ACTIN-BINDING PROTEINS AND DROSOPHILA EMBRYOS

早期的果蝇胚胎被用来研究蛋白质的作用 与肌动蛋白合作，在这个巨大的细胞中产生有组织的细胞质， 高度活跃的合胞体细胞 肌动蛋白丝结构参与 在细胞质的组织和所需的动态事件中， 在正常的发展过程中。 因此，这些研究将提供深入了解 肌动蛋白介导的过程，有助于形成一个适当的 有组织的细胞胚盘作为分化的基质。 以来 肌动蛋白结构和过程与胚胎中的重要结构和过程相似 毫无疑问，在其他细胞的功能中发挥着重要作用， 对该模型系统的研究可能会揭示 肌动蛋白的组织和功能，将适用于所有细胞。 两种肌动蛋白相关蛋白，在早期胚胎中使用F-肌动蛋白鉴定 亲和层析和使用单克隆抗体的体内定位， 将详细研究。 将任一单克隆抗体注射到 早期胚胎会破坏正常发育。 的机理 使用荧光标记的细胞骨架 蛋白质，组蛋白和抗体，使许多主要的可视化 胚胎成分在抗体作用期间。 的 蛋白质将从早期胚胎中纯化， 涉及肌动蛋白的体外活性的方法和特征 细丝。 将使用抗体筛选表达文库， 检测表达这些蛋白质cDNA克隆。 一旦克隆编码 序列将被改变，以确定蛋白质的哪些区域 重要的功能。 将尝试体外和体内试验 以评估序列变化的影响。 双折射标记和 注射改变的蛋白质将允许蛋白质的功能和定位。 突变体蛋白在体内进行测定。 使用过表达的蛋白质， 将构建亲和矩阵来识别相互作用的组件 这些蛋白质在大分子组装体中的作用 在体内控制肌动蛋白聚合位点和时间。 研究这些和其他控制细胞周期协调的蛋白质 肌动蛋白重排发生在早期发展的 果蝇胚胎可以提供肌动蛋白协调的见解 与细胞周期中其他事件的重排， 肌动蛋白重排发生的机制。 自从重新安排 肌动蛋白在所有细胞中都是至关重要的，这些实验应该有助于 我们对肌动蛋白丝结构在空间中的定位的理解 并在适当的时候调节至稳定。

The early Drosophila embryo is being used to study the role of proteins that cooperate with actin to generate an organized cytoplasm in this large and highly dynamic syncytial cell. Actin filament structures participate in both the organization of the cytoplasm and the dynamic events required during normal development. Thus, these studies will provide insight into the actin-mediated process that contribute to the formation of a properly organized cellular blastoderm as a substrate of differentiation. Since actin structures and processes similar to those important in the embryo undoubtedly play important roles in the function of other cells, the studies of this model system will likely uncover general principles of actin organization and function that will apply to all cells. Two actin-associated proteins, identified in early embryos using F-actin affinity chromatography and located in vivo using monoclonal antibodies, will be studied in detail. Injections of either monoclonal antibody into early embryos causes disruption of normal development. The mechanism of disruption is being studied using fluorescently-labeled cytoskeletal proteins, histones and antibodies, allowing the visualization of many major embryonic components during the period when the antibody acts. The proteins will be purified from early embryos using standard biochemical methods and characterized for in vitro activities involving actin filaments. Expression libraries will be screened using the antibodies to detect cDNA clone that express these proteins. Once cloned coding sequences will be altered to determine which regions of the protein are important for function. Both in vitro and in vivo assays will be attempted to assess the impact of sequence changes. Fluorescent-labeling and injection of the altered protein will allow function and localization of mutant proteins to be assayed in vivo. Using overexpressed protein, affinity matricies will be constructed to identify componants that interact with these proteins in the macromolecular assemblies that are thought control actin polymerization sites and times in vivo. Study of these and other proteins that control the cell-cycle coordinated rearrangements of actin that occur during early development of the Drosophila embryo could provide insight into the coordination of actin rearrangements with the other events during the cell cycle and the mechanism by which actin rearrangements take place. Since rearrangements of actin are critical in all cells, these experiments should contribute to our understanding of how actin filament structures are positioned in space and regulated to polymerize at appropriate times.