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 lethal (2) Giant Discs (lgd) 编码内体途径的一个组成部分，这是跨膜蛋白通过内体途径正确降解所必需的。它与 ESRT-III 复合物的核心成分 Shrub (Shrb) 相互作用，ESCRT-III 复合物是在成熟内涵体 (ME) 的管腔中产生管腔内囊泡 (ILV) 的四种复合物之一。此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传导至关重要。 lgd 功能的丧失会导致 Notch 通路不受控制的、不依赖于配体的激活。这是由 Notch 受体运输缺陷引起的。将 Lgd 连接到内体途径的大部分工作是在果蝇中完成的。 Lgd 的两个直向同源物存在于哺乳动物的基因组中，Lgd1/CC2D1B/FREUD-2 和 Lgd2/Aki/CC2D1A/FREUD-1/TAPE。虽然 Lgd2 具有多种功能，但尚未建立任何 Lgd 与内体途径的明确联系。在上一个资助期间，我们牢固地建立了这种联系，并表征了细胞超微结构水平和肠道上皮细胞中 Lgd1 和 Lgd2 的表型。此外，我们还利用人类 LGD 在果蝇中进行了功能分析。我们在小鼠中以及从这些常规等位基因中生成了两种lgd直系同源物的条件等位基因。我们可以确认 Lgd2 突变小鼠在出生后会因呼吸缺陷而死亡。这种缺陷是由大脑中 Lgd2 功能丧失引起的。 Lgd1 功能的丧失不会引起明显的可检测表型，并且突变体保持为纯合子。我们的结果表明，它们在内体途径中具有与果蝇中发现的功能相似的功能。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，为了精确确定哺乳动物中 Lgd 的功能，这两个基因都必须失活。因此我们产生了双突变动物。双突变体表型的分析是第二个资助期的目标。我们将精确确定双突变体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细胞中 Lgd 功能完全丧失的后果。我们将分析我们生成的双突变 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。