Myosin Va and VI Cargo Transport: In Vitro Model Systems

肌球蛋白 Va 和 VI 货物运输：体外模型系统

• 批准号: 8641390
• 负责人: David M Warshaw
• 金额: $ 29.43万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8641390
• 关键词: Actins; Adopted; Behavior; Biological Models; Cell physiology; Cells; Color; Cytoskeleton; Data; Dependence; Dimerization; Endocytic Vesicle; Endocytosis; Equilibrium; Exocytosis; Gene Mutation; Genes; Head; Hereditary Disease; Hypertrophic Cardiomyopathy; Immunologic Deficiency Syndromes; Impairment; In Vitro; Individual; Intracellular Transport; Kinetics; Label; Lasers; Lead; Link; MYO5A gene; Medial; Microfilaments; Microscope; Modeling; Molecular; Molecular Motors; Monitor; Mothers; Motion; Motor; Movement; Mutagenesis; Myosin ATPase; Nature; Neurologic; Outcome; Polystyrenes; Positioning Attribute; Quantum Dots; Resolution; Subarachnoid Hemorrhage; Tail; Techniques; Therapeutic; Total Internal Reflection Fluorescent; Travel; War; arm; base; biophysical techniques; design; in vitro Model; insulin granule; myosin VI; public health relevance; response; single molecule; triple helix; virtual

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.

描述（由申请人提供）：肌球蛋白 Va (myoVa) 和肌球蛋白 VI (myoVI) 是双头持续分子马达，可通过肌动蛋白丝轨道长距离运输细胞内货物。由于 myoVa 能够沿着极化肌动蛋白细胞骨架向相反方向移动，因此 myoVa 运输对于胞吐作用至关重要，而 myoVI 则与胞吞作用相关。为了成功运送货物，myoVa 和 myoVI 都必须克服细胞内环境以及与附着在同一货物上的其他马达的相互作用带来的物理挑战。为了评估这些电机固有的传输能力，我们将使用具有高空间（> 6nm）和时间（<2ms）分辨率的最先进的单分子生物物理技术来独立表征单个 myoVa 或 myoVI 电机的步进动力学或者在拔河比赛中连在一起时。目标#1 将确定 myoVa 分子的各个头如何协调和调整其步进行为，以维持响应阻力和辅助力的持续运动。这将通过在激光陷阱-TIRF 显微镜组合中对电机施加力时监控标有不同颜色量子点 (Qdot) 的各个头来实现。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/或内侧尾是否充当替代杠杆臂以允许 myoVI 逐步步进。最后，在目标 3 中，我们将通过将 myoVa 和 myoVI 电机连接到相同的 Qdot 货物来构建体外复杂性，作为多个电机进行细胞内货物运输的模型。当两个电机试图运输相同的货物时，我们将同时明确地确定它们的步进动态。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，因为它与分子马达集合的细胞内双向运输有关。