Abstract：
The microtubule cytoskeleton is essential for various biological processes such as the intracellular distribution of molecules and organelles, cell morphogenesis, chromosome segregation, and specification of the location of contractile ring formation. Distinct cell types contain microtubules with different extents of stability. For example, microtubules in neurons are highly stabilized to support organelle (or vesicular) transport over large distances, and microtubules in motile cells are more dynamic. In some cases, such as the mitotic spindle, both dynamic and stable microtubules coexist. Alteration of microtubule stability is connected to disease states, making understanding microtubule stability an important area of research. Methods to measure microtubule stability in mammalian cells are described here. Together, these approaches allow microtubule stability to be measured qualitatively or semiquantitatively following staining for post-translational modifications of tubulin or treating cells with microtubule destabilizing agents such as nocodazole. Microtubule stability can also be measured quantitatively by performing fluorescence recovery after photobleaching or fluorescence photoactivation of tubulin in live cells. These methods should be helpful for those seeking to understand microtubule dynamics and stabilization. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Fixing and staining cells for tubulin post-translational modifications Basic Protocol 2: Evaluating microtubule stability following treatment with nocodazole in live or fixed cells Basic Protocol 3: Measurement of microtubule dynamic turnover by quantification of fluorescence recovery after photobleaching Basic Protocol 4: Measurement of microtubule dynamic turnover by quantification of dissipation of fluorescence after photoactivation.
摘要：
微管细胞骨架对于各种生物过程至关重要，例如分子和细胞器的细胞内分布，细胞形态发生，染色体分离,以及收缩环形成位置的规范。不同的细胞类型包含具有不同稳定性程度的微管。例如,神经元中的微管高度稳定，以支持细胞器（或囊泡）远距离运输，活动细胞中的微管更具活力。在某些情况下,如有丝分裂纺锤体，动态和稳定的微管共存。微管稳定性的改变与疾病状态有关，使理解微管稳定性成为一个重要的研究领域。本文描述了测量哺乳动物细胞中微管稳定性的方法。一起,这些方法允许在微管蛋白的翻译后修饰染色或用微管去稳定剂如诺考达唑处理细胞后定性或半定量地测量微管稳定性。微管稳定性也可以通过在活细胞中光漂白或微管蛋白的荧光光活化后进行荧光恢复来定量测量。这些方法应该有助于那些寻求了解微管动力学和稳定性的人。©2023威利期刊有限责任公司。基本方案1:针对微管蛋白翻译后修饰对细胞进行固定和染色基本方案2:在活细胞或固定细胞中评价用诺科达唑处理后的微管稳定性基本方案3:通过定量光漂白后的荧光恢复来测量微管动态周转基本方案4:通过定量光活化后的荧光耗散来测量微管动态周转。
