Monachino Enrico, Spenkelink Lisanne M, van Oijen Antoine M
Centre for Medical and Molecular Bioscience, Illawarra Health and Medical Research Institute and University of Wollongong, New South Wales 2522, Australia.
Zernike Institute for Advanced Materials, University of Groningen, Groningen 9747 AG, Netherlands.
J Cell Biol. 2017 Jan 2;216(1):41-51. doi: 10.1083/jcb.201610025. Epub 2016 Dec 15.
Single-molecule manipulation and imaging techniques have become important elements of the biologist's toolkit to gain mechanistic insights into cellular processes. By removing ensemble averaging, single-molecule methods provide unique access to the dynamic behavior of biomolecules. Recently, the use of these approaches has expanded to the study of complex multiprotein systems and has enabled detailed characterization of the behavior of individual molecules inside living cells. In this review, we provide an overview of the various force- and fluorescence-based single-molecule methods with applications both in vitro and in vivo, highlighting these advances by describing their applications in studies on cytoskeletal motors and DNA replication. We also discuss how single-molecule approaches have increased our understanding of the dynamic behavior of complex multiprotein systems. These methods have shown that the behavior of multicomponent protein complexes is highly stochastic and less linear and deterministic than previously thought. Further development of single-molecule tools will help to elucidate the molecular dynamics of these complex systems both inside the cell and in solutions with purified components.
单分子操作与成像技术已成为生物学家工具库中的重要组成部分，有助于深入了解细胞过程的机制。通过消除总体平均效应，单分子方法为研究生物分子的动态行为提供了独特途径。近来，这些方法的应用范围已扩展至复杂多蛋白系统的研究，并能够详细表征活细胞内单个分子的行为。在本综述中，我们概述了基于力和荧光的各种单分子方法，及其在体外和体内的应用，并通过描述它们在细胞骨架马达和DNA复制研究中的应用来突出这些进展。我们还讨论了单分子方法如何增进我们对复杂多蛋白系统动态行为的理解。这些方法表明，多组分蛋白复合物的行为具有高度随机性，其线性和确定性低于先前的认知。单分子工具的进一步发展将有助于阐明这些复杂系统在细胞内以及纯化组分溶液中的分子动力学。
