provided us with valuable biophysical and chemo-mechanical insights of this biomolecular motor system. Visualization of MTs in an in vitro gliding assay has been mainly dependent on optical microscopes, limited resolution of which often render them insufficient sources of desired information. In this work, using high speed atomic force microscopy (HS-AFM), which allows imaging with higher resolution, we monitored MTs and protofilaments (PFs) of tubulins while gliding on kinesins. Moreover, under the HS-AFM, we also observed splitting of gliding MTs into single PFs at their leading ends. The split single PFs interacted with kinesins and exhibited translational motion, but with a slower velocity than the MTs. Our investigation at the molecular level, using the HS-AFM, would provide new insights to the mechanics of MTs in dynamic systems and their interaction with motor proteins.
体外微管（MTs）在驱动蛋白上滑行的实验为我们提供了这种生物分子马达系统的宝贵生物物理和化学机械学见解。在体外滑行实验中，MTs 的可视化主要依赖于光学显微镜，其有限的分辨率往往使得它们无法提供所需信息。在这项工作中，我们使用高速原子力显微镜（HS-AFM）来监测在驱动蛋白上滑行的 MTs 和微管蛋白原丝（PFs），HS-AFM 具有更高的分辨率，可以进行成像。此外，在 HS-AFM 下，我们还观察到滑行的 MTs 在其前端分裂成单个 PFs。分裂的单个 PFs 与驱动蛋白相互作用并表现出平移运动，但速度比 MTs 慢。我们使用 HS-AFM 在分子水平上的研究将为动态系统中 MTs 的力学及其与马达蛋白的相互作用提供新的见解。
