Ayloo Swathi, Holzbaur Erika L F
Department of Physiology and the Pennsylvania Muscle Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA; Department of Biology Graduate Group, School of Arts and Sciences at the University of Pennsylvania, Philadelphia, PA, USA.
Department of Physiology and the Pennsylvania Muscle Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.
Methods Cell Biol. 2015;128:57-68. doi: 10.1016/bs.mcb.2015.02.002. Epub 2015 Apr 8.
Long-range intracellular transport of organelles driven by kinesin and dynein motor proteins depends on additional cellular factors including adaptors and scaffolding proteins. While single-molecule studies of the motility of purified motor proteins have been a powerful approach, these assays are not fully representative of the complex interactions that occur in a cellular environment. To gain insights into the functioning of adaptor proteins that work in concert with motors proteins, motility assays in cell extracts have been developed. These assays are an attractive means to begin to dissect the roles of additional factors in motor-driven transport. Further, this system can be easily manipulated to study this process in different physiological environments. Here we describe in vitro reconstitution of motor-driven motility along microtubules in cell extracts, followed by considerations for data analysis and how these assays can be powerful in informing our understanding of basic cellular processes.
由驱动蛋白和动力蛋白驱动的细胞器长距离细胞内运输依赖于包括衔接蛋白和支架蛋白在内的其他细胞因子。虽然对纯化的驱动蛋白运动性的单分子研究是一种强大的方法，但这些检测方法并不能完全代表细胞环境中发生的复杂相互作用。为了深入了解与驱动蛋白协同工作的衔接蛋白的功能，人们开发了细胞提取物中的运动性检测方法。这些检测方法是开始剖析其他因素在驱动蛋白运输中作用的一种有吸引力的手段。此外，该系统可以很容易地进行操作，以研究不同生理环境中的这一过程。在这里，我们描述了在细胞提取物中沿微管进行驱动蛋白运动性的体外重建，随后考虑了数据分析以及这些检测方法如何能够有力地增进我们对基本细胞过程的理解。
