RUI: Dynamics of Actin Interactions and Structure

9316025纽曼将结合动态光散射、流变学和荧光显微镜技术研究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。动态光散射和流变学实验将确定溶剂(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和细胞松弛蛋白D的两个轻链亚型)对肌动蛋白自结合的动力学和状态的影响。在不同浓度的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细丝的动力学、成束、稳态尺寸和动态性质的影响。在拥挤的条件下，使用高浓度的蔗糖或聚乙二醇，在人工制备的小泡内和某些类型的上皮细胞内，肌动蛋白的自我结合也将使用荧光成像显微镜技术进行研究。这些研究将提供关于G-肌动蛋白的自身相互作用的详细信息，以及使用各种实验技术作为溶剂和肌动蛋白特异性结合分子的函数在体外和体内的寡聚体、细丝和肌动蛋白束的动态性质。将使用三种不同的物理技术来研究肌动蛋白的结构和相互作用，肌动蛋白是自然界中发现的最常见的蛋白质之一。肌动蛋白在决定细胞结构和机械性能中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量以改变细胞的形状或允许细胞主动移动方面发挥着独特的作用，就像许多细胞所能做到的那样。已经发现了许多其他蛋白质，它们与肌动蛋白特异地相互作用，或者调节或改变许多肌动蛋白在一起的结合，形成能够维持力量的长丝。这项拟议的研究将在一定程度上试图通过研究它们对试管中肌动蛋白的影响、在由内含肌动蛋白的脂泡组成的“人造”细胞中以及在活细胞中对肌动蛋白的影响来阐明几种更突出的此类蛋白质的作用。使用的方法包括动态激光光散射、粘弹性测量和荧光显微镜技术。计划进行两个主要领域的实验。第一个将探索特定蛋白质、盐或药物对肌动蛋白自结合的影响。这样的实验将帮助我们理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对实际自结合率和最终状态的影响，例如细胞内发现的情况。大多数溶液实验都是在只有其他小分子存在的情况下用纯化的蛋白质进行的。最近的研究表明，在其他大分子的存在下，不仅会对自相互作用的速率产生很大的影响，而且最终的聚集态也会受到很大影响。为了模拟细胞内的实际情况，添加不直接与肌动蛋白相互作用的惰性大分子将产生拥挤。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简单得多的环境中研究具有良好特性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。

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.