Department of Physiology and Biophysics, University of Washington, Seattle, WA, USA.
Departments of Biophysics and Biochemistry, UT Southwestern, Dallas, TX, USA.
Methods Mol Biol. 2022;2478:653-676. doi: 10.1007/978-1-0716-2229-2_23.
Optical traps have enabled foundational studies of how mechanoenzymes such as kinesins and dynein motors walk along microtubules, how myosins move along F-actin, and how nucleic acid enzymes move along DNA or RNA. Often the filamentous substrates serve merely as passive tracks for mechanoenzymes but microtubules and F-actin are themselves dynamic protein polymers, capable of generating movement and force independently of conventional motors. Microtubule-driven forces are particularly important during mitosis, when they align duplicated chromosomes at the metaphase plate and then pull them apart during anaphase. These vital movements depend on specialized protein assemblies called kinetochores that couple the chromosomes to the tips of dynamic microtubule filaments, thereby allowing filament shortening to produce pulling forces. Although great strides have been made toward understanding the structures and functions of many kinetochore subcomplexes, the biophysical basis for their coupling to microtubule tips remains unclear. During tip disassembly, strain energy is released when straight protofilaments in the microtubule lattice curl outward, creating a conformational wave that propagates down the microtubule. A popular viewpoint is that the protofilaments as they curl outward hook elements of the kinetochore and tug on them, transferring some of their curvature strain energy to the kinetochore. As a first step toward testing this idea, we recently developed a laser trap assay to directly measure the working strokes generated by curling protofilaments. Our "wave" assay is based on an earlier pioneering study, with improvements that allow measurement of curl-driven movements as functions of force and quantification of their conformational strain energy. In this chapter, we provide a detailed protocol for our assay and describe briefly our instrument setup and data analysis methods.
光学陷阱使人们能够深入研究肌球蛋白和动力蛋白等机械酶如何沿着微管行走，肌球蛋白如何沿着 F-肌动蛋白移动，以及核酸酶如何沿着 DNA 或 RNA 移动。通常，丝状底物仅仅作为机械酶的被动轨道，但微管和 F-肌动蛋白本身就是动态的蛋白质聚合物，能够独立于传统的马达产生运动和力。微管驱动的力在有丝分裂过程中尤为重要，此时它们将复制的染色体排列在中期板上，然后在后期将它们拉开。这些重要的运动依赖于称为动粒的特殊蛋白质组装体，它将染色体与动态微管纤维的尖端连接起来，从而允许纤维缩短产生拉力。尽管在理解许多动粒亚基复合物的结构和功能方面已经取得了很大进展，但它们与微管尖端耦合的生物物理基础仍然不清楚。在尖端解体过程中，当微管晶格中的直原纤维向外卷曲时，应变能被释放出来，从而产生一个沿着微管传播的构象波。一个流行的观点是，当原纤维向外卷曲时，它会钩住动粒的元件并拉动它们，将它们的曲率应变能的一部分传递给动粒。作为验证这一观点的第一步，我们最近开发了一种激光陷阱测定法来直接测量卷曲原纤维产生的工作冲程。我们的“波”测定法基于早期的开创性研究，有了改进，可以测量作为力的函数的卷曲驱动运动，并对其构象应变能进行定量。在本章中，我们提供了我们测定法的详细方案，并简要描述了我们的仪器设置和数据分析方法。
