Myosin Va and VI Cargo Transport: In Vitro Model Systems

描述（由申请人提供）：肌凝蛋白Va （myoVa）和肌凝蛋白VI （myoVI）是双头、过程性分子马达，通过肌动蛋白丝轨道远距离运输细胞内货物。由于它们能够沿着极化的肌动蛋白细胞骨架向相反方向移动，myoVa的运输对于胞吐作用至关重要，而myoVI则与内吞作用有关。为了成功地运送货物，myoVa和myoVI都必须克服细胞内环境和与附着在同一货物上的其他马达的相互作用所带来的物理挑战。为了评估这些马达的固有运输能力，我们将使用最先进的单分子生物物理技术，具有高空间（bbb6nm）和时间（<2ms）分辨率来表征单个myoVa或myoVI马达的步进动力学，无论是独立的还是在拉锯赛中连接在一起的。目标1将确定myoVa分子的单个头部如何协调并调整其步进行为，以保持对阻力和辅助力的响应的进程运动。这将通过在组合激光陷阱- tirf显微镜中监测标记有不同颜色量子点（Qdots）的单个头部来完成。在Aim #2中，myoVI将具有类似的特征。然而，myoVI的独特之处在于它需要意想不到的大步骤，挑战“摆动杠杆臂”模型。通过结构突变，我们将确定是否近端和/或内侧尾巴作为替代杠杆臂，以使myoVI逐步发展。最后，在Aim #3中，我们将通过将myoVa和myoVI马达连接到相同的Qdot货物，作为多个马达的细胞内货物运输模型，在体外构建复杂性。我们将明确地确定两个电机在试图运输相同货物时同时的步进动力学。利用目标1和目标2中收集的数据，我们将定量建模并预测这些拉锯战场景的结果，因为它与分子马达组合的细胞内双向运输有关。

DESCRIPTION (provided by applicant): Myosin Va (myoVa) and myosin VI (myoVI) are double-headed, processive molecular motors that transport intracellular cargo over long distances by way of actin filament tracks. With their ability to travel in opposite directions along the polarized actin cytoskeleton, myoVa transport is critical for exocytosis while myoVI is associated with endocytosis. To successfully deliver cargo, both myoVa and myoVI must overcome physical challenges presented by the intracellular milieu and the interactions with other motors that are attached to the same cargo. To assess these motors' inherent transport capabilities, we will use state-of-the- art single molecule biophysical techniques with high spatial (> 6nm) and temporal (<2ms) resolution to characterize the stepping dynamics of single myoVa or myoVI motors, independently or when linked together in a tug of war. Aim #1 will determine how the individual heads of a myoVa molecule coordinate and adjust their stepping behavior to maintain processive motion in response to both resistive and assistive forces. This will be accomplished by monitoring the individual heads that are labeled with different color quantum dots (Qdots) as force is applied to the motor in a combination laser trap-TIRF microscope. In Aim #2, myoVI will be similarly characterized. However, myoVI is unique in that it takes unexpectedly large steps, challenging the "swinging lever arm" model. Through structural mutagenesis, we will determine whether the proximal and/or medial tail serve as surrogate lever arms to allow myoVI to step processively. Finally in Aim #3, we will build complexity in vitro by linking myoVa and myoVI motors to the same Qdot cargo, as a model for intracellular cargo transport by multiple motors. We will unambiguously determine the stepping dynamics of both motors simultaneously as they attempt to transport the same cargo. With the data gathered in Aims #1 and #2, we will quantitatively model and predict the outcome of these tug of war scenarios as it relates to intracellular bidirectional transport by ensembles of molecular motors.