Determining the function of Lgd/CC2D1 in mouse

确定 Lgd/CC2D1 在小鼠中的功能

• 批准号: 188145095
• 负责人: Professor Dr. Thomas Klein
• 金额: --
• 依托单位: Institut für Genetik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2010
• 资助国家: 德国
• 起止时间: 2009-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/188145095?language=en
• 关键词: Determining; function; Lgd; CC2D1; mouse

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.

肿瘤抑制基因致死(2)巨盘(LGD)编码内体途径的一个组成部分，该成分是通过内体途径正确降解跨膜蛋白所必需的。它与灌木(SURB)相互作用，灌木是ESCRT-III复合体的核心成分，ESCRT-III复合体是在成熟内体(MES)的管腔中产生腔内小泡(ILV)的四个复合体之一。这一步骤对于跨膜蛋白的正确降解和通过激活的受体终止信号至关重要。LGD功能的丧失会导致Notch通路失控的非受控配体激活。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。在哺乳动物基因组中存在两个LGD的同源基因：Lgd1/CC2D1B/Freud-2和Lgd2/Aki/CC2D1A/Freud-1/Tape。虽然LGD2具有多种功能，但尚未发现任何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，并表征了Lgd1和Lgd2在细胞直至超微结构水平和肠道上皮中的表型。此外，我们还对果蝇和人类LGDS进行了功能分析。我们在小鼠体内产生了LGD同源基因的条件等位基因，并从这些传统等位基因中产生了条件等位基因。我们可以证实，Lgd2突变小鼠出生后死于呼吸缺陷。这种缺陷是由于大脑中Lgd2功能的丧失造成的。Lgd1的功能丧失没有引起明显的可检测到的表型，突变体保持纯合状态。我们的结果表明，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LGDS的功能分析表明，LGDS和LGDS都可以替代LGD的功能。这些结果有力地表明，LGD蛋白的功能是进化保守的，LGD1和LGD2之间存在功能冗余。因此，为了准确地确定LGD在哺乳动物中的功能，这两个基因都必须被灭活。因此，我们产生了双重突变的动物。对双突变表型的分析是第二个资助期的目标。我们将准确地确定双突变株死亡的阶段，并分析其表型。使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。我们将分析我们产生的双突变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。