Fees Colby P, Estrem Cassi, Moore Jeffrey K
Department of Cell and Developmental Biology, University of Colorado School of Medicine.
Department of Cell and Developmental Biology, University of Colorado School of Medicine;
J Vis Exp. 2017 Apr 20(122):55610. doi: 10.3791/55610.
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.
动态微管对于许多细胞过程至关重要，准确测量微管动力学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量化存在许多相关挑战，包括微管密度高和基因操作的局限性。相比之下，芽殖酵母模型具有克服这些挑战的优势。本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。表达荧光标记微管蛋白的细胞附着在组装好的载玻片室上，以便进行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，以及共聚焦图像堆栈中动态微管特性量化的方案。这种方法与传统酵母遗传学相结合，提供了一种独特适合定量评估微管调节剂或改变微管蛋白亚基活性的突变的影响的方法。
