Kinesins and microtubule associated proteins (MAPs) are critical to sustain life, facilitating cargo transport, cell division, and motility. To interrogate the mechanistic underpinnings of their function, these microtubule-based motors and proteins have been studied extensively at the single molecule level. However, a long-standing issue in the single molecule biophysics field has been how to investigate motors and associated proteins within a physiologically relevant environment in vitro. While the one motor/one filament orientation of a traditional optical trapping assay has revolutionized our knowledge of motor protein mechanics, this reductionist geometry does not reflect the structural hierarchy in which many motors work within the cellular environment. Here, we review approaches that combine the precision of optical tweezers with reconstituted ensemble systems of microtubules, MAPs, and kinesins to understand how each of these unique elements work together to perform large scale cellular tasks, such as but not limited to building the mitotic spindle. Not only did these studies develop novel techniques for investigating motor proteins in vitro, but they also illuminate ensemble filament and motor synergy that helps bridge the mechanistic knowledge gap between previous single molecule and cell level studies.

中文翻译：

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使用光镊测量重组微管束组件内的力产生

驱动蛋白和微管相关蛋白 (MAP) 对于维持生命、促进货物运输、细胞分裂和运动至关重要。为了探究它们功能的机制基础，这些基于微管的马达和蛋白质已在单分子水平上进行了广泛的研究。然而，单分子生物物理学领域的一个长期存在的问题是如何在体外生理相关环境中研究马达和相关蛋白。虽然传统光学捕获分析的一个电机/一个灯丝方向已经彻底改变了我们对电机蛋白力学的认识，但这种简化几何学并不能反映许多电机在细胞环境中工作的结构层次。这里，我们回顾了将光学镊子的精度与微管、MAP 和驱动蛋白的重组集成系统相结合的方法，以了解这些独特元素中的每一个如何协同工作以执行大规模细胞任务，例如但不限于构建有丝分裂纺锤体。这些研究不仅开发了在体外研究运动蛋白的新技术，而且还阐明了整体细丝和运动协同作用，有助于弥合先前单分子和细胞水平研究之间的机制知识差距。