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: 8248783
• 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 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8248783
• 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; 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. 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.

描述（由申请人提供）：肌球蛋白VA（Myova）和肌球蛋白VI（Myovi）是双头的，过程的分子电动机，通过肌动蛋白细丝轨迹在长距离上运输细胞内货物。凭借其沿极化肌动蛋白细胞骨架的相反方向行进的能力，Myova转运对于胞吐作用至关重要，而MYOVI与内吞作用有关。要成功交付货物，Myova和Myovi都必须克服细胞内环境所带来的身体挑战，以及与同一货物附加的其他电动机的互动。为了评估这些电动机的固有运输能力，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表征单个Myova或Myovi Motors的阶梯动力学，或者是独立的，或者是在战争中链接在一起的。 AIM＃1将确定Myova分子的单个头如何坐标并调整其垫脚行为，以保持对电阻和辅助力的响应过程。这将通过监视用不同颜色量子点（QDOT）标记的单个头部来实现，因为将力在组合激光陷阱-TIRF显微镜中施加到电动机上。在AIM＃2中，Myovi将同样表征。但是，Myovi是独一无二的，因为它采取了出乎意料的大步，挑战了“摆动杠杆臂”模型。通过结构诱变，我们将确定近端和/或内侧尾部是否充当替代杆臂，以使Myovi进行过程上踩踏。最后，在AIM＃3中，我们将通过将Myova和Myovi电动机与同一QDOT货物联系起来，以在体外建立复杂性，这是多个电动机的细胞内货物运输模型。当他们试图运输同一货物时，我们将明确地确定两个电动机的垫脚动力学。随着数据收集在目标＃1和＃2中的数据，我们将定量建模并预测这些战争场景的结果，因为它与分子电动机集合的细胞内双向转运有关。 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，依赖于微小的分子电机，例如肌球蛋白VA和肌球蛋白VI，可分别提供从胰岛素颗粒到内吞囊泡的货物。由于肌5a基因的遗传突变导致神经系统障碍或免疫缺陷以及MyO6基因突变导致肥厚性心肌病，因此了解这些电机的正常功能对这些遗传疾病的治疗管理具有重大影响。