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、连续的分子马达，通过肌动蛋白细丝轨迹长距离运输细胞内的货物。由于它们能够沿着极化的肌动蛋白细胞骨架向相反的方向移动，所以myoVa转运对胞吞作用至关重要，而myoVI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带来的物理挑战，以及与连接到同一货物上的其他发动机的相互作用。为了评估这些马达的内在运输能力，我们将使用最先进的单分子生物物理技术，以高空间(&gt；6 nm)和时间(&lt；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拔河比赛中连接在一起的。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，以保持前进运动，以响应阻力和辅助力。这将通过监测在激光陷阱-TIRF组合显微镜中对电机施加压力时标记有不同颜色量子点(Qdots)的单个磁头来实现。在目标2中，myoVI将具有类似的特征。然而，myoVI的独特之处在于，它采取了出人意料的大步，挑战了“摆动杠杆臂”的模式。通过结构突变，我们将确定近端和/或内侧的尾巴是否作为代理杠杆臂，允许myoVI连续前进。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货物来建立体外复杂性，作为多个马达在细胞内运输货物的模型。我们将毫不含糊地同时确定两个电机在试图运输相同货物时的步进动力学。有了在AIMS#1和#2中收集的数据，我们将对这些拉锯战的结果进行定量建模和预测，因为它与分子马达集合在细胞内的双向运输有关。 公共卫生相关性：细胞内货物的运输是最基本的细胞过程之一，它分别依靠肌球蛋白Va和肌球蛋白VI等微小分子马达来运送从胰岛素颗粒到内吞囊泡的各种货物。随着myo5a基因突变导致神经功能障碍或免疫缺陷，myo6基因突变导致肥厚型心肌病，了解这些运动的正常功能对于这些遗传性疾病的治疗具有重要意义。

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. PUBLIC HEALTH RELEVANCE: The transport of intracellular cargo is one of the most basic cellular processes that relies on tiny molecular motors such as myosin Va and myosin VI to deliver cargoes ranging from insulin granules to endocytic vesicles, respectively. With genetic mutations in the myo5a gene resulting in neurological impairment or immunodeficiency and myo6 gene mutations leading to hypertrophic cardiomyopathy, understanding the normal function of these motors has major implications for therapeutic management of these genetic disorders.