Identification and characterization of new mutants affecting Notch trafficking

这个项目致力于通过细胞内膜隔间，对影响Notch和其他潜在分泌底物运输的新突变体进行遗传筛选。尽管对影响细胞增殖、极性和模式的突变体的筛选已经导致了对影响内体Notch运输的基因的鉴定，但对直接改变Notch细胞分布的突变还没有进行系统的筛选。基于我们之前对钙-ATPase突变对Notch通过分泌途径运输的影响的分析，果蝇盘细胞的高度极化结构，以及可用于在发育中的果蝇组织中产生致命突变克隆的基因技术，我们认为果蝇是尝试这种类型的细胞生物学筛选的合适模型。为了进行筛选，我们对雄性果蝇进行诱变，建立平衡种群，诱导对单个诱变染色体臂纯合的组织克隆，并分析Notch蛋白在突变克隆中相对于邻近野生型组织的分布。目前，我们正在完成第二条染色体的筛选。已经筛选了大约4000条突变的染色体臂，导致了91个突变的恢复，这些突变定义了20多个不同的基因(每个基因由2-8个突变等位基因代表)。不同的突变体可以根据它们对Notch转运和受影响的细胞膜间隔的影响进行分类。对其中几个新突变的进一步鉴定，包括作图和分子克隆，目前正在进行中。

This project is devoted to forward genetic screens for new mutants that affect trafficking of Notch, and potentially other γ-secretase substrates, through intracellular membrane compartments. Although screens for mutants affecting cell proliferation, polarity, and patterning have led to the identification of genes influencing endosomal Notch trafficking, systematic screens for mutations that directly alter the cellular distribution of Notch have not been performed. Based on our previous analysis of the effects of Ca2+-ATPase mutations on the trafficking of Notch through the secretory pathway, the highly polarized structure of Drosophila disc cells, and the genetic techniques available for producing clones of lethal mutations in developing fly tissues, we reason that Drosophila is a suitable model for attempting this type of cell biological screen. To perform the screen, we mutagenize male flies, establish balanced stocks, induce tissue clones that are homozygous for a single mutagenized chromosome arm, and analyze the distribution of Notch protein in the mutant clone relative to adjacent wildtype tissue. At present, we are completing our screen of the second chromosome. Approximately 4000 mutagenized chromosome arms have been screened, resulting in the recovery of 91 mutations, which define over 20 separate genes (each represented by 2-8 mutant alleles). The different mutants can be classified with respect to their effects on Notch trafficking and the intracellular membrane compartment affected. Further characterization of several of these new mutants, including mapping and molecular cloning, is now underway.