High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast
High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast
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DOI：
10.3791/55610
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发表时间：
2017-04-01
影响因子：
1.2
通讯作者：
Moore, Jeffrey K.
中科院分区：
文献类型：
--
作者：
Fees, Colby P.;Estrem, Cassi;Moore, Jeffrey K.
动态微管是许多细胞过程的基础，微管动力学的准确测量可以深入了解细胞如何调节这些过程以及基因突变如何影响调节。后生动物模型中微管动力学的定量具有许多相关的挑战，包括高微管密度和遗传操作的限制。相比之下，芽殖酵母模型提供了克服这些挑战的优势。该方案描述了一种测量活酵母细胞中单个微管动力学的方法。表达荧光标记的微管蛋白的细胞粘附到组装的载玻片室，允许稳定的延时图像采集。还提供了高速四维图像采集的详细指南，以及用于量化共聚焦图像堆栈中动态微管特性的协议。这种方法，与传统的酵母遗传学相结合，提供了一种方法，是唯一适合于定量评估微管调节剂或突变，改变微管蛋白亚基的活性的影响。
Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.