Ultrafast Force-Clamp Spectroscopy of Microtubule-Binding Proteins.
Ultrafast Force-Clamp Spectroscopy of Microtubule-Binding Proteins.
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DOI：
10.1007/978-1-0716-2229-2_22
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发表时间：
2022
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光捕获已被用于破译力产生的细胞骨架蛋白的易位机制。然而，微管（MT）和MT相关蛋白（MAP）之间的动态相互作用的研究没有运动活动是滞后的。研究可以沿沿着MT壁扩散的MAP的运动性对于光学捕获测定是一个特别的挑战，因为热驱动的运动依赖于弱且高度瞬时的相互作用。三珠，超快速力钳（UFFC）光谱具有解决不同的MAP的静态和扩散易位的潜力，具有亚毫秒的时间分辨率和亚纳米的空间精度。在这份报告中，我们提出了实现UFFC的详细程序，包括设置的光学仪器和反馈控制，固定和功能化的基座珠，和MT哑铃的制备。在AMP-PNP存在下使用驱动蛋白-7马达CENP-E产生强静态相互作用的示例性结果。在UFFC测定中MAP-MT相互作用的时间分辨率受到MT哑铃松弛时间的限制，该时间显著长于使用肌动蛋白丝的类似实验所报道的时间。然而，UFFC提供了一个独特的机会，定量研究地图上的滑动沿着MT的拖曳力下，如所示，使用kinetochore-associated斯卡复杂。
Optical trapping has been instrumental for deciphering translocation mechanisms of the force-generating cytoskeletal proteins. However, studies of the dynamic interactions between microtubules (MTs) and MT-associated proteins (MAPs) with no motor activity are lagging. Investigating the motility of MAPs that can diffuse along MT walls is a particular challenge for optical-trapping assays because thermally driven motions rely on weak and highly transient interactions. Three-bead, ultrafast force-clamp (UFFC) spectroscopy has the potential to resolve static and diffusive translocations of different MAPs with sub-millisecond temporal resolution and sub-nanometer spatial precision. In this report, we present detailed procedures for implementing UFFC, including setup of the optical instrument and feedback control, immobilization and functionalization of pedestal beads, and preparation of MT dumbbells. Example results for strong static interactions were generated using the Kinesin-7 motor CENP-E in the presence of AMP-PNP. Time resolution for MAP–MT interactions in the UFFC assay is limited by the MT dumbbell relaxation time, which is significantly longer than reported for analogous experiments using actin filaments. UFFC, however, provides a unique opportunity for quantitative studies on MAPs that glide along MTs under a dragging force, as illustrated using the kinetochore-associated Ska complex.