PDF(Pubmed)
Abstract：
Kinesin-1 ensembles maneuver vesicular cargoes through the three-dimensional (3D) intracellular microtubule (MT) network. To define how such cargoes navigate MT intersections, we first determined how many kinesins from an ensemble on a lipid-based cargo simultaneously engage a MT, and then determined the directional outcomes (straight, turn, terminate) for liposome cargoes at perpendicular MT intersections. Run lengths of 350-nm diameter liposomes decorated with up to 20, constitutively active, truncated kinesin-1 KIF5B (K543) were longer than single motor transported cargo, suggesting multiple motor engagement. However, detachment forces of lipid-coated beads with ~20 kinesins, measured using an optical trap, showed no more than three simultaneously engaged motors, with a single engaged kinesin predominating, indicating anticooperative MT binding. At two-dimensional (2D) and 3D in vitro MT intersections, liposomes frequently paused (~2 s), suggesting kinesins simultaneously bind both MTs and engage in a tug-of-war. Liposomes showed no directional outcome bias in 2D (1.1 straight:turn ratio) but preferentially went straight (1.8 straight:turn ratio) in 3D intersections. To explain these data, we developed a mathematical model of liposome transport incorporating the known mechanochemistry of kinesins, which diffuse on the liposome surface, and have stiff tails in both compression and extension that impact how motors engage the intersecting MTs. Our model predicts the ~3 engaged motor limit observed in the optical trap and the bias toward going straight in 3D intersections. The striking similarity of these results to our previous study of liposome transport by myosin Va suggests a \"universal\" mechanism by which cargoes navigate 3D intersections.
摘要：
Kinesin-1合奏通过三维（3D）细胞内微管（MT）网络操纵囊泡。要定义此类货物如何在MT交叉路口中导航，我们首先确定了来自基于脂质的货物上的合奏中的多少驱动蛋白同时参与MT，然后确定方向性结果(直，转,终止)用于垂直MT交叉点处的脂质体货物。350nm直径的脂质体的运行长度装饰有多达20个组成型活性，截短驱动蛋白-1KIF5B(K543)比单电机运输货物长，建议多马达接合。然而,具有约20种驱动蛋白的脂质涂层珠子的分离力，使用光学陷阱测量，显示不超过三个同时接合的电机，单一参与驱动蛋白占主导地位，表明反合作MT结合。在二维(2D)和3D体外MT交叉点，脂质体经常停顿（~2s），提示驱动蛋白同时结合两个MT并进行拔河。脂质体在2D（1.1直线：转弯比）中没有方向结果偏差，但在3D交叉点中优先直行（1.8直线：转弯比）。为了解释这些数据，我们建立了脂质体运输的数学模型，结合已知的驱动蛋白的机械化学，在脂质体表面扩散，并且在压缩和延伸方面都有坚硬的尾部，这影响了电机如何与相交的MT接合。我们的模型预测了在光阱中观察到的〜3接合运动极限，以及在3D交叉点中直行的偏向。这些结果与我们先前通过肌球蛋白Va进行的脂质体转运研究的惊人相似性表明了一种“通用”机制，通过该机制，货物可以导航3D交叉点。
