Microtubules are highly dynamic filaments with dramatic structural rearrangements and length changes during the cell cycle. An accurate control of the microtubule length is essential for many cellular processes, in particular during cell division. Motor proteins from the kinesin-8 family depolymerize microtubules by interacting with their ends in a collective and length-dependent manner. However, it is still unclear how kinesin-8 depolymerizes microtubules. Here, we tracked the microtubule end-binding activity of yeast kinesin-8, Kip3, under varying loads and nucleotide conditions using high-precision optical tweezers. We found that single Kip3 motors spent up to 200 s at the microtubule end and were not stationary there but took several 8-nm forward and backward steps that were suppressed by loads. Interestingly, increased loads, similar to increased motor concentrations, also exponentially decreased the motors' residence time at the microtubule end. On the microtubule lattice, loads also exponentially decreased the run length and time. However, for the same load, lattice run times were significantly longer compared to end residence times, suggesting the presence of a distinct force-dependent detachment mechanism at the microtubule end. The force dependence of the end residence time enabled us to estimate what force must act on a single motor to achieve the microtubule depolymerization speed of a motor ensemble. This force is higher than the stall force of a single Kip3 motor, supporting a collective force-dependent depolymerization mechanism that unifies the so-called "bump-off" and "switching" models. Understanding the mechanics of kinesin-8's microtubule end activity will provide important insights into cell division with implications for cancer research.

中文翻译：

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kinesin-8 Kip3 以集体力依赖机制解聚微管

微管是高度动态的细丝，在细胞周期中具有显着的结构重排和长度变化。微管长度的准确控制对于许多细胞过程至关重要，尤其是在细胞分裂过程中。来自驱动蛋白 8 家族的运动蛋白通过以集体和长度依赖的方式与其末端相互作用来解聚微管。然而，目前尚不清楚 kinesin-8 如何解聚微管。在这里，我们使用高精度光镊在不同负载和核苷酸条件下跟踪了酵母驱动蛋白 8、Kip3 的微管末端结合活性。我们发现单个 Kip3 电机在微管末端花费了长达 200 s 并且在那里不是静止的，而是进行了几个 8 nm 的向前和向后步骤，这些步骤受到负载的抑制。有趣的是，增加了负载，类似于增加的运动浓度，也呈指数减少了马达在微管末端的停留时间。在微管晶格上，负载也以指数方式减少运行长度和时间。然而，对于相同的负载，与末端停留时间相比，晶格运行时间明显更长，这表明微管末端存在明显的力依赖性分离机制。最终停留时间的力依赖性使我们能够估计必须作用在单个电机上的力才能达到电机整体的微管解聚速度。该力高于单个 Kip3 电机的失速力，支持一种集体力依赖解聚机制，该机制统一了所谓的“撞击”和“切换”模型。了解kinesin-8'的机制