RUI: Dynamics of Actin Interactions and Structure

RUI：肌动蛋白相互作用和结构的动力学

• 批准号: 9316025
• 负责人: Jay Newman
• 金额: $ 15.8万
• 依托单位: Union College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-03-15 至 1998-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9316025&HistoricalAwards=false
• 关键词: RUI; Dynamics; Actin; Interactions; Structure

9316025 Newman The effect of the state of G-actin on its interactions, polymerization kinetics and polymer structure will be investigated by a combination of dynamic light scatting, rheology, and fluorescence microscopy techniques. Dynamic light scattering and rheology experiments will be performed to determine the effects of the solvent (divalent cation, nucleotide, and temperature) and the presence of certain actin binding moieties (including the two light chain isoforms of myosin S-1, profilin, and cytochalasin D) on the kinetics and state of actin self-association. Further measurements in the presence of various high concentrations of an inert low molecular weight polymer (polyethylene glycol, PEG) will study the effects of crowding on the kinetics, bundling, and steady-state sizes and dynamic properties of actin filaments. The self- association of actin under crowded conditions, using high concentrations of sucrose or PEG, within artificially-prepared vesicles and within certain epithelial cell types will also be studied using fluorescence imaging microscopy techniques. These studies will provide detailed information on the self-interactions of G-actin and the dynamic properties of the oligomers, filaments, and actin bundles, both in vitro and in vivo, as functions of the solvent and actin-specific binding molecules using a variety of experimental techniques. %%% Three different physical techniques will be used to study the structure and interactions of the protein actin, one of the most common proteins found in nature. Actin's role in determining the structure and mechanical properties of cells is incompletely known. It is clear, however, that actin plays a unique role in generating forces within a cell in order to change the cell's shape or to allow the cell to move actively, as many cells can. A variety of other proteins have been discovered which interact specifically with actin and either regulate or modify the association of many actins togethe r to form long filaments which are capable to sustaining a force. The proposed research will, in part, attempt to elucidate the role of several of the more prominent such proteins by studying their effects on actin in a test tube, in an "artificial" cell constructed from lipid vesicles with actin incorporated inside, and in a living cell. Methods used will include dynamic laser light scattering, viscoelasticity measurements, and fluorescence microscopy techniques. Two main areas of experiments are planned. The first will probe the effects of specific proteins, salts, or drugs on the self- association of actins. Such experiments will help us to understand the details of filament formation in cells leading to eventual force generation, and some of the control mechanisms involved. The second area will study the effects of crowded conditions, such as are found within cells, on the actual self-association rates and final states. Most solution experiments are carried out with purified proteins in the presence of only other small molecules. Recently it has been demonstrated that in the presence of other large macromolecules, not only the rates of self-interaction, but also the final aggregation state can be greatly influenced. Crowding will be produced by the addition of inert macromolecules, which do not directly interact with actin, in order to simulate the actual conditions within cells. These experiments will be carried out both in solutions and in artificial cells, and will be compared to experiments performed in living cells using fluorescence methods. In this way we will attempt to study the interactions of well-characterized proteins in much simpler environments that those of cells, but be able to draw meaningful conclusions about the role of these interactions in living cells.

9316025 Newman 将通过动态光散射、流变学和荧光显微镜技术的结合来研究 G-肌动蛋白的状态对其相互作用、聚合动力学和聚合物结构的影响。 将进行动态光散射和流变学实验，以确定溶剂（二价阳离子、核苷酸和温度）和某些肌动蛋白结合部分（包括肌球蛋白 S-1、profilin 和细胞松弛素 D 的两种轻链亚型）的存在对肌动蛋白自缔合动力学和状态的影响。 在各种高浓度惰性低分子量聚合物（聚乙二醇，PEG）存在下的进一步测量将研究拥挤对肌动蛋白丝的动力学、成束、稳态尺寸和动态特性的影响。 肌动蛋白在拥挤条件下的自缔合，使用高浓度的蔗糖或PEG，在人工制备的囊泡内和某些上皮细胞类型内，也将使用荧光成像显微镜技术进行研究。 这些研究将使用各种实验技术，提供关于 G-肌动蛋白的自相互作用以及体外和体内低聚物、丝和肌动蛋白束的动态特性的详细信息，作为溶剂和肌动蛋白特异性结合分子的功能。 %%% 将使用三种不同的物理技术来研究肌动蛋白（自然界中最常见的蛋白质之一）的结构和相互作用。 肌动蛋白在决定细胞结构和机械特性方面的作用尚不完全清楚。 然而，很明显，肌动蛋白在细胞内产生力方面发挥着独特的作用，以改变细胞的形状或允许细胞像许多细胞一样主动移动。 已经发现了多种其他蛋白质，它们与肌动蛋白特异性相互作用，并调节或修饰许多肌动蛋白在一起的缔合，以形成能够维持力的长丝。 拟议的研究将在一定程度上试图通过研究试管中、由内含肌动蛋白的脂质囊泡构建的“人造”细胞以及活细胞中的肌动蛋白的影响来阐明几种更重要的此类蛋白质的作用。 使用的方法包括动态激光散射、粘弹性测量和荧光显微镜技术。 计划进行两个主要实验领域。 第一个将探讨特定蛋白质、盐或药物对肌动蛋白自缔合的影响。 此类实验将帮助我们了解细胞中导致最终力产生的细丝形成的细节，以及涉及的一些控制机制。 第二个领域将研究拥挤条件（例如细胞内的拥挤条件）对实际自关联率和最终状态的影响。 大多数溶液实验都是在仅存在其他小分子的情况下使用纯化的蛋白质进行的。 最近已经证明，在存在其他大分子的情况下，不仅自相互作用的速率，而且最终的聚集状态都会受到很大的影响。 通过添加不与肌动蛋白直接相互作用的惰性大分子来产生拥挤，以模拟细胞内的实际条件。 这些实验将在溶液和人造细胞中进行，并将与使用荧光方法在活细胞中进行的实验进行比较。 通过这种方式，我们将尝试在比细胞更简单的环境中研究特征明确的蛋白质的相互作用，但能够就这些相互作用在活细胞中的作用得出有意义的结论。