STRUCTURE/INTERACTIONS OF ACTINS & ACTIN-BINDING PROTEIN

肌动蛋白在所有真核细胞中大量存在， 在收缩过程中的作用，以及在维护以及 细胞形状和细胞质一致性的动力学。 应用以企图 最终了解这些肌动蛋白为基础的过程的分子基础， 我们建议使用高分辨率电子显微镜和三维 (3D)图像分析和处理以开发一致的3D分子 模型的功能重要的肌动蛋白丝，并研究 肌动蛋白与其自身和 各种收缩、调节和细胞骨架蛋白。 使用 结构、蛋白质化学、免疫学和显微注射方法， 我还建议探讨突变肌动蛋白的作用，从稳定分离， 转化的人成纤维细胞。 具体而言，我们建议 进行如下操作： (1)为了在1-2 nm处确定肌动蛋白分子的一致3D模型， 不同类型的负染色和冷冻的分辨率水平 水合结晶肌动蛋白阵列。 (2)为了建立一个一致的肌动蛋白丝的3D分子模型， 各种负染色和冷冻水合肌动蛋白丝阵列， 在最终的细丝3D中排列分子肌动蛋白模型， 重建。 (3)绘制收缩、调节和细胞质的结合位点， 蛋白质的肌动蛋白分子通过三维结构分析， 化学计量修饰的结晶肌动蛋白阵列或来自共晶体 由肌动蛋白和肌动蛋白相互作用之间的特定复合物诱导 proteins. (4)对几种交联聚乙烯的结构和功能进行了分析， 肌动蛋白二聚体（a），以帮助开发一个一致的分子模型， 肌动蛋白丝（见2）和（B），以了解它们在调节肌动蛋白中的作用 聚合法 (5)为了研究体外聚合性能，三维结构， 和原位分布（即通过突变体的免疫荧光 肌动蛋白特异性抗体）和相互作用（即通过显微注射 突变肌动蛋白混合物）的突变β-肌动蛋白 人成纤维细胞。

Actin is found abundantly in all eukaryotic cells, where it plays a key role in contractile processes, and in the maintenance as well as the dynamics of cell shape and cytoplasmic consistency. In attempts to ultimately understand the molecular basis of these actin-based processes, we propose to use high resolution electron microscopy and three-dimensional (3D) image analysis and processing to develop a consistent 3D molecular model of the functionally important actin filament, and to investigate the structural basis of actin's specific interactions with itself and with various contractile, regulatory and cytoskeletal proteins. Using structural, proteinchemical, immunological and microinjection methods, we also propose to explore the role of mutant actins isolated from stably transformed human fibroblasts in neoplasia. Specifically, we propose to proceed as follows: (1) To determine a consistent 3D model of the actin molecule at the 1-2 nm resolution level from different types of negatively stained and frozen hydrated crystalline actin arrays. (2) To develop a consistent 3D molecular model of the actin filament from a variety of negatively stained and frozen hydrated actin filament arrays and to align molecular actin models within the resulting filament 3D reconstructions. (3) To map binding sites for contractile, regulatory and cytoplasmic proteins on the actin molecule via 3D structural analysis of stoichiometrically decorated crystalline actin arrays or from co-crystals induced from specific complexes between actin and actin-interacting proteins. (4) To structurally and functionally analyze several types of crosslinked actin dimers (a) to aid in developing a consistent molecular model of the actin filament (see 2) and (b) to understand their role in regulating actin polymerization. (5) To investigate the in vitro polymerization properties, 3D structure, and in situ distribution (i.e. via immunofluorescence with mutant actin-specific antibody) and interactions (i.e. via microinjection of mutant actin mixtures) of mutant beta-actins isolated from transformed human fibroblasts.