MUTATIONAL STUDIES OF PROCESSIVE MYOSIN MOTORS

描述(由申请人提供)：肌球蛋白在解离前沿肌动蛋白轨迹运输细胞内的货物，如细胞器和信使核糖核酸几微米(即加工性)。我们将探索双头(V类)和单头(IXb类)肌球蛋白马达的前进运动的基础，并研究基于肌动蛋白和微管的马达如何相互作用。突变的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，它将被用来直接评估单个肌球蛋白分子的前进长度和速度。其他分析方法包括体外动力、动力学分析和流体力学技术。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(小鼠肌球蛋白V)。这两个V类肌球蛋白之间的许多差异聚集在运动域的关键区域。颈部区域被认为在前进运动中调节两个头之间的协调。我们将改变其遵从性，并评估其对游行运动的影响。最后，我们将测试单体-二聚体平衡调节酵母V类肌球蛋白加工性的想法。在目标2中，我们将确定单头肌球蛋白IXb是否需要在环2中独特的大插入和N末端延伸来实现加工能力。肌球蛋白IX结合的肌球蛋白IX将通过电子冷冻显微镜可视化，以确定两个独特插入片段的结构作用，以及不同核苷酸状态下肌球蛋白IX与肌动蛋白结合的方式(与Hanein联合)。在目标3中，我们将测试肌球蛋白V和肌动蛋白直接相互作用和协作的假设，以促进货物从微管到肌动蛋白轨道的运输。使用TIRF显微镜，将在单分子水平上跟踪进程运动。结构方法(与K.Taylor合作)将用于深入了解发动机在没有或存在轨迹的情况下如何相互作用。肌球蛋白V的突变会导致Griscelli综合征(色素减少和神经功能障碍)。肌球蛋白IX在细胞信号转导中发挥作用，因为其尾部的间隙结构域(其“货物”)被认为调节运动细胞中依赖于Rho的细胞骨架重塑以及突触中肌动蛋白的动态排列。许多人类疾病是由于沿肌动蛋白或微管的货物运输中断，或由于疾病蛋白充当这些运输蛋白的货物。更好地了解分子马达沿其轨迹移动的机制对于进一步了解这些疾病的基础至关重要。

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.