Thomas Ezekiel C, Yue Yang, Pimm Morgan L, Hotta Takashi, Ohi Ryoma, Verhey Kristen J
Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA.
Cytoskeleton (Hoboken). 2025 Jul 12. doi: 10.1002/cm.70005.
Microtubules play essential roles in numerous cellular processes. All microtubules are built from the protein tubulin, yet individual microtubules can differ spatially and temporally due to their tubulin isotype composition and post-translational modifications (PTMs). The tubulin code hypothesis posits that these differences can regulate microtubule function. However, investigating the properties of specific tubulin PTMs in vitro has been challenging because most reconstitution assays rely on tubulin purified from brain tissue that contains highly heterogeneous and modified microtubules. In this study, we present an optimized method for the purification of milligram quantities of unmodified tubulin from large-scale cultures of HeLa S3 cells. We also describe steps for efficient chemical labeling of tubulin and the generation of controlled tubulin PTMs. These tubulins can be used in microscopy or biochemistry-based experiments to investigate how the tubulin code influences microtubule properties and functions. Overall, our method is easily adaptable, highly reproducible, and broadly accessible to labs with general equipment.
微管在众多细胞过程中发挥着至关重要的作用。所有微管均由微管蛋白构建而成，但由于其微管蛋白异构体组成和翻译后修饰（PTM）的不同，单个微管在空间和时间上可能存在差异。微管蛋白编码假说认为，这些差异能够调节微管功能。然而，在体外研究特定微管蛋白PTM的特性具有挑战性，因为大多数重组实验依赖于从脑组织中纯化的微管蛋白，而脑组织中含有高度异质且经过修饰的微管。在本研究中，我们提出了一种优化方法，可从大规模培养的HeLa S3细胞中纯化出毫克级的未修饰微管蛋白。我们还描述了微管蛋白高效化学标记以及可控微管蛋白PTM生成的步骤。这些微管蛋白可用于显微镜观察或基于生物化学的实验，以研究微管蛋白编码如何影响微管的特性和功能。总体而言，我们的方法易于调整、高度可重复，并且一般实验室借助通用设备即可广泛采用。
