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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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Disruption of Cc2d1a in the nervous system results in perinatal death.(A) PCR genotyping of different tissues (tb: tail biopsy, br: brain, lu: lung) of Nestin-Cre mediated wild type and heterozygous Cc2d1a mutants using primers P1,P3 and P5. DNA from heterozygous and homozygous Cc2d1a deficient mice was used as control. Cre mediated recombination of Cc2d1a is only detected in the brain of mutant mice. (B) Immunoblotting of protein lysates from abdominal organs (ao) and brain (br) isolated from Nestin-Cretg/+;Cc2d1aflox/flox and control mice. The 130kDa CC2D1A band is absent in the lysates of brain of homozygous mutant mice but not in lysates of abdominal organs or control siblings. (C, D) Litter of breedings of full knockout animals (C) and of conditional brain specific knockout animals (D). Homozygous mutants from full knockouts could not start breathing and turned cyanotic (marked by an arrow). One third of homozygous conditional mutants developed the same phenotype after delivery, while about two-thirds of homozygous conditional mutants started breathing and were undistinguishable from littermates but died within a day.
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pgen.1005749.g003: Disruption of Cc2d1a in the nervous system results in perinatal death.(A) PCR genotyping of different tissues (tb: tail biopsy, br: brain, lu: lung) of Nestin-Cre mediated wild type and heterozygous Cc2d1a mutants using primers P1,P3 and P5. DNA from heterozygous and homozygous Cc2d1a deficient mice was used as control. Cre mediated recombination of Cc2d1a is only detected in the brain of mutant mice. (B) Immunoblotting of protein lysates from abdominal organs (ao) and brain (br) isolated from Nestin-Cretg/+;Cc2d1aflox/flox and control mice. The 130kDa CC2D1A band is absent in the lysates of brain of homozygous mutant mice but not in lysates of abdominal organs or control siblings. (C, D) Litter of breedings of full knockout animals (C) and of conditional brain specific knockout animals (D). Homozygous mutants from full knockouts could not start breathing and turned cyanotic (marked by an arrow). One third of homozygous conditional mutants developed the same phenotype after delivery, while about two-thirds of homozygous conditional mutants started breathing and were undistinguishable from littermates but died within a day.

Mentions: Heterozygous animals are viable, fertile and display no obvious abnormalities. Genotyping of litter from heterozygous breedings revealed no homozygous littermates, suggesting that no homozygous animal survive until weaning age (3–4 weeks). Closer examination of newborn pubs revealed that homozygous mice die within a few hours after delivery, similar to recently published studies [15,17,21]. Size and weight of the homozygous pups did not differ from their littermates but they displayed severe difficulties in breathing and turned cyanotic within a few minutes, suggesting that these pups suffer from respiratory distress (see Fig 3C).


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)

Disruption of Cc2d1a in the nervous system results in perinatal death.(A) PCR genotyping of different tissues (tb: tail biopsy, br: brain, lu: lung) of Nestin-Cre mediated wild type and heterozygous Cc2d1a mutants using primers P1,P3 and P5. DNA from heterozygous and homozygous Cc2d1a deficient mice was used as control. Cre mediated recombination of Cc2d1a is only detected in the brain of mutant mice. (B) Immunoblotting of protein lysates from abdominal organs (ao) and brain (br) isolated from Nestin-Cretg/+;Cc2d1aflox/flox and control mice. The 130kDa CC2D1A band is absent in the lysates of brain of homozygous mutant mice but not in lysates of abdominal organs or control siblings. (C, D) Litter of breedings of full knockout animals (C) and of conditional brain specific knockout animals (D). Homozygous mutants from full knockouts could not start breathing and turned cyanotic (marked by an arrow). One third of homozygous conditional mutants developed the same phenotype after delivery, while about two-thirds of homozygous conditional mutants started breathing and were undistinguishable from littermates but died within a day.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005749.g003: Disruption of Cc2d1a in the nervous system results in perinatal death.(A) PCR genotyping of different tissues (tb: tail biopsy, br: brain, lu: lung) of Nestin-Cre mediated wild type and heterozygous Cc2d1a mutants using primers P1,P3 and P5. DNA from heterozygous and homozygous Cc2d1a deficient mice was used as control. Cre mediated recombination of Cc2d1a is only detected in the brain of mutant mice. (B) Immunoblotting of protein lysates from abdominal organs (ao) and brain (br) isolated from Nestin-Cretg/+;Cc2d1aflox/flox and control mice. The 130kDa CC2D1A band is absent in the lysates of brain of homozygous mutant mice but not in lysates of abdominal organs or control siblings. (C, D) Litter of breedings of full knockout animals (C) and of conditional brain specific knockout animals (D). Homozygous mutants from full knockouts could not start breathing and turned cyanotic (marked by an arrow). One third of homozygous conditional mutants developed the same phenotype after delivery, while about two-thirds of homozygous conditional mutants started breathing and were undistinguishable from littermates but died within a day.
Mentions: Heterozygous animals are viable, fertile and display no obvious abnormalities. Genotyping of litter from heterozygous breedings revealed no homozygous littermates, suggesting that no homozygous animal survive until weaning age (3–4 weeks). Closer examination of newborn pubs revealed that homozygous mice die within a few hours after delivery, similar to recently published studies [15,17,21]. Size and weight of the homozygous pups did not differ from their littermates but they displayed severe difficulties in breathing and turned cyanotic within a few minutes, suggesting that these pups suffer from respiratory distress (see Fig 3C).

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