微管是细胞骨架的重要成员，在细胞的结构支持，胞内运输和细胞内力的产生等方面都起着至关重要的作用。微管在细胞内是高度动态变化的，可以在生长和回缩之间随机切换，这一动态过程被称为微管动态不稳定性或微管动力学。微管动态不稳定性受到细胞内多种微管相关蛋白的调控。MCAK（Mitotic centromere-associated kinesin）是一种重要的微管相关蛋白，它参与了纺锤体的组装，染色体的集会和分离等过程。MCAK的生理功能与其对微管的调控是相关的，主要表现为解聚微管和增加微管崩塌频率。为了实现这些功能，MCAK需要到达微管末端并通过直接与之相互作用而施加调控作用。之前的研究表明，MCAK可能是通过直接结合或沿微管一维扩散的方式到达稳定微管的末端，或者通过与微管生长端结合蛋白EBs相互作用到达微管末端。但MCAK识别微管末端的分子机制及其调控微管动态不稳定性的特点仍然有待探究。 在本研究中，我们发现MCAK能够优先结合到微管生长末端，而且这种结合不具有协同性。然后，我们探究了MCAK识别微管末端的分子机制。通过对微管末端的GTP帽（GTP/GDP-Pi微管区域）和弯曲的原丝结构这两个耦合在一起的特征进行分别模拟，我们发现MCAK会优先结合弯曲的微管（即弯曲的原丝），并且对GTPγS（即GDP-Pi）微管有轻微的偏向性。这些结果表明MCAK主要是通过识别微管末端的弯曲结构的机制而特异性结合微管生长末端的。MCAK特异性的loop2结构在该识别机制中发挥着至关重要的作用。此外，MCAK的二聚体形式比MCAK的单体突变体对微管生长末端具有更高的亲和力。当研究MCAK在微管生长末端的定位时，我们发现MCAK与EB1具有相似的定位，位于GDP-Pi区域，而该区域距离微管最末端具有一定距离。因此，我们推测MCAK在解聚微管过程中，能够从微管末端解离原丝，而不是持续性解离单个的微管蛋白二聚体。在了解了MCAK的工作机制之后，我们进一步探究了它对微管动态不稳定性的影响。结果表明MCAK的调控作用随着微管生长速度的增加而减弱。这就说明MCAK是微管生长速度依赖型的崩塌因子。MCAK对于微管崩塌的调控作用表明，MCAK可能倾向于优先促使生长速度慢的微管崩塌，从而调节微管的数量与长度。 综上所述，我们的研究结果揭示了MCAK识别微管末端弯曲结构的分子机制并发现了MCAK作为微管生长速度依赖型崩塌因子参与微管调控的新机制。这些结果加深了我们对微管动态不稳定性调控机制的了解。
Microtubule is an important class of cytoskeleton that plays critical roles in structural support, intracellular transport and force generation in cells. Microtubules can switch between growing and shrinking phases in a stochastic manner, a process termed microtubule dynamic instability or microtubule dynamics. Microtubule dynamic instability is often regulated by microtubule-associated proteins (MAPs) in cells. MCAK (Mitotic centromere-associated kinesin) is an important MAP which participates in the processes of spindle assembly, chromosome congression, and chromosome segregation. The physiological functions of MCAK is related to regulating microtubules by serving as a microtubule depolymerase and/or a catastrophe factor. To achieve these functions, MCAK is expected to reach and work at microtubule tips. Previous studies have shown that MCAK can reach the tips of stable microtubules through direct binding or one-dimensional diffusion along the microtubules, and it can interact with EBs (end-binding proteins) to reach the microtubule growing tips. However, it still remains unclear how MCAK could directly recognize the microtubule growing tips and regulate microtubule dynamic instability? In the present study, we found that MCAK preferentially binds to microtubule growing tips by itself. This tip binding of MCAK shows a non-cooperative manner. Investigating how MCAK recognizes microtubule tips, we mimicked the two coupled features of microtubule growing tips: the GTP cap (the GTP/GDP-Pi microtubule region) and the curved protofilament, respectively. We found that MCAK preferentially binds to curved microtubules and has slight preference for GTPγS (i.e. GDP-Pi) microtubules, mainly suggesting a curvature recognition mechanism. The specific loop2 of MCAK play a crucial role in the curvature recognition mechanism. Moreover, Dimeric MCAK is more effective in identifying microtubule tips than the monomeric MCAK mutant. While investigating the location of MCAK at microtubule growing tips, we found that MCAK has a similar location to EB1, where is the GDP-Pi region, suggesting that MCAK can dissociate the protofilament from tips rather than removing tubulin dimers in a processive depolymerization. To investigate how MCAK regulates microtubule dynamic instability, we found that the effect is weaker while the microtubule growth rate is larger, suggesting that MCAK is a microtubule growth rate-dependent catastrophe factor. In this way, MCAK might selectively induce catastrophe of slowly growing microtubules, through which MCAK could in turn regulate the number and length of microtubules in cells. In all, we find the curvature recognition mechanism underlying the specific tip-binding behavior of MCAK and that the effects of MCAK on microtubule dynamic instability depend on the growth rate of microtubules. These findings provide further insight into the mechanism of MCAK regulating microtubule dynamic instability.
