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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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Subcellular localisation of CC2D1A and CHMP4B in wild type cells and Vps4a+/-,Vps4b+/- cells.(A) Immunocytochemical staining was performed on wild type and Cc2d1a deficient MEFs with antibodies directed against CC2D1A, CHMP4B and the late endosomal marker RAB7. No significant co-localisation of the proteins was detected. Lack of CC2D1A did not alter the distribution of CHMP4B and RAB7. (B) Immunocytochemical staining was performed on wild type and Vps4a+/-,Vps4b+/- MEFs with antibodies directed against CC2D1A, CHMP4B and the late endosomal/lysosomal marker LAMP1. In Vps4a+/-,Vps4b+/- cells CC2D1A, CHMP4B and LAMP1 co-localise on enlarged vesicles.(A`, B`) Colocalisation was assessed by measuring the Pearson`s correlation coefficient (PCC) (n≥4). Scale bars are 20 μm.
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pgen.1005749.g007: Subcellular localisation of CC2D1A and CHMP4B in wild type cells and Vps4a+/-,Vps4b+/- cells.(A) Immunocytochemical staining was performed on wild type and Cc2d1a deficient MEFs with antibodies directed against CC2D1A, CHMP4B and the late endosomal marker RAB7. No significant co-localisation of the proteins was detected. Lack of CC2D1A did not alter the distribution of CHMP4B and RAB7. (B) Immunocytochemical staining was performed on wild type and Vps4a+/-,Vps4b+/- MEFs with antibodies directed against CC2D1A, CHMP4B and the late endosomal/lysosomal marker LAMP1. In Vps4a+/-,Vps4b+/- cells CC2D1A, CHMP4B and LAMP1 co-localise on enlarged vesicles.(A`, B`) Colocalisation was assessed by measuring the Pearson`s correlation coefficient (PCC) (n≥4). Scale bars are 20 μm.

Mentions: Moreover, we found little co-localisation of CC2D1A and CHMP4B and neither of them could be detected on RAB7 positive late endosomes (Fig 7A and 7A`). CC2D1 proteins have a C2 binding domain that has been shown to be able to bind phospholipids present in endosomal membranes [4]. To test whether CC2D1A and CC2D1B can associate with membranes in general, we performed a cell fractionation assay. We detected CC2D1A and CC2D1B in the cytosolic as well as in the membrane fraction, in contrast to control proteins, which were correctly restricted to only one fraction (S5A Fig). This result indicates that a fraction of CC2D1A and also CC2D1B is associated with membranes. CHMP4 proteins cycle between the endosomal membrane and the cytosol. An endosomal localisation of the CHMP4 yeast ortholog Snf7 could only be observed in the absence of Vps4p function [28]. Loss of Vps4p leads to an accumulation of ESCRT-III on endosomal membranes and disrupts proper ILV formation. We wondered whether this is also the case for CHMP4 proteins in mammals and, since CC2D1 proteins physically interact with CHMP4 proteins (see below), whether CC2D1A also accumulates at the endosomal membrane upon reduction of VPS4 function. The mammalian genome contains two Vps4 genes, Vps4a and Vps4b [45]. Homozygous Vps4a-/- and Vps4b-/- mice die during early embryonic stages. To analyse the influence of the two mammalian orthologs VPS4A and VPS4B on subcellular localisation of CC2D1A, we generated MEFs that are double heterozygous for Vps4a and Vps4b. Strikingly, we observed dramatically enlarged LAMP1 positive MEs/lysosomes in these cells, although the corresponding mice were healthy and displayed no detectable phenotype (Fig 7B). Moreover, CHMP4B accumulated on the enlarged MEs of the double heterozygous cells (Fig 7B and 7B`). This indicates that a reduction of VPS4 function results in the accumulation of CHMP4 proteins on the endosomal membrane also in mammals. Importantly, also CC2D1A accumulated on these CHMP4B/LAMP1 positive vesicles (Fig 7B and 7B`). In a complementary experiment, we over-expressed a dominant negative VPS4B (VPS4BE235Q) that lacks its ATPase activity leading to aberrant endosomal structures [46]. Indeed, we could confirm that expression of VPS4BE235Q in wild type MEFs leads to the formation of enlarged endosomal structures while expression of normal VPS4B did not lead to any changes (S5B and S5B` Fig). Moreover, FK2, a marker for ubiquitinated proteins, strongly labelled the VPS4BE235Q induced aberrant MEs, suggesting that the removal of CHMP4 from the endosomal membrane is impaired. As in Vps4a+/-,Vps4b+/- cells, CHMP4A and CC2D1A accumulated on the enlarged MEs (S5B and S5B` Fig). These results indicate that CC2D1A might function at the endosome and cycles between the cytosol and the endosomal membrane, just like its interaction partner CHMP4B. They also indicate that CC2D1A appears to be removed from the endosomal membrane by VPS4 and suggest that CC2D1A is involved in ESCRT-III function in mammals.


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)

Subcellular localisation of CC2D1A and CHMP4B in wild type cells and Vps4a+/-,Vps4b+/- cells.(A) Immunocytochemical staining was performed on wild type and Cc2d1a deficient MEFs with antibodies directed against CC2D1A, CHMP4B and the late endosomal marker RAB7. No significant co-localisation of the proteins was detected. Lack of CC2D1A did not alter the distribution of CHMP4B and RAB7. (B) Immunocytochemical staining was performed on wild type and Vps4a+/-,Vps4b+/- MEFs with antibodies directed against CC2D1A, CHMP4B and the late endosomal/lysosomal marker LAMP1. In Vps4a+/-,Vps4b+/- cells CC2D1A, CHMP4B and LAMP1 co-localise on enlarged vesicles.(A`, B`) Colocalisation was assessed by measuring the Pearson`s correlation coefficient (PCC) (n≥4). Scale bars are 20 μm.
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pgen.1005749.g007: Subcellular localisation of CC2D1A and CHMP4B in wild type cells and Vps4a+/-,Vps4b+/- cells.(A) Immunocytochemical staining was performed on wild type and Cc2d1a deficient MEFs with antibodies directed against CC2D1A, CHMP4B and the late endosomal marker RAB7. No significant co-localisation of the proteins was detected. Lack of CC2D1A did not alter the distribution of CHMP4B and RAB7. (B) Immunocytochemical staining was performed on wild type and Vps4a+/-,Vps4b+/- MEFs with antibodies directed against CC2D1A, CHMP4B and the late endosomal/lysosomal marker LAMP1. In Vps4a+/-,Vps4b+/- cells CC2D1A, CHMP4B and LAMP1 co-localise on enlarged vesicles.(A`, B`) Colocalisation was assessed by measuring the Pearson`s correlation coefficient (PCC) (n≥4). Scale bars are 20 μm.
Mentions: Moreover, we found little co-localisation of CC2D1A and CHMP4B and neither of them could be detected on RAB7 positive late endosomes (Fig 7A and 7A`). CC2D1 proteins have a C2 binding domain that has been shown to be able to bind phospholipids present in endosomal membranes [4]. To test whether CC2D1A and CC2D1B can associate with membranes in general, we performed a cell fractionation assay. We detected CC2D1A and CC2D1B in the cytosolic as well as in the membrane fraction, in contrast to control proteins, which were correctly restricted to only one fraction (S5A Fig). This result indicates that a fraction of CC2D1A and also CC2D1B is associated with membranes. CHMP4 proteins cycle between the endosomal membrane and the cytosol. An endosomal localisation of the CHMP4 yeast ortholog Snf7 could only be observed in the absence of Vps4p function [28]. Loss of Vps4p leads to an accumulation of ESCRT-III on endosomal membranes and disrupts proper ILV formation. We wondered whether this is also the case for CHMP4 proteins in mammals and, since CC2D1 proteins physically interact with CHMP4 proteins (see below), whether CC2D1A also accumulates at the endosomal membrane upon reduction of VPS4 function. The mammalian genome contains two Vps4 genes, Vps4a and Vps4b [45]. Homozygous Vps4a-/- and Vps4b-/- mice die during early embryonic stages. To analyse the influence of the two mammalian orthologs VPS4A and VPS4B on subcellular localisation of CC2D1A, we generated MEFs that are double heterozygous for Vps4a and Vps4b. Strikingly, we observed dramatically enlarged LAMP1 positive MEs/lysosomes in these cells, although the corresponding mice were healthy and displayed no detectable phenotype (Fig 7B). Moreover, CHMP4B accumulated on the enlarged MEs of the double heterozygous cells (Fig 7B and 7B`). This indicates that a reduction of VPS4 function results in the accumulation of CHMP4 proteins on the endosomal membrane also in mammals. Importantly, also CC2D1A accumulated on these CHMP4B/LAMP1 positive vesicles (Fig 7B and 7B`). In a complementary experiment, we over-expressed a dominant negative VPS4B (VPS4BE235Q) that lacks its ATPase activity leading to aberrant endosomal structures [46]. Indeed, we could confirm that expression of VPS4BE235Q in wild type MEFs leads to the formation of enlarged endosomal structures while expression of normal VPS4B did not lead to any changes (S5B and S5B` Fig). Moreover, FK2, a marker for ubiquitinated proteins, strongly labelled the VPS4BE235Q induced aberrant MEs, suggesting that the removal of CHMP4 from the endosomal membrane is impaired. As in Vps4a+/-,Vps4b+/- cells, CHMP4A and CC2D1A accumulated on the enlarged MEs (S5B and S5B` Fig). These results indicate that CC2D1A might function at the endosome and cycles between the cytosol and the endosomal membrane, just like its interaction partner CHMP4B. They also indicate that CC2D1A appears to be removed from the endosomal membrane by VPS4 and suggest that CC2D1A is involved in ESCRT-III function in mammals.

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