Serikbaeva Anara, Tvorogova Anna, Kauanova Sholpan, Vorobjev Ivan A
Department of Biology, School of Science and Technology, Nazarbayev University, Astana, Kazakhstan.
Department of Biology, School of Sciences and Technology, Nazarbayev University, Astana, Kazakhstan.
Methods Mol Biol. 2018;1745:181-204. doi: 10.1007/978-1-4939-7680-5_11.
Microtubules (MTs) are dynamic components of the cytoskeleton playing an important role in a large number of cell functions. Individual MTs in living cells undergo stochastic switching between alternate states of growth, shortening and attenuated phase, a phenomenon known as tempered dynamic instability. Dynamic instability of MTs is usually analyzed by labeling MTs with +TIPs, namely, EB proteins. Tracking of +TIP trajectories allows analyzing MT growth in cells with a different density of MTs. Numerous labs now use +TIP to track growing MTs in a variety of cell cultures. However, heterogeneity of MT dynamics is usually underestimated, and rather small sampling for the description of dynamic instability parameters is often used. The strategy described in this chapter is the method for repetitive quantitative analysis of MT growth rate within the same cell that allows minimization of the variation in MT dynamics measurement. We show that variability in MT dynamics within a cell when using repeated measurements is significantly less than between different cells in the same chamber. This approach allows better estimation of the heterogeneity of cells' responses to different treatments. To compare the effects of different MT inhibitors, the protocol using normalized values for MT dynamics and repetitive measurements for each cell is employed. This chapter provides detailed methods for analysis of MT dynamics in tissue cultures. We describe protocols for imaging MT dynamics by fluorescent microscopy, contrast enhancement technique, and MT dynamics analysis using triple color-coded display based on sequential subtraction analysis.
微管（MTs）是细胞骨架的动态组成部分，在大量细胞功能中发挥着重要作用。活细胞中的单个微管会在生长、缩短和衰减阶段的交替状态之间进行随机切换，这种现象被称为缓和动态不稳定性。微管的动态不稳定性通常通过用+TIPs（即EB蛋白）标记微管来分析。追踪+TIP轨迹可以分析不同微管密度细胞中的微管生长情况。现在许多实验室都使用+TIP来追踪各种细胞培养物中正在生长的微管。然而，微管动力学的异质性通常被低估，而且在描述动态不稳定性参数时经常使用相当小的样本量。本章所述的策略是一种在同一细胞内对微管生长速率进行重复定量分析的方法，它能够将微管动力学测量中的变化最小化。我们表明，使用重复测量时，细胞内微管动力学的变异性明显小于同一培养皿中不同细胞之间的变异性。这种方法能够更好地估计细胞对不同处理的反应的异质性。为了比较不同微管抑制剂的效果，采用了对微管动力学使用归一化值并对每个细胞进行重复测量的方案。本章提供了在组织培养中分析微管动力学的详细方法。我们描述了通过荧光显微镜成像微管动力学、对比度增强技术以及基于顺序减法分析的三色编码显示进行微管动力学分析的方案。
