Combining Structure-Function and Single-Molecule Studies on Cytoplasmic Dynein.
Combining Structure-Function and Single-Molecule Studies on Cytoplasmic Dynein.
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DOI：
10.1007/978-1-4939-7271-5_4
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Gennerich A
中科院分区：
文献类型：
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作者：
Rao L;Hülsemann M;Gennerich A
细胞质动力蛋白是最大和最复杂的细胞骨架运动蛋白。它负责大量的生物学功能，从细胞器和mRNA的运输到神经元迁移期间的细胞核运动以及细胞分裂期间有丝分裂纺锤体的形成和定位。尽管其大道尔顿的大小和复杂的设计，最近成功的重组表达的动力蛋白重链推进了我们的理解，通过结合结构-功能和单分子研究的动力蛋白的分子机制。单分子荧光分析提供了详细的见解，动力蛋白如何前进沿着其微管轨道在没有负载，而光镊产生的力量产生和失速行为的动力蛋白的见解。在这里，使用S。在酿酒酵母表达系统中，我们提供了用于产生动力蛋白突变体以及用于表达和纯化突变和/或标记的蛋白质的改进的方案。为了促进单分子荧光和光捕获分析，我们进一步描述了更新的，易于使用的协议，用于连接微管盖玻片表面。所提出的协议连同最近解决的动力蛋白马达结构域的晶体结构将进一步简化和加速动力蛋白的假设驱动的诱变和结构-功能的研究。
Cytoplasmic dynein is the largest and most intricate cytoskeletal motor protein. It is responsible for a vast array of biological functions, ranging from the transport of organelles and mRNAs to the movement of nuclei during neuronal migration and the formation and positioning of the mitotic spindle during cell division. Despite its megadalton size and its complex design, recent success with the recombinant expression of the dynein heavy chain has advanced our understanding of dynein’s molecular mechanism through the combination of structure-function and single-molecule studies. Single-molecule fluorescence assays have provided detailed insights into how dynein advances along its microtubule track in the absence of load, while optical tweezers have yielded insights into the force generation and stalling behavior of dynein. Here, using the S. cerevisiae expression system, we provide improved protocols for the generation of dynein mutants and for the expression and purification of the mutated and/or tagged proteins. To facilitate single-molecule fluorescence and optical trapping assays, we further describe updated, easy-to-use protocols for attaching microtubules to coverslip surfaces. The presented protocols together with the recently solved crystal structures of the dynein motor domain will further simplify and accelerate hypothesis-driven mutagenesis and structure-function studies on dynein.