PROTEOMIC ANALYSIS OF DROSOPHILA GASTRULATION

果蝇原肠胚形成的蛋白质组学分析

• 批准号: 6706343
• 负责人: JONATHAN S MINDEN
• 金额: $ 21.72万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6706343
• 关键词: Drosophilidae; biotechnology; developmental genetics; embryo /fetus protein; embryogenic cleavage; fluorescent dye /probe; gel electrophoresis; gene targeting; invertebrate embryology; mass spectrometry; mesoderm; molecular cloning; proteasome; protein structure function; proteomics; time resolved data; transferrin

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.

描述（逐字附加研究者摘要）：我们感兴趣 了解参与细胞形状维持的机制和蛋白质 并使用果蝇中胚层形态发生作为模型系统进行改变。这 中胚层是由一大片矩形细胞内陷形成的 胚胎腹侧表面的形成过程称为腹侧沟形成。 这种内陷是由一系列细胞形状在 15 分钟内产生的 柱状上皮细胞转变为楔形的变化 沟细胞最终迁移并形成中胚层。 遗传分析揭示了参与信号级联的基因 导致腹侧沟细胞的规格。最终的基因在这个 信号级联是两个转录调节因子，twist 和 snail。胚胎 任一基因的突变体都无法形成腹侧皱纹。遗传 解剖无法识别任何细胞骨架相关或 结构蛋白作为细胞形状变化的机械介质。这是 可能是由于母体贡献、多效性的综合影响 功能和功能冗余。我们开发了一种新型生化药物 称为差异凝胶电泳 (DIGE) 的方法，用于鉴定蛋白质 腹沟形成过程中发生的变化。这项新技术是 改进的二维聚丙烯酰胺凝胶电泳方法 涉及在电泳前用不同颜色标记蛋白质 荧光染料。来自两种不同细胞类型的蛋白质被标记为 将不同的染料组合起来，然后在相同的电泳凝胶上运行。后 电泳，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显示为 双色斑点；仅一种细胞类型或另一种细胞类型独有的蛋白质 含有一种颜色的染料。这可以快速检测蛋白质差异， 所谓的“差异蛋白”。然后鉴定这些差异蛋白 通过质谱（MS）。迄今为止，已有二十多种差异蛋白 被检测到。这些包括母源供应和翻译后供应 修饰的蛋白质。 MS 已鉴定出其中三种蛋白质：两种 蛋白酶体亚基和转铁蛋白。该提案的目的是使用 DIGE/MS 鉴定剩余的 VF 差异蛋白用于克隆和 抗体的产生并确定这些差异蛋白在其中所起的作用 细胞形状调节和腹侧沟形态发生。对于后者，我们将 将延时显微镜与基因敲除和异位结合使用 克隆的差异蛋白的表达。

项目成果

Drosophila ventral furrow morphogenesis: a proteomic analysis.

果蝇腹沟形态发生：蛋白质组分析。

• 发表时间: 2004-02
• 作者: Gong, Lei;Puri, Mamta;Unlu, Mustafa;Young, Margaret;Robertson, Katherine;Viswanathan, Surya;Krishnaswamy, Arun;Dowd, Susan R;Minden, Jonathan S
• 通讯作者: Minden, Jonathan S