Regulations and Interactions amoung Molecular Motors

非常规肌球蛋白的结构变化正开始被结合使用来阐明 单分子力学和光谱方法以及整体生物化学和结构 生物学。它们的调节和与其他分子马达的相互作用的机制在很大程度上仍然是 未知。我们假设，颈椎IQ模体结合的钙调蛋白分子的结构变化 当钙离子结合时，肌球蛋白V和I改变了杠杆臂的机械结构。我们以前使用的技术 设计用于研究基本的运动特性，将应用于这种调制的机理。 结合光学陷阱和单分子荧光显微镜将确定刚性的变化， 钙调蛋白亚基的旋转运动和旋转迁移率及其参数之间的关系 到调节运动能力。细胞质动力蛋白是一种分子马达，它利用三磷酸腺苷水解的自由能 以推动货物沿微管移动。对于广泛的细胞功能，激活剂复合体， 同时与动力蛋白和微管结合的动力蛋白是必需的。动力蛋白在货物结合中起作用， 但也可能在力产生的机械力化学中起到更积极的作用。动力蛋白，动力蛋白， 非传统的肌球蛋白和动蛋白通过相互结合相互作用，与单个囊泡货物和 通过它们在细胞质中的相互作用。通过将几种类型的分子马达整合到 在体外可操纵的货物，我们将试图了解这些相互作用。我们将在体外开发检测方法 实现了细胞复杂性的各个方面，例如肌动蛋白细丝和 靠近表面和远离任何表面的微管。这项工作开始了一条自下而上的路线 理解细胞运动的复杂性。我们将研究这些系统的分子机制。 使用新开发的技术，结合光学陷阱和偏振TIRF显微镜，以及3-D单 在体外和活细胞中以纳米精度进行分子跟踪。这些研究是相辅相成的。 通过提供适用于细胞生物学和 纯化的细胞骨架和运动部件的简单体外组装的结构研究。

The structural changes in unconventional myosins are beginning to be elucidated using a combination of single-molecule mechanical and spectroscopic approaches along with ensemble biochemistry and structural biology. The mechanisms of their regulation and interaction with other molecular motors are still largely unknown. We hypothesize that structural changes in calmodulin molecules bound to IQ motifs in the neck of myosin V and I alter the mechanics of the lever arm when Ca2+ ions bind. Techniques we previously devised for investigation of the basic motility properties will be applied to the mechanism of this modulation. Combined optical trap and single molecule fluorescence microscopy will determine the stiffness changes, rotational motions and rotational mobility of the calmodulin subunits and the relationship of these parameters to modulating motility. Cytoplasmic dynein is a molecular motor that uses the free energy of ATP hydrolysis to drive movement of cargo along microtubules. For a wide range of cellular functions, an activator complex, dynactin, which binds both to dynein and to microtubules is necessary. Dynactin has a role in cargo-binding, but also may also have a more active role in the mechano-chemistry of force generation. Dynein, dynactin, unconventional myosins, and kinesins interact by binding to each other, to individual vesicle cargoes and through their mutual interactions in the cytoplasm. By incorporating several molecular motor types onto manipulatable cargoes in vitro, we will seek to understand these interactions. We will develop assays in vitro that implement aspects of cellular complexity, such as intersections between actin filaments and microtubules near to a surface and away from any surfaces. This work begins a 'bottom up' route to understanding the complexities of cellular motility. We will study the molecular mechanisms of these systems using newly developed technologies, combined optical trap and polarized TIRF microscopy, and 3-D single molecule tracking at nanometer accuracy in vitro and in live cells. These studies complement and link strongly to all of the other sections and cores by providing mechanisms that apply to the cell biological and structural studies with simpler in vitro assemblies of purified cytoskeletal and motor components.