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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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Endogenous CC2D1A and CHMP4B interact in wild type MEFs.PLA (Proximity Ligation Assay) was performed on wild type and Cc2d1a deficient MEFs. Positive signals were abundant only in wild type cells. Data are mean ± SD values from 2 independent experiments (** p < 0.001). Scale bars are 20 μm.
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pgen.1005749.g008: Endogenous CC2D1A and CHMP4B interact in wild type MEFs.PLA (Proximity Ligation Assay) was performed on wild type and Cc2d1a deficient MEFs. Positive signals were abundant only in wild type cells. Data are mean ± SD values from 2 independent experiments (** p < 0.001). Scale bars are 20 μm.

Mentions: So far only in vitro data showed physical interactions of CHMP4B with CC2D1A. In order to test whether this can also be observed between the corresponding endogenous proteins in a cell, we performed the proximity ligation assay (PLA). PLA enables the in situ detection of endogenous protein protein interactions, using protein specific antibodies [47]. For the assay, we compared wild type and Cc2d1a deficient MEF cells and used our specific CC2D1A antibody in combination with a commercially available CHMP4B antibody. As expected, only few PLA signals were visible in Cc2d1a deficient cells (0.6±1.07), confirming the accuracy of the method (Fig 8). In contrast, abundant PLA signals were detected in wild type MEFs (6.47±3.18) indicating that endogenous CC2D1A and CHMP4B interact (Fig 8). To exclude the possibility that CHMP4B and CC2D1A interact in the cytosol randomly by freely diffusing cytosolic proteins we performed PLA with CHMP4B and the cytosolic protein PGK1 (S6 Fig). Again, we could detect a clear interaction only in the control and not between CHMP4B and PGK1 (5.61±2.39 for CHMP4B with CC2D1A compared to 0.64± 1.01 for CHMP4B with PGK1). Taken together, these experiments further support the assumption that CC2D1A and possibly also CC2D1B are involved in ESCRT-III mediated events.


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)

Endogenous CC2D1A and CHMP4B interact in wild type MEFs.PLA (Proximity Ligation Assay) was performed on wild type and Cc2d1a deficient MEFs. Positive signals were abundant only in wild type cells. Data are mean ± SD values from 2 independent experiments (** p < 0.001). Scale bars are 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005749.g008: Endogenous CC2D1A and CHMP4B interact in wild type MEFs.PLA (Proximity Ligation Assay) was performed on wild type and Cc2d1a deficient MEFs. Positive signals were abundant only in wild type cells. Data are mean ± SD values from 2 independent experiments (** p < 0.001). Scale bars are 20 μm.
Mentions: So far only in vitro data showed physical interactions of CHMP4B with CC2D1A. In order to test whether this can also be observed between the corresponding endogenous proteins in a cell, we performed the proximity ligation assay (PLA). PLA enables the in situ detection of endogenous protein protein interactions, using protein specific antibodies [47]. For the assay, we compared wild type and Cc2d1a deficient MEF cells and used our specific CC2D1A antibody in combination with a commercially available CHMP4B antibody. As expected, only few PLA signals were visible in Cc2d1a deficient cells (0.6±1.07), confirming the accuracy of the method (Fig 8). In contrast, abundant PLA signals were detected in wild type MEFs (6.47±3.18) indicating that endogenous CC2D1A and CHMP4B interact (Fig 8). To exclude the possibility that CHMP4B and CC2D1A interact in the cytosol randomly by freely diffusing cytosolic proteins we performed PLA with CHMP4B and the cytosolic protein PGK1 (S6 Fig). Again, we could detect a clear interaction only in the control and not between CHMP4B and PGK1 (5.61±2.39 for CHMP4B with CC2D1A compared to 0.64± 1.01 for CHMP4B with PGK1). Taken together, these experiments further support the assumption that CC2D1A and possibly also CC2D1B are involved in ESCRT-III mediated events.

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