PROTEOMIC ANALYSIS OF DROSOPHILA GASTRULATION

描述（从研究者摘要中逐字附上）：我们感兴趣 在理解维持细胞形状的机制和蛋白质方面， 并使用果蝇中胚层形态发生作为模型系统进行改变。的 中胚层是由一大片矩形细胞内陷形成的， 在一个叫做腹沟形成的过程中胚胎的腹面。 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 沟细胞，最终成为迁移和形成中胚层。 遗传分析揭示了参与信号级联的基因， 导致腹沟细胞的特化。最后一个基因 信号级联是两种转录调控因子，twist和snail。胚胎 不能形成腹沟。遗传 解剖还不能鉴定出任何与细胞学相关的或 结构蛋白作为细胞形状变化的机械介质。这是 可能是由于母亲的贡献，多效性， 功能和功能冗余。我们开发了一种新的生化 一种用于鉴定蛋白质的方法，称为差异凝胶电泳（DIGE） 在腹沟形成过程中发生的变化。这项新技术是 改良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 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，然后在相同的电泳凝胶上运行。后 电泳时，以大格式记录一对荧光图像 荧光成像仪两种细胞类型共有的蛋白质表现为 双色点;蛋白质是唯一的一种细胞类型或其他只 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”。“这些差异蛋白质 质谱（MS）。到目前为止，已经有二十多种差异蛋白 被发现。这些包括母亲提供的和产后提供的 改性蛋白质其中三种蛋白质已被MS鉴定： 蛋白酶体亚基和转铁蛋白。本提案的目的是利用 DIGE/MS以鉴定用于克隆的剩余VF差异蛋白， 抗体的产生，并确定这些差异蛋白在 细胞形状调节和腹沟形态发生。对于后者，我们将 使用延时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.