Regulations and Interactions amoung Molecular Motors

分子马达之间的调节和相互作用

• 批准号: 8063028
• 负责人: YALE E GOLDMAN
• 金额: $ 32.19万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8063028
• 关键词: 3-Dimensional; ATP Hydrolysis; Actins; Activator Appliances; Address; Adipocytes; Affinity; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Area; Binding; Biochemical; Biochemistry; Biological; Biological Assay; Calmodulin; Cell physiology; Cells; Characteristics; Charcot-Marie-Tooth Disease; Chemistry; Collaborations; Complement; Complex; Cytoplasm; Cytoskeleton; Data; Development; Diagnostic; Disease; Dissociation; Dynein ATPase; Electron Microscopy; Environment; Family; Fluorescence Microscopy; Free Energy; 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; Physiological; Plus End of the Microtubule; Polycystic Kidney Diseases; Power stroke; Principal Investigator; Process; Property; Regulation; Relative (related person); Research; Resolution; Role; Route; Stroke; Structure; Surface; System; Techniques; Technology; Total Internal Reflection Fluorescent; Tweens; Vesicle; War; Work; arm; base; behavior observation; cell motility; cellular imaging; dynactin; fundamental research; in vitro Assay; light microscopy; lissencephaly; motor neuron degeneration; nanometer; operation; 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通过结合以下方法来阐明： 单分子机械和光谱方法以及整体生物化学和结构 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。我们假设钙调蛋白分子的结构变化与颈部的 IQ 基序结合。 当 Ca2+ 离子结合时，肌球蛋白 V 和 I 会改变杠杆臂的力学结构。我们之前的技术 为研究基本运动特性而设计的技术将应用于这种调制的机制。 结合光阱和单分子荧光显微镜将确定刚度变化， 钙调蛋白亚基的旋转运动和旋转迁移率以及这些参数的关系 来调节运动。细胞质动力蛋白是一种分子马达，利用 ATP 水解的自由能 驱动货物沿着微管运动。对于广泛的细胞功能，激活剂复合物， 动力蛋白（dynactin）既与动力蛋白又与微管结合，是必需的。 Dynactin 在货物结合中发挥作用， 但也可能在力产生的机械化学中发挥更积极的作用。动力蛋白、动力蛋白、 非常规肌球蛋白和驱动蛋白通过彼此结合、与单个囊泡货物和 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结合到 体外可操纵的货物，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，例如肌动蛋白丝和肌动蛋白丝之间的交叉点 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 了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，结合光阱和偏振 TIRF 显微镜，以及 3D 单 在体外和活细胞中以纳米精度进行分子追踪。这些研究相互补充和联系 通过提供适用于细胞生物和细胞的机制，强烈地影响所有其他部分和核心 通过更简单的纯化细胞骨架和运动组件的体外组装进行结构研究。