Department of Physiology, The Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
Eberhard Karls Universität Tübingen, Cellular Nanoscience (ZMBP), Auf der Morgenstelle, Tübingen, Germany.
Methods Mol Biol. 2022;2478:559-583. doi: 10.1007/978-1-0716-2229-2_20.
The cytoskeletal motors myosin, kinesin, and dynein and their corresponding tracks, actin and microtubules, are force generating ATPases responsible for motility and morphological changes at the intracellular, cellular, and tissue levels. The pioneering application of optical tweezers to measure the force-producing properties of cytoskeletal motors has provided an unparalleled understanding of their mechanochemistry. The mechanosensitivity of processive, microtubule-based motors has largely been studied in the optical trap using the "single-bead" assay, where a bead-attached motor is held adjacent to a cytoskeletal filament as it processively steps along it. However, because of the geometrical constraints in the conventional single-bead assay, the motor-filament bond is not only loaded parallel to the long axis of the filament, but also perpendicular to the long axis of the filament. This perpendicular force, which is inherent in the conventional single-bead assay, accelerates the motor-filament detachment and has not been carefully considered in prior experiments. An alternative approach is the "three-bead" assay, which was developed for the study of non-processive myosin motors. The vertical force component is minimized in this assay, and the total opposing force is mainly parallel to the microtubule. Experiments with kinesin show that microtubule attachment durations can be highly variable and last for up to tenfold longer times in the three-bead assay, compared to the single-bead assay. Thus, the ability of kinesin to bear mechanical load and remain attached to microtubules depends on the forces in more than one dimension. In this chapter, we provide detailed methods for preparing the proteins, buffers, flow chambers, and bead-filament assemblies for performing the three-bead assay with microtubules and their motors.
细胞骨架马达肌球蛋白、驱动蛋白和动力蛋白及其相应的轨道肌动蛋白和微管是产生力的 ATP 酶，负责细胞内、细胞和组织水平的运动和形态变化。光学镊子的开创性应用，用于测量细胞骨架马达的产生力的特性，为它们的机械化学提供了无与伦比的理解。基于微管的进行性马达的机械敏感性在光学陷阱中主要使用“单珠”测定法进行研究，其中，附着有珠子的马达与附着在其上的微管相邻，同时沿着它进行进行性步骤。然而，由于传统单珠测定法中的几何约束，马达-细丝键不仅沿细丝的长轴加载，而且还垂直于细丝的长轴加载。这种垂直力是传统单珠测定法固有的，在以前的实验中没有被仔细考虑。另一种方法是“三珠”测定法，该方法是为研究非进行性肌球蛋白马达而开发的。在该测定法中，垂直力分量最小，并且总相反力主要与微管平行。用驱动蛋白进行的实验表明，与单珠测定法相比，在三珠测定法中，微管附着持续时间可能高度变化并且持续长达十倍以上的时间。因此，驱动蛋白承受机械负荷并保持与微管附着的能力取决于一个以上维度的力。在本章中，我们提供了详细的方法，用于制备蛋白质、缓冲液、流动室和珠丝组件，以进行带有微管及其马达的三珠测定法。
