End-binding proteins are capable of tracking the plus-ends of growing microtubules (MTs). The motor protein Ncd, a member of the kinesin-14 family, interacts with EB1 protein and becomes a non-autonomous tip-tracker. Here, we attempted to find out whether at least for Ncd, the efficient EB1-mediated tip-tracking involves the interaction of the kinesin with the MT surface. We prepared a series of Ncd tail mutants in which the MT-binding sites were altered or eliminated. Using TIRF microscopy, we characterized their behavior as tip-trackers and measured the dwell times of single molecules of EB1 and Ncd tail or its mutated forms. The mutated forms of Ncd tail exhibited tip-tracking in the presence of EB1 and the effectiveness of this process was proportional to the affinity of the mutant's tail to MT. Even though the interaction of Ncd with EB1 was weak (Kd∼9μM) the half saturating concentration of EB1 for tip-tracking was 7nM. The dwell time of Ncd tail in the presence of EB1 was ∼1s. The dwell time of EB1 alone was shorter (∼0.3s) and increased considerably in the presence of a large excess of Ncd tail. We demonstrated that tip-tracking of kinesin-14 occurs through several concurrent mechanisms: binding of kinesin only to EB1 located at the MT end, interaction of the kinesin molecules with a composite site formed by EB1 and the MT tip, and probably surface diffusion of the tail along MT. The second mechanism seems to play a crucial role in efficient tip-tracking.

中文翻译：

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深入了解EB1依赖的驱动蛋白14 Ncd的末端跟踪过程。微管的作用。

末端结合蛋白能够追踪生长中的微管（MT）的正末端。运动蛋白Ncd是kinesin-14家族的成员，与EB1蛋白相互作用并成为非自主的末端追踪剂。在这里，我们试图找出至少对于Ncd而言，有效的EB1介导的末端追踪是否涉及驱动蛋白与MT表面的相互作用。我们准备了一系列Ncd尾突变体，其中MT结合位点被改变或消除。使用TIRF显微镜，我们将它们的行为表征为尖端跟踪器，并测量了EB1和Ncd尾部单分子或其突变形式的停留时间。Ncd尾巴的突变形式在EB1的存在下表现出尖端跟踪，并且此过程的有效性与突变体尾巴对MT的亲和力成正比。尽管Ncd与EB1的相互作用较弱（Kd〜9μM），但用于尖端跟踪的EB1的半饱和浓度为7nM。在EB1存在的情况下，Ncd尾部的停留时间约为1s。单独的EB1的停留时间较短（〜0.3s），并且在存在大量Ncd尾巴的情况下显着增加了停留时间。我们证明了驱动蛋白14的末端跟踪是通过几种同时发生的机制发生的：驱动蛋白仅与位于MT末端的EB1结合，驱动蛋白分子与EB1和MT末端形成的复合位点的相互作用，以及可能的表面扩散。 MT的尾巴。第二种机制似乎在有效的技巧跟踪中起着至关重要的作用。单独的EB1的停留时间较短（〜0.3s），并且在存在大量Ncd尾巴的情况下显着增加了停留时间。我们证明了驱动蛋白14的末端跟踪是通过几种同时发生的机制发生的：驱动蛋白仅与位于MT末端的EB1结合，驱动蛋白分子与EB1和MT末端形成的复合位点的相互作用，以及可能的表面扩散。 MT的尾巴。第二种机制似乎在有效的技巧跟踪中起着至关重要的作用。单独的EB1的停留时间较短（〜0.3s），并且在存在大量Ncd尾巴的情况下显着增加了停留时间。我们证明了驱动蛋白14的末端跟踪是通过几种同时发生的机制发生的：驱动蛋白仅与位于MT末端的EB1结合，驱动蛋白分子与EB1和MT末端形成的复合位点的相互作用，以及可能的表面扩散。 MT的尾巴。第二种机制似乎在有效的技巧跟踪中起着至关重要的作用。