Determining the function of Lgd/CC2D1 in mouse

• Applicant: Professor Dr. Thomas Klein
• Subject Area: Developmental Biology ; Cell Biology
• Term: from 2010 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 188145095

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.

• DFG Programme: Research Grants