STRUCTURE/INTERACTIONS OF ACTINS & ACTIN-BINDING PROTEIN

肌动蛋白广泛存在于所有真核细胞中，在真核细胞中起着关键作用。 在收缩过程中的作用，以及在维护以及 细胞形态和细胞质一致性的动态变化。在试图 最终了解这些基于肌动蛋白的过程的分子基础， 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 功能上重要的肌动蛋白细丝的模型，并研究 肌动蛋白与自身及与肌动蛋白特定相互作用的结构基础 各种收缩、调节和细胞骨架蛋白。vbl.使用 结构、蛋白质化学、免疫学和微量注射方法，我们 还建议探索从稳定株中分离的突变肌动蛋白的作用。 肿瘤中转化的人成纤维细胞。具体来说，我们建议 请按以下步骤操作： (1)确定在1-2 nm处的肌动蛋白分子的一致3D模型 从不同类型的负染和冻结的分辨率 水合晶体肌动蛋白阵列。 (2)开发一个一致的肌动蛋白细丝的3D分子模型 各种负染和冷冻的水合肌动蛋白细丝阵列和 在生成的细丝3D内对齐分子肌动蛋白模型 重建。 (3)绘制收缩、调节和细胞质的结合部位图 肌动蛋白分子上蛋白质的三维结构分析 化学计量学修饰的晶体肌动蛋白阵列或来自共晶体 肌动蛋白与肌动蛋白相互作用的特异性复合体诱导 蛋白质。 (4)对几种类型的交联剂进行了结构和功能分析 肌动蛋白二聚体(A)，以帮助建立一个一致的分子模型 肌动蛋白细丝(见2)和(B)，以了解它们在调节肌动蛋白中的作用 聚合反应。 (5)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(即通过微量注射 突变肌动蛋白混合物)从转化的 人类成纤维细胞。

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.