MUTATIONAL STUDIES OF PROCESSIVE MYOSIN MOTORS

进行性肌球蛋白运动的突变研究

• 批准号: 7910491
• 负责人: KATHLEEN M TRYBUS
• 金额: $ 32.26万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7910491
• 关键词: Actins; Baculoviruses; Binding; Biological Assay; Calmodulin; Carrier Proteins; Cells; Collaborations; Complex; Cryoelectron Microscopy; Cytoskeleton; Disease; Drosophila genus; Electron Microscopy; Electrons; Engineering; Equilibrium; Fluorescence; Fluorescence Microscopy; Griscelli Syndrome; Head; Hypopigmentation; Impairment; In Vitro; Indium; Insecta; Intracellular Transport; Kinesin; Kinetics; Lead; Length; Mediating; Messenger RNA; Microtubules; Molecular Motors; Motion; Motor; Movement; Mus; Mutate; Mutation; Myosin ATPase; Myosin Type V; N-terminal; Neck; Neurologic; Nucleotides; Organelles; Pattern; Play; Property; Proteins; Reporting; Role; Running; Signal Transduction; Synapses; System; Tail; Techniques; Testing; Time; Vertebral column; Walking; Yeasts; arm; base; cell motility; dimer; human disease; insight; monomer; mutant; retinal rods; rho; single molecule

DESCRIPTION (provided by applicant): Processive myosins transport intracellular cargo, such as organelles and mRNA, along actin tracks for several micrometers before dissociating (i.e. processivity). We will probe the basis for processive motion in a double-headed (class V) and a single-headed (class IXb) myosin motor, and investigate how actin- and microtubule-based motors interact with each other. Mutant myosins will be expressed in the baculovirus/insect cell expression system. A key new technique is a TIRF (total internal reflectance fluorescence)-microscopy assay which will be used to directly assess processive run lengths and velocities of single-myosin molecules. Other assays include in vitro motility, kinetic analysis, and hydrodynamic techniques. In Aim #1 we seek to convert a non-processive class V myosin (Drosophila) into a processive motor (murine myosin V). Many differences between these two class V myosins are clustered in key regions in the motor domain. The neck region has been proposed to mediate coordination between the two-heads during processive motion. We will alter its compliance and assess the impact on processive movement. Lastly, we will test the idea that a monomer-dimer equilibrium regulates processivity in yeast class V myosins. In Aim #2 we will determine if the unique large insertion in loop 2 and the N-terminal extension is necessary for single-headed myosin IXb to achieve processivity. Actin-bound myosin IX will be visualized by electron cryomicroscopy to identify the structural role of the two unique insertions, and the mode of myosin IX binding to actin in different nucleotide states (consortium with Hanein). In Aim #3 we will test the hypothesis that myosin V and kinesin interact directly and co-operate to facilitate transport of cargo from microtubules to actin tracks. Processive motion will be followed at the single molecule level using TIRF-microscopy. Structural approaches (collaboration with K. Taylor) will be used to gain insight into how the motors interact in the absence or presence of their tracks. Mutations in myosin V lead to Griscelli syndrome (hypopigmentation and neurological impairment). Myosin IX plays a role in cell-signaling, because the GAP domain in its tail (its "cargo") is believed to regulate Rho-dependent remodeling of the cytoskeleton in motile cells and in the dynamic arrays of actin in synapses. A number of human diseases result from disruption of cargo transport along actin or microtubules, or from disease proteins acting as cargo for these transport proteins. A better understanding of the mechanism by which molecular motors move along their track is critical to further understand the basis of these diseases.

描述（由申请人提供）：进行性肌球蛋白在解离（即持续合成能力）之前沿着肌动蛋白轨道运输细胞内货物，如细胞器和mRNA。我们将探讨双头（V类）和单头（IXb类）肌球蛋白马达进行性运动的基础，并研究肌动蛋白和微管马达如何相互作用。突变肌球蛋白将在杆状病毒/昆虫细胞表达系统中表达。一项关键的新技术是TIRF（全内反射荧光）显微镜分析，将用于直接评估单个肌球蛋白分子的进行性运行长度和速度。其他测定包括体外运动性、动力学分析和流体动力学技术。在目标#1中，我们试图将非进行性V类肌球蛋白（果蝇）转化为进行性运动（鼠肌球蛋白V）。这两种V类肌球蛋白之间的许多差异集中在运动域的关键区域。颈部区域被认为是在进行性运动期间调节两个头部之间的协调。我们将改变其顺应性，并评估对进行性运动的影响。最后，我们将测试的想法，单体-二聚体平衡调节酵母V类肌球蛋白的持续合成能力。在目标#2中，我们将确定环2中独特的大插入和N-末端延伸是否是单头肌球蛋白IXb实现持续合成能力所必需的。肌动蛋白结合的肌球蛋白IX将通过电子冷冻显微镜观察，以确定两种独特插入的结构作用，以及肌球蛋白IX在不同核苷酸状态下与肌动蛋白结合的模式（与Hanein的联合体）。在目标#3中，我们将检验肌球蛋白V和驱动蛋白直接相互作用并合作促进货物从微管运输到肌动蛋白轨道的假设。将使用TIRF显微镜在单分子水平跟踪进行性运动。结构方法（与K.泰勒）将被用来深入了解电机如何相互作用，在没有或存在的轨道。肌球蛋白V的突变导致Griscelli综合征（色素减退和神经损伤）。肌球蛋白IX在细胞信号传导中起作用，因为其尾部差距结构域（其“货物”）被认为调节运动细胞和突触中肌动蛋白动态阵列中细胞骨架的Rho依赖性重塑。许多人类疾病是由于货物运输沿着肌动蛋白或微管的破坏，或者是由于疾病蛋白充当这些运输蛋白的货物。更好地理解分子马达沿其轨道沿着运动的机制对于进一步理解这些疾病的基础至关重要。