Myosin Va and VI Cargo Transport: In Vitro Model Systems

描述（由申请人提供）：肌球蛋白Va（myoVa）和肌球蛋白VI（myoVI）是双头、进行性分子马达，其通过肌动蛋白丝轨道长距离运输细胞内货物。由于它们能够沿极化肌动蛋白细胞骨架在相反方向沿着行进，myoVa转运对于胞吐作用是关键的，而myoVI与胞吞作用相关。为了成功地递送货物，myoVa和myoVI都必须克服由细胞内环境以及与连接到相同货物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，我们将使用具有高空间（> 6 nm）和时间（<2 ms）分辨率的最先进的单分子生物物理技术来表征单个myoVa或myoVI马达的步进动力学，独立地或当在拔河中连接在一起时。目标#1将确定myoVa分子的各个头部如何协调和调整它们的步进行为，以响应于阻力和辅助力两者而保持进行性运动。这将通过在组合激光陷阱-TIRF显微镜中向电机施加力时监测标记有不同颜色量子点（Qdot）的各个头来实现。在目标#2中，myoVI将被类似地表征。然而，myoVI的独特之处在于它采取了意想不到的大步骤，挑战了“摆动杠杆臂”模型。通过结构诱变，我们将确定近端和/或内侧尾是否用作替代杠杆臂以允许myoVI进行前突。最后，在目标#3中，我们将通过将myoVa和myoVI马达连接到相同的Qdot货物来构建体外复杂性，作为多个马达的细胞内货物运输的模型。我们将明确地确定步进动力学的两个电机同时，因为他们试图运输相同的货物。利用目标#1和#2中收集的数据，我们将定量建模和预测这些拔河场景的结果，因为它涉及分子马达集合的细胞内双向转运。

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.