The microtubule (MT) cytoskeleton underlies processes such as intracellular transport and cell division. Immunolabeling for posttranslational modifications of tubulin has revealed the presence of different MT subsets, which are believed to differ in stability and function. Whereas dynamic MTs can readily be studied using live-cell plus-end markers, the dynamics of stable MTs have remained obscure due to a lack of tools to directly visualize these MTs in living cells. Here, we present StableMARK (Stable Microtubule-Associated Rigor-Kinesin), a live-cell marker to visualize stable MTs with high spatiotemporal resolution. We demonstrate that a rigor mutant of Kinesin-1 selectively binds to stable MTs without affecting MT organization and organelle transport. These MTs are long-lived, undergo continuous remodeling, and often do not depolymerize upon laser-based severing. Using this marker, we could visualize the spatiotemporal regulation of MT stability before, during, and after cell division. Thus, this live-cell marker enables the exploration of different MT subsets and how they contribute to cellular organization and transport.

中文翻译：

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活细胞标记物，可可视化整个细胞周期中稳定微管的动态

微管 (MT) 细胞骨架是细胞内运输和细胞分裂等过程的基础。微管蛋白翻译后修饰的免疫标记揭示了不同 MT 亚群的存在，这些亚群被认为在稳定性和功能上存在差异。虽然可以使用活细胞加端标记轻松研究动态 MT，但由于缺乏直接可视化活细胞中这些 MT 的工具，稳定 MT 的动态仍然模糊。在这里，我们提出了 StableMARK（稳定微管相关刚性驱动蛋白），这是一种活细胞标记物，可以以高时空分辨率可视化稳定的 MT。我们证明，Kinesin-1 的严格突变体选择性地结合稳定的 MT，而不影响 MT 组织和细胞器运输。这些 MT 寿命长，可连续重塑，并且在激光切割时通常不会解聚。使用这个标记，我们可以可视化细胞分裂之前、期间和之后 MT 稳定性的时空调节。因此，这种活细胞标记物能够探索不同的 MT 子集以及它们如何促进细胞组织和运输。