Regulations and Interactions amoung Molecular Motors

分子马达之间的调节和相互作用

• 批准号: 7691972
• 负责人: YALE E GOLDMAN
• 金额: $ 31.58万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7691972
• 关键词: 3-Dimensional; Actins; Activator Appliances; Address; Adipocytes; Affinity; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Area; Binding; Biochemical; Biochemistry; Biological Assay; Calmodulin; Cell physiology; Cells; Cellular biology; Characteristics; Charcot-Marie-Tooth Disease; Chemistry; Collaborations; Complex; Cytoplasm; Cytoskeleton; Data; Development; Diagnostic; Disease; Dissociation; Dynein ATPase; Electron Microscopy; Environment; Family; Generations; Head; Huntington Disease; Image; In Vitro; Individual; Investigation; Ions; Kinesin; Lateral; Learning; Length; Life; Light; Link; Measures; Mechanics; Microfilaments; Microtubules; Minus End of the Microtubule; Molecular; Molecular Motors; Motion; Motor; Motor Activity; Movement; Myosin ATPase; Myosin Type V; Neck; Neurons; Operative Surgical Procedures; Physiological; Plus End of the Microtubule; Polycystic Kidney Diseases; Power stroke; Principal Investigator; Process; Property; Regulation; Relative (related person); Research; Resolution; Role; Stroke; Structure; System; Techniques; Technology; Total Internal Reflection Fluorescent; Tweens; Upper arm; Vesicle; War; base; behavior observation; cell motility; cellular imaging; dynactin; fundamental research; light microscopy; lissencephaly; motor neuron degeneration; nanometer; optical traps; programs; research study; response; single molecule; structural biology; therapeutic target

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.

非常规肌球蛋白的结构变化开始使用以下组合来阐明： 单分子力学和光谱学方法沿着系综生物化学和结构 生物学它们的调节机制以及与其他分子马达的相互作用在很大程度上仍然是未知的。 未知我们假设，在颈部的IQ基序结合钙调素分子的结构变化， 肌球蛋白V和I在Ca 2+离子结合时改变杠杆臂的力学。我们以前的技术 设计用于调查的基本运动特性将被应用到这种调制的机制。 结合光学陷阱和单分子荧光显微镜将确定刚度变化， 钙调素亚基的旋转运动和旋转迁移率以及这些参数之间的关系 调节运动性。细胞质动力蛋白是一种利用ATP水解自由能的分子马达 以驱动货物沿着微管移动。对于广泛的细胞功能，激活剂复合物， 结合动力蛋白和微管的动力蛋白是必需的。Dynactin在货物结合中起作用， 而且还可以在力产生的机械化学中具有更积极的作用。动力蛋白，动力肌动蛋白， 非常规肌球蛋白和驱动蛋白通过彼此结合、与单个囊泡货物结合而相互作用， 通过它们在细胞质中的相互作用通过将几种分子马达类型整合到 可操纵的货物在体外，我们将寻求了解这些相互作用。我们将开发体外检测方法， 实现细胞复杂性的各个方面，例如肌动蛋白丝之间的交叉， 微管靠近表面和远离任何表面。这项工作开始了一个“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， 在体外和活细胞中以纳米精度进行分子跟踪。这些研究补充并联系了 通过提供适用于细胞生物学的机制， 结构研究与纯化的细胞骨架和电机组件的更简单的体外组装。