Determining the function of Lgd/CC2D1 in mouse

肿瘤抑制基因致命巨盘（lgd）编码内体途径的一个组分，该组分是通过内体途径正确降解跨膜蛋白所必需的。它与ESCRT-III复合物的核心成分灌木（Shrb）相互作用，ESCRT-III复合物是在成熟核内体（MEs）的管腔中产生腔内囊泡（ILVs）的四种复合物之一。这一步对于跨膜蛋白的适当降解和通过激活受体的信号终止是必不可少的。lgd功能的丧失导致Notch通路不受控制的不依赖配体的激活。这是由Notch受体的运输缺陷引起的。大多数将Lgd与内体途径联系起来的工作都是在果蝇身上完成的。哺乳动物基因组中存在Lgd的两个同源基因Lgd1/CC2D1B/FREUD-2和Lgd2/Aki/CC2D1A/FREUD-1/TAPE。虽然Lgd2具有多种功能，但尚未确定任何Lgd与内体途径的明确联系。在上一个资助期，我们坚定地建立了这种联系，并在细胞超微结构水平和肠上皮中表征了Lgd1和Lgd2的表型。此外，我们对果蝇与人类LGDs进行了功能分析。我们从小鼠和这些常规等位基因中生成lgd同源基因的条件等位基因。我们可以确认Lgd2突变小鼠在出生后死于呼吸缺陷。这种缺陷是由大脑中Lgd2的功能丧失引起的。Lgd1功能的丧失没有引起明显的可检测表型，突变体保持为纯合子。我们的研究结果表明，它们在内体途径中具有类似于在果蝇中发现的功能。我们对果蝇lgd的功能分析表明，两者都可以替代lgd的功能。这些结果强烈表明，Lgd蛋白的功能是进化保守的，并且LGD1和LGD2之间存在功能冗余。因此，为了精确地确定哺乳动物中Lgd的功能，两个基因都必须灭活。因此，我们产生了双突变动物。双突变表型的分析是第二个资助期的目标。我们将精确地确定双突变体死亡的阶段并分析其表型。利用我们的条件等位基因，我们将分析肠道上皮中Lgd功能完全丧失的后果。我们将分析已经生成的双突变mef。我们将结合光学和电子显微镜技术，分子生物学，以及免疫组织化学来实现这一目标。

The tumorsuppressor gene lethal (2) giant discs (lgd) encodes a component of the endosomal pathway that is required for the correct degradation of transmembrane proteins through the endosomal pathway. It interacts with Shrub (Shrb), the core component of the ESCRT-III complex, one of four complexes that generate the intraluminal vesicles (ILVs) in the lumen of maturing endosomes (MEs). This step is essential for the proper degradation of transmembrane proteins and termination of signalling through activated receptors. Loss of lgd function results in the uncontrolled ligand-independent activation of the Notch pathway. This is caused by a defect in the trafficking of the Notch receptor. Most of work that connects Lgd to the endosomal pathway was done in Drosophila. Two orthologs of Lgd exist in the genomes of mammals, Lgd1/CC2D1B/FREUD-2 and Lgd2/Aki/CC2D1A/FREUD-1/TAPE. While several functions had been attributed to Lgd2, a definitive link with the endosomal pathway was not established for any Lgd. In the last funding period we firmly established this link and characterised the phenotype of Lgd1 and Lgd2 in cells up to the ultra-structural level and in the gut epithelium. Moreover, we conducted a functional analysis in Drosophila with the human LGDs. We generated conditional alleles of both lgd orthologs in mouse and from these conventional alleles. We could confirm that Lgd2 mutant mice die after birth due to a breathing defect. This defect is caused by the loss of function of Lgd2 in the brain. The loss of function of Lgd1 did not cause an obvious detectable phenotype and the mutant is maintained as homozygotes. Our results suggest that they have a function in the endosomal pathway that is similar to that discovered in Drosophila. Our functional analysis of LGDs in Drosophila showed that both can replace the function of lgd. These results strongly suggested that the function of Lgd proteins is evolutionary conserved and that a functional redundancy between LGD1 and LGD2 exists. Thus, to precisely determine the function of Lgd in mammals both genes have to be inactivated. We therefore generated double mutant animals. The analysis of the double mutant phenotype is the aim of the second funding period. We will precisely determine the stage where double mutants die and analyse their phenotype. Using our conditional alleles, we will analyse the consequences of complete loss of the Lgd function in the gut epithelium. We will analyse double mutant MEFs, we have generated. We will combine light and electron microscopic techniques, molecular biology, as well as immunohistochemistry to achieve this aim.