The microtubule cytoskeleton is a major structural element inside cells that directs self‐organization using microtubule‐associated proteins and motors. It has been shown that finite‐sized, spindle‐like microtubule organizations, called “tactoids,” can form in vitro spontaneously from mixtures of tubulin and the antiparallel crosslinker, MAP65, from the MAP65/PRC1/Ase family. Here, we probe the ability of MAP65 to form tactoids as a function of the ionic strength of the buffer to attempt to break the electrostatic interactions binding MAP65 to microtubules and inter‐MAP65 binding. We observe that, with increasing monovalent salts, the organizations change from finite tactoids to unbounded length bundles, yet the MAP65 binding and crosslinking appear to stay intact. We further explore the effects of ionic strength on the dissociation constant of MAP65 using both microtubule pelleting and single‐molecule binding assays. We find that salt can reduce the binding, yet salt never negates it. Instead, we believe that the salt is affecting the ability of the MAP65 to form phase‐separated droplets, which cause the nucleation and growth of tactoids, as recently demonstrated.

中文翻译：

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离子强度改变交联剂驱动的微管自组织

微管细胞骨架是细胞内的主要结构元件，它利用微管相关蛋白和马达指导自组织。研究表明，有限尺寸的纺锤状微管组织（称为“类晶团簇”）可以在体外由微管蛋白和来自 MAP65/PRC1/Ase 家族的反向平行交联剂 MAP65 的混合物自发形成。在这里，我们探讨了 MAP65 形成类晶团簇的能力，作为缓冲液离子强度的函数，以试图打破 MAP65 与微管的静电相互作用以及 MAP65 间的结合。我们观察到，随着单价盐的增加，组织从有限的类晶团簇转变为无限长度的束，但 MAP65 结合和交联似乎保持完整。我们使用微管沉淀和单分子结合测定进一步探讨离子强度对 MAP65 解离常数的影响。我们发现盐可以减少结合，但盐永远不会消除它。相反，我们认为盐会影响 MAP65 形成相分离液滴的能力，从而导致类晶团聚体的成核和生长，正如最近所证明的那样。