我们详细介绍了使用计算机控制的光学陷阱来研究动粒组件与动态微管之间的相互作用。在过去的二十年中,光学陷阱帮助揭示了传统分子马达的工作原理,如驱动蛋白和动力蛋白,但直到最近才被应用于研究动粒功能。最有用的陷阱结合了灵敏的位置探测器和伺服控制,允许它们作为力夹操作,在物体移动时保持恒定的载荷。我们的乐器,这是允许力夹紧的最简单的设计之一,依靠计算机控制的压电平台和用于捕获和位置检测的单个激光器。我们将其应用于运动性测定,其中将涂有纯微管结合动粒成分的珠子附着在单个动态微管的尖端。就像体内的动静脉一样,珠子保持尖端连接,经历与灯丝组装和拆卸耦合的运动。该力夹钳提供了许多优于缺乏反馈控制的器械的益处。它允许在组装和拆卸驱动运动期间连续施加张力,提供与生理状况的紧密匹配。它还可以实现高分辨率跟踪,并通过消除分子顺应性引起的伪影简化了数据解释。持久性的形成,动态微管尖端的承重附件是所有动核活动的基础。我们的直接,因此,对动粒-微管耦合的物理研究可以提供对许多重要动粒功能的见解,包括纠正异常附件和产生延迟有丝分裂的“等待后期”信号,直到所有动体正确连接。
We detail our use of computer-controlled optical traps to
study interactions between kinetochore components and dynamic microtubules. Over the last two decades optical traps have helped uncover the working principles of conventional molecular motors, such as kinesin and dynein, but only recently have they been applied to
study kinetochore function. The most useful traps combine sensitive position detectors and servo-control, allowing them to be operated as force clamps that maintain constant loads on objects as they move. Our instrument, which is among the simplest designs that permits force clamping, relies on a computer-controlled piezoelectric stage and a single laser for trapping and position detection. We apply it in motility assays where beads coated with pure microtubule-binding kinetochore components are attached to the tips of individual dynamic microtubules. Like
kinetochores in vivo, the beads remain tip-attached, undergoing movements coupled to filament assembly and disassembly. The force clamp provides many benefits over instruments that lack feedback control. It allows tension to be applied continuously during both assembly- and disassembly-driven movement, providing a close match to the physiological situation. It also enables tracking with high resolution, and simplifies data interpretation by eliminating artifacts due to molecular compliance. The formation of persistent, load-bearing attachments to dynamic microtubule tips is fundamental to all kinetochore activities. Our direct, physical
study of kinetochore-microtubule coupling may therefore furnish insights into many vital kinetochore functions, including correction of aberrant attachments and generation of the \'wait-anaphase\' signals that delay mitosis until all
kinetochores are properly attached.