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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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CC2D1A appears not to regulate centrosome function.(A) Immunocytochemical staining of wild type, Cc2d1a+/- and Cc2d1a-/- MEF cells with an affinity purified anti-guinea-pig-CC2D1A antibody. No staining could be detected in Cc2d1a deficient cells. (B) Immunocytochemical staining of wild type, CC2D1A-dsRed or EGFP-CC2D1B overexpressing cells revealed 1 or 2 γ-Tubulin positive centrosomes (marked by arrows). CC2D1A and γ-Tubulin co-localise only in over-expression. (C, D) Neither the mitotic index (in 1478 mutant cell and 974 wild type cells) (C) nor cell proliferation (D) is significantly altered in Cc2d1a deficient MEF cells compared to wild type MEF cells. Two to three different passages were analysed per genotype. Scale bars are 20 μm.
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pgen.1005749.g004: CC2D1A appears not to regulate centrosome function.(A) Immunocytochemical staining of wild type, Cc2d1a+/- and Cc2d1a-/- MEF cells with an affinity purified anti-guinea-pig-CC2D1A antibody. No staining could be detected in Cc2d1a deficient cells. (B) Immunocytochemical staining of wild type, CC2D1A-dsRed or EGFP-CC2D1B overexpressing cells revealed 1 or 2 γ-Tubulin positive centrosomes (marked by arrows). CC2D1A and γ-Tubulin co-localise only in over-expression. (C, D) Neither the mitotic index (in 1478 mutant cell and 974 wild type cells) (C) nor cell proliferation (D) is significantly altered in Cc2d1a deficient MEF cells compared to wild type MEF cells. Two to three different passages were analysed per genotype. Scale bars are 20 μm.

Mentions: CC2D1A was recently described to localise to centrosomes and regulate centriole cohesion in HeLa cells. siRNA mediated knockdown of CC2D1A in these cells was shown to cause formation of multipolar spindles accompanied by separase dependent centriole splitting [18]. To review these finding in murine cells we established murine embryonic fibroblast (MEF) cell lines isolated from Cc2d1a-/-, Cc2d1a+/- and wild type embryos and stained them with CC2D1A specific antibodies. The commercially available antibodies used in previous studies gave strong signals in our assay in all genotypes suggesting unspecific binding of the antibodies to other cellular antigens (see S2 Fig). To generate a specific polyclonal CC2D1A antibody we immunized guinea pigs with a murine antigen that comprises the 4th DM14 domain (aa 481 to 640) fused to the extended C-Terminus of CC2D1A (aa 788 to 943) that is absent in CC2D1B. Immunocytochemical staining of MEF cells revealed a specific signal of CC2D1A in wild type MEFs, a weaker signal in heterozygous Cc2d1a+/- cells and no signal in homozygous Cc2d1a-/- cells (Fig 4A), confirming the specificity of the antibody.


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)

CC2D1A appears not to regulate centrosome function.(A) Immunocytochemical staining of wild type, Cc2d1a+/- and Cc2d1a-/- MEF cells with an affinity purified anti-guinea-pig-CC2D1A antibody. No staining could be detected in Cc2d1a deficient cells. (B) Immunocytochemical staining of wild type, CC2D1A-dsRed or EGFP-CC2D1B overexpressing cells revealed 1 or 2 γ-Tubulin positive centrosomes (marked by arrows). CC2D1A and γ-Tubulin co-localise only in over-expression. (C, D) Neither the mitotic index (in 1478 mutant cell and 974 wild type cells) (C) nor cell proliferation (D) is significantly altered in Cc2d1a deficient MEF cells compared to wild type MEF cells. Two to three different passages were analysed per genotype. Scale bars are 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005749.g004: CC2D1A appears not to regulate centrosome function.(A) Immunocytochemical staining of wild type, Cc2d1a+/- and Cc2d1a-/- MEF cells with an affinity purified anti-guinea-pig-CC2D1A antibody. No staining could be detected in Cc2d1a deficient cells. (B) Immunocytochemical staining of wild type, CC2D1A-dsRed or EGFP-CC2D1B overexpressing cells revealed 1 or 2 γ-Tubulin positive centrosomes (marked by arrows). CC2D1A and γ-Tubulin co-localise only in over-expression. (C, D) Neither the mitotic index (in 1478 mutant cell and 974 wild type cells) (C) nor cell proliferation (D) is significantly altered in Cc2d1a deficient MEF cells compared to wild type MEF cells. Two to three different passages were analysed per genotype. Scale bars are 20 μm.
Mentions: CC2D1A was recently described to localise to centrosomes and regulate centriole cohesion in HeLa cells. siRNA mediated knockdown of CC2D1A in these cells was shown to cause formation of multipolar spindles accompanied by separase dependent centriole splitting [18]. To review these finding in murine cells we established murine embryonic fibroblast (MEF) cell lines isolated from Cc2d1a-/-, Cc2d1a+/- and wild type embryos and stained them with CC2D1A specific antibodies. The commercially available antibodies used in previous studies gave strong signals in our assay in all genotypes suggesting unspecific binding of the antibodies to other cellular antigens (see S2 Fig). To generate a specific polyclonal CC2D1A antibody we immunized guinea pigs with a murine antigen that comprises the 4th DM14 domain (aa 481 to 640) fused to the extended C-Terminus of CC2D1A (aa 788 to 943) that is absent in CC2D1B. Immunocytochemical staining of MEF cells revealed a specific signal of CC2D1A in wild type MEFs, a weaker signal in heterozygous Cc2d1a+/- cells and no signal in homozygous Cc2d1a-/- cells (Fig 4A), confirming the specificity of the antibody.

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