PROTEOMIC ANALYSIS OF DROSOPHILA GASTRULATION

描述(逐字摘自调查人员摘要)：我们感兴趣 了解细胞形状维持的机制和蛋白质 并以果蝇中胚层形态发生为模式系统进行变化。这个 中胚层是由一大片长方形细胞内陷而成。 胚胎的腹面在称为腹面皱纹形成的过程中。 这种内陷是在15分钟内由一系列细胞形状引起的 将柱状上皮细胞转变为楔形上皮细胞的变化 皱纹细胞，最终变成迁徙并形成中胚层。 遗传分析揭示了参与信号级联反应的基因 导致腹侧沟细胞的特征。这里面的最终基因 信号级联有两个转录调控因子，twist和Snail。胚胎 任何一种基因的突变都不能形成腹侧皱纹。遗传 解剖未能发现任何与细胞骨架相关的或 结构蛋白作为细胞形态变化的机械中介。这是 可能是由于母亲的贡献，多效性的综合作用 功能和功能冗余。我们开发了一种新型的生物化学 一种称为差示凝胶电泳法(DGE)的蛋白质鉴定方法 在腹侧沟形成过程中发生的变化。这种新技术是一种 改进的双向聚丙烯酰胺凝胶电泳法 包括在电泳前用不同颜色的蛋白质进行标记 荧光染料。来自两种不同细胞类型的蛋白质用 不同的染料混合在一起，然后在同一电泳胶上运行。之后 在电泳法中，一对荧光图像以大格式记录 荧光成像仪。两种细胞共有的蛋白质表现为 双色点；只有一种或另一种细胞所特有的蛋白质 含有一种颜色的染料。这允许快速检测蛋白质差异， 所谓的“差异蛋白质”。然后对这些差异蛋白质进行鉴定 用质谱仪(MS)分析。到目前为止，有20多种差异蛋白质 已被检测到。这些包括母亲提供的和翻译后提供的 修饰的蛋白质。其中三种蛋白质已被MS鉴定：两种 蛋白酶体亚基和转铁蛋白。这项提议的目的是利用 DGE/MS鉴定剩余的VF差异-蛋白质用于克隆和 产生抗体，并确定这些差异蛋白在 细胞形态调控与腹侧沟形态发生。对于后者，我们将 使用时间推移显微镜结合基因敲除和异位 克隆的差异蛋白的表达。

DESCRIPTION (appended verbatim from investigator's abstract): We are interested in understanding the mechanisms and proteins involved in cell shape maintenance and change using Drosophila mesoderm morphogenesis as a model system. The mesoderm is formed by the invagination of a large rectangular patch of cells on the ventral surface of the embryo in a process called ventral furrow formation. This invagination is brought about in 15 minutes by a series of cell shape changes that transform the columnar-shaped epithelial cells into wedge-shaped furrow cells, which eventually become migratory and form the mesoderm layer. Genetic analysis has revealed the genes involved in the signaling cascade that leads to the specification of the ventral furrow cells. The final genes in this signaling cascade are two transcriptional regulators, twist and snail. Embryos that are mutant for either gene fail to form a ventral furrow. Genetic dissection has not been able to identify any cytoskeletal-associated or structural proteins as mechanical mediators of the cell shape changes. This is probably due to the combined effects of maternal contribution, pleiotropic function and functional redundancy. We have developed a novel biochemical approach, called difference gel electrophoresis (DIGE), for identifying protein changes that occur during ventral furrow formation. This new technique is a modified two-dimensional polyacrylamide gel electrophoresis method that involves labeling of proteins prior to electrophoresis with differently-colored fluorescent dyes. Proteins from two different cell types are labeled with the different dyes, combined, and then run on the same electrophoresis gel. After electrophoresis, a pair of fluorescence images is recorded with a large format fluorescence imager. Proteins that are common to both cell types appear as two-colored spots; proteins that are unique to one cell type or the other only contain one color dye. This allows for rapid detection of protein differences, so called "difference-proteins." These difference-proteins are then identified by mass spectrometry (MS). To date, more than twenty difference-proteins have been detected. These include maternally supplied and post-translationally modified proteins. Three of these proteins have been identified by MS: two proteasome subunits and transferrin. The aims of this proposal are to use DIGE/MS to identify the remaining VF difference-proteins for cloning and antibody production and to determine the role these difference-proteins play in cell shape modulation and ventral furrow morphogenesis. For the latter, we will use time-lapse-microscopy in conjunction with gene knock-out and ectopic expression of the cloned difference-proteins.