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The Mammalian Orthologs of Drosophila Lgd, CC2D1A and CC2D1B, Function in the Endocytic Pathway, but Their Individual Loss of Function Does Not Affect Notch Signalling.

Drusenheimer N, Migdal B, Jäckel S, Tveriakhina L, Scheider K, Schulz K, Gröper J, Köhrer K, Klein T - PLoS Genet. (2015)

Bottom Line: We could show that CC2D1A and its interaction partner CHMP4B are localised on endosomes in MEF cells, when the activity of the endosomal protein VPS4 is reduced.Additionally, in rescue experiments in D. melanogaster, CC2D1A and CC2D1B were able to functionally replace Lgd.Altogether our data suggest a functional conservation of the Lgd protein family in the ESCRT-III mediated process in metazoans.

View Article: PubMed Central - PubMed

Affiliation: Institut für Genetik, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany.

ABSTRACT
CC2D1A and CC2D1B belong to the evolutionary conserved Lgd protein family with members in all multi-cellular animals. Several functions such as centrosomal cleavage, involvement in signalling pathways, immune response and synapse maturation have been described for CC2D1A. Moreover, the Drosophila melanogaster ortholog Lgd was shown to be involved in the endosomal trafficking of the Notch receptor and other transmembrane receptors and physically interacts with the ESCRT-III component Shrub/CHMP4. To determine if this function is conserved in mammals we generated and characterized Cc2d1a and Cc2d1b conditional knockout mice. While Cc2d1b deficient mice displayed no obvious phenotype, we found that Cc2d1a deficient mice as well as conditional mutants that lack CC2D1A only in the nervous system die shortly after birth due to respiratory distress. This finding confirms the suspicion that the breathing defect is caused by the central nervous system. However, an involvement in centrosomal function could not be confirmed in Cc2d1a deficient MEF cells. To analyse an influence on Notch signalling, we generated intestine specific Cc2d1a mutant mice. These mice did not display any alterations in goblet cell number, proliferating cell number or expression of the Notch reporter Hes1-emGFP, suggesting that CC2D1A is not required for Notch signalling. However, our EM analysis revealed that the average size of endosomes of Cc2d1a mutant cells, but not Cc2d1b mutant cells, is increased, indicating a defect in endosomal morphogenesis. We could show that CC2D1A and its interaction partner CHMP4B are localised on endosomes in MEF cells, when the activity of the endosomal protein VPS4 is reduced. This indicates that CC2D1A cycles between the cytosol and the endosomal membrane. Additionally, in rescue experiments in D. melanogaster, CC2D1A and CC2D1B were able to functionally replace Lgd. Altogether our data suggest a functional conservation of the Lgd protein family in the ESCRT-III mediated process in metazoans.

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Generation of a conditional knockout mouse of the Cc2d1a gene locus.(A) Scheme for generating animals carrying the conditional knockout allele (Cc2d1aflox) and the recombined allele (Cc2d1a-). LoxP sites are depicted as yellow triangles, FRT sites as orange triangles. The DM14 and C2 protein domains are marked in green and red, respectively. Primers for genotyping are numbered P1-P5 and are depicted as black arrows. (B) PCR genotyping of wild type and heterozygous and homozygous Cc2d1a- mice using primers P1, P3 and P5. (C) RT-PCR analysis of total RNA extracts of brain of newborn animals with exon specific primer pairs that confirm lack of expression of the deleted segment. (D) Immunoblotting of protein lysates from MEF cells isolated from Cc2d1a-/- and control mice. The 130kDa CC2D1A band is lacking in the lysates of homozygous Cc2d1a-/- mice.
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pgen.1005749.g002: Generation of a conditional knockout mouse of the Cc2d1a gene locus.(A) Scheme for generating animals carrying the conditional knockout allele (Cc2d1aflox) and the recombined allele (Cc2d1a-). LoxP sites are depicted as yellow triangles, FRT sites as orange triangles. The DM14 and C2 protein domains are marked in green and red, respectively. Primers for genotyping are numbered P1-P5 and are depicted as black arrows. (B) PCR genotyping of wild type and heterozygous and homozygous Cc2d1a- mice using primers P1, P3 and P5. (C) RT-PCR analysis of total RNA extracts of brain of newborn animals with exon specific primer pairs that confirm lack of expression of the deleted segment. (D) Immunoblotting of protein lysates from MEF cells isolated from Cc2d1a-/- and control mice. The 130kDa CC2D1A band is lacking in the lysates of homozygous Cc2d1a-/- mice.

Mentions: To characterise the function of CC2D1A in vivo we generated a Cc2d1a conditional allele where the exons 7 and 14 are flanked by loxP sites (Fig 2A). Cre-mediated recombination leads to deletion of the floxed DNA segment. In the resulting Cc2d1a transcript exon 6 is spliced to exon 15, leading to a frame-shift mutation and a premature stop codon after 120bp of missense sequence. The truncated protein consists of 284 amino acids and contains just the first DM14 domain (Fig 2A). Homologous recombination of ES cells was analysed by Southern Blotting and subsequently functionality of loxP sites in ES cell clones was confirmed by a cell permeable His-TAT-NLS-Cre [38] prior to blastocyst injection. Cc2d1aneoflox mice were bred with Flp deleter and Cre deleter lines and Cc2d1a deficiency was confirmed by genotyping PCR, RT-PCR and Immunoblotting (Fig 2B–2D). The truncated protein could not be detected.


The Mammalian Orthologs of Drosophila Lgd, CC2D1A and CC2D1B, Function in the Endocytic Pathway, but Their Individual Loss of Function Does Not Affect Notch Signalling.

Drusenheimer N, Migdal B, Jäckel S, Tveriakhina L, Scheider K, Schulz K, Gröper J, Köhrer K, Klein T - PLoS Genet. (2015)

Generation of a conditional knockout mouse of the Cc2d1a gene locus.(A) Scheme for generating animals carrying the conditional knockout allele (Cc2d1aflox) and the recombined allele (Cc2d1a-). LoxP sites are depicted as yellow triangles, FRT sites as orange triangles. The DM14 and C2 protein domains are marked in green and red, respectively. Primers for genotyping are numbered P1-P5 and are depicted as black arrows. (B) PCR genotyping of wild type and heterozygous and homozygous Cc2d1a- mice using primers P1, P3 and P5. (C) RT-PCR analysis of total RNA extracts of brain of newborn animals with exon specific primer pairs that confirm lack of expression of the deleted segment. (D) Immunoblotting of protein lysates from MEF cells isolated from Cc2d1a-/- and control mice. The 130kDa CC2D1A band is lacking in the lysates of homozygous Cc2d1a-/- mice.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4697852&req=5

pgen.1005749.g002: Generation of a conditional knockout mouse of the Cc2d1a gene locus.(A) Scheme for generating animals carrying the conditional knockout allele (Cc2d1aflox) and the recombined allele (Cc2d1a-). LoxP sites are depicted as yellow triangles, FRT sites as orange triangles. The DM14 and C2 protein domains are marked in green and red, respectively. Primers for genotyping are numbered P1-P5 and are depicted as black arrows. (B) PCR genotyping of wild type and heterozygous and homozygous Cc2d1a- mice using primers P1, P3 and P5. (C) RT-PCR analysis of total RNA extracts of brain of newborn animals with exon specific primer pairs that confirm lack of expression of the deleted segment. (D) Immunoblotting of protein lysates from MEF cells isolated from Cc2d1a-/- and control mice. The 130kDa CC2D1A band is lacking in the lysates of homozygous Cc2d1a-/- mice.
Mentions: To characterise the function of CC2D1A in vivo we generated a Cc2d1a conditional allele where the exons 7 and 14 are flanked by loxP sites (Fig 2A). Cre-mediated recombination leads to deletion of the floxed DNA segment. In the resulting Cc2d1a transcript exon 6 is spliced to exon 15, leading to a frame-shift mutation and a premature stop codon after 120bp of missense sequence. The truncated protein consists of 284 amino acids and contains just the first DM14 domain (Fig 2A). Homologous recombination of ES cells was analysed by Southern Blotting and subsequently functionality of loxP sites in ES cell clones was confirmed by a cell permeable His-TAT-NLS-Cre [38] prior to blastocyst injection. Cc2d1aneoflox mice were bred with Flp deleter and Cre deleter lines and Cc2d1a deficiency was confirmed by genotyping PCR, RT-PCR and Immunoblotting (Fig 2B–2D). The truncated protein could not be detected.

Bottom Line: We could show that CC2D1A and its interaction partner CHMP4B are localised on endosomes in MEF cells, when the activity of the endosomal protein VPS4 is reduced.Additionally, in rescue experiments in D. melanogaster, CC2D1A and CC2D1B were able to functionally replace Lgd.Altogether our data suggest a functional conservation of the Lgd protein family in the ESCRT-III mediated process in metazoans.

View Article: PubMed Central - PubMed

Affiliation: Institut für Genetik, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany.

ABSTRACT
CC2D1A and CC2D1B belong to the evolutionary conserved Lgd protein family with members in all multi-cellular animals. Several functions such as centrosomal cleavage, involvement in signalling pathways, immune response and synapse maturation have been described for CC2D1A. Moreover, the Drosophila melanogaster ortholog Lgd was shown to be involved in the endosomal trafficking of the Notch receptor and other transmembrane receptors and physically interacts with the ESCRT-III component Shrub/CHMP4. To determine if this function is conserved in mammals we generated and characterized Cc2d1a and Cc2d1b conditional knockout mice. While Cc2d1b deficient mice displayed no obvious phenotype, we found that Cc2d1a deficient mice as well as conditional mutants that lack CC2D1A only in the nervous system die shortly after birth due to respiratory distress. This finding confirms the suspicion that the breathing defect is caused by the central nervous system. However, an involvement in centrosomal function could not be confirmed in Cc2d1a deficient MEF cells. To analyse an influence on Notch signalling, we generated intestine specific Cc2d1a mutant mice. These mice did not display any alterations in goblet cell number, proliferating cell number or expression of the Notch reporter Hes1-emGFP, suggesting that CC2D1A is not required for Notch signalling. However, our EM analysis revealed that the average size of endosomes of Cc2d1a mutant cells, but not Cc2d1b mutant cells, is increased, indicating a defect in endosomal morphogenesis. We could show that CC2D1A and its interaction partner CHMP4B are localised on endosomes in MEF cells, when the activity of the endosomal protein VPS4 is reduced. This indicates that CC2D1A cycles between the cytosol and the endosomal membrane. Additionally, in rescue experiments in D. melanogaster, CC2D1A and CC2D1B were able to functionally replace Lgd. Altogether our data suggest a functional conservation of the Lgd protein family in the ESCRT-III mediated process in metazoans.

Show MeSH
Related in: MedlinePlus