Using Budding Yeast as a Model to Understand Dynein-Mediated Cargo Transport.
Using Budding Yeast as a Model to Understand Dynein-Mediated Cargo Transport.
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DOI：
10.1007/978-1-0716-2958-1_2
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发表时间：
2023
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细胞质动力蛋白-1是一种负端定向微管运动蛋白，在整个进化谱中运输多种细胞类型的货物。动力蛋白受各种运动内在和外在因素的调节，这些因素可以增强其加工能力，将其招募到不同的细胞部位，或促进或限制其活性。在高等真核生物中研究动力蛋白的活性由于各种因素而变得复杂，包括这种马达参与的无数功能，以及随之而来的与破坏其活性相关的多效性效应。出芽酵母长期以来一直是了解这种巨大的运动蛋白复合物的强大模型系统，它在酵母和人类之间的初级序列和结构水平上高度保守。对出芽酵母的研究简化了，因为动力蛋白在这个生物体中只执行一个已知的功能：将有丝分裂纺锤体定位在细胞分裂的位置。监测出芽酵母中动力蛋白介导的纺锤体运动为定量测量各种运动参数提供了有力的工具，也是评估动力蛋白或其调节因子突变后果的系统。在这里，我们提供了详细的方案来执行动态蛋白活性的定量测量活细胞使用荧光显微镜和计算方法的组合来跟踪和定量动态蛋白介导的纺锤体运动。这些方法广泛适用于任何希望对出芽酵母进行荧光显微镜检查的人。
Cytoplasmic dynein-1 is a minus end-directed microtubule motor protein that transports numerous cargoes in cell types throughout the evolutionary spectrum. Dynein is regulated by various motor-intrinsic and extrinsic factors that enhance its processivity, recruit it to various cellular sites, or otherwise promote or restrict its activity. Studying dynein activity in higher eukaryotes is complicated by various factors, including the myriad functions in which this motor participates, and the consequential pleotropic effects associated with disrupting its activity. Budding yeast has long been a powerful model system for understanding this enormous motor protein complex, which is highly conserved between yeast and humans at the primary sequence and structural levels. Studies in budding yeast are simplified by the fact that dynein only performs one known function in this organism: to position the mitotic spindle at the site of cell division. Monitoring dynein-mediated spindle movements in budding yeast provides a powerful tool for the quantitative measurements of various motility parameters, and a system with which to assess the consequence of mutations in dynein or its regulators. Here we provide detailed protocols to perform quantitative measurements of dynein activity in live cells using a combination of fluorescence microscopy and computational methods to track and quantitate dynein-mediated spindle movements. These methods are broadly applicable to anyone that wishes to perform fluorescence microscopy on budding yeast.