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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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Ultra-structural characterisation of endo/lysosomal compartments in wild type, Cc2d1a-/-, Cc2d1b-/- and Vps4a+/-,Vps4b+/- MEFs.(A-A”) wild type, (B-B”) Cc2d1a-/- mutant, (C-C”) Cc2d1b-/- mutant and (D-D”) Vps4a+/-,Vps4b+/- double heterozygous MEFs. The comparison reveals that Cc2d1a-/- and Cc2d1b-/- contain endo/lysosomal organelles that are similar to wild type endo/lysosomal organelles in appearance. In contrast, the appearance of the organelles is dramatically changed in Vps4a+/-,Vps4b+/- double heterozygous cells. These cells contain massively enlarged MEs with a class E like phenotype. The MEs partially lose their normal round shape and contain many intraluminal vesicles or membrane layers (arrowheads). (E) Statistical analysis of the endo/lysosomal perimeter revealed that the organelles in Cc2d1a-/- and Vps4a+/-,Vps4b+/- MEFs are significantly enlarged compared to wild type cells. Data are mean ± SD values from 4 independent experiments (** p < 0.001). Arrows highlight individual endosomes. Scale bars are 5 μm (A-D) and 0.5 μm (A`-D`, A”-D”).
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pgen.1005749.g009: Ultra-structural characterisation of endo/lysosomal compartments in wild type, Cc2d1a-/-, Cc2d1b-/- and Vps4a+/-,Vps4b+/- MEFs.(A-A”) wild type, (B-B”) Cc2d1a-/- mutant, (C-C”) Cc2d1b-/- mutant and (D-D”) Vps4a+/-,Vps4b+/- double heterozygous MEFs. The comparison reveals that Cc2d1a-/- and Cc2d1b-/- contain endo/lysosomal organelles that are similar to wild type endo/lysosomal organelles in appearance. In contrast, the appearance of the organelles is dramatically changed in Vps4a+/-,Vps4b+/- double heterozygous cells. These cells contain massively enlarged MEs with a class E like phenotype. The MEs partially lose their normal round shape and contain many intraluminal vesicles or membrane layers (arrowheads). (E) Statistical analysis of the endo/lysosomal perimeter revealed that the organelles in Cc2d1a-/- and Vps4a+/-,Vps4b+/- MEFs are significantly enlarged compared to wild type cells. Data are mean ± SD values from 4 independent experiments (** p < 0.001). Arrows highlight individual endosomes. Scale bars are 5 μm (A-D) and 0.5 μm (A`-D`, A”-D”).

Mentions: To further elucidate a function of CC2D1 proteins in endosome maturation we analysed the endo/lysosomal compartments of wild type and mutant MEFs with the transmission electron microscope (TEM) (Fig 9). Endo/lysosomal organelles were identified by their ultrastructural characteristics proposed by others [48–50]. Exemplary pictures of the analysed endo/lysosomal organelles are depicted in S7A–S7D Fig. Cc2d1b-/- cells show no differences in size or morphology of the endo/lysosomal compartments compared to wild type cells (Figs 9A–9A”, 9C–9C”, 9E and S7E). In contrast and in accordance with the fluorescence microscopy data, Vps4a+/-,Vps4b+/- cells had enlarged endo/lysosomal compartments. We detected large multi-membrane structures that resembled the class E compartment described for loss of ESCRT function in yeast and mammalian cells [51,52]. We did not observe these structures in Cc2d1a-/- cells, but found that the average size of their endo/lysosomal organelles was significantly increased (Figs 9B–9B”, 9E and S7E). The endo/lysosomal phenotype of Cc2d1a-/- cells resembled that observed in lgd mutant cells of D. melanogaster [53]. When we allocated the endo/lysosomal organelles to different size classes, we found classes in Cc2d1a-/- and Vps4a+/-,Vps4b+/- MEFs that were not present in Cc2d1b-/- or wild type MEFs (S7E Fig). Thus, the loss of Cc2d1a function results in a moderate enlargement of the endo/lysosomal compartment, indicating that it is involved in endosomal maturation. Moreover, already the double heterozygosity of Vps4a and Vps4b results in a dramatic morphological defect of the endo/lysosomal phenotype, which appears to have no direct consequences for the viability of mice in captivity.


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)

Ultra-structural characterisation of endo/lysosomal compartments in wild type, Cc2d1a-/-, Cc2d1b-/- and Vps4a+/-,Vps4b+/- MEFs.(A-A”) wild type, (B-B”) Cc2d1a-/- mutant, (C-C”) Cc2d1b-/- mutant and (D-D”) Vps4a+/-,Vps4b+/- double heterozygous MEFs. The comparison reveals that Cc2d1a-/- and Cc2d1b-/- contain endo/lysosomal organelles that are similar to wild type endo/lysosomal organelles in appearance. In contrast, the appearance of the organelles is dramatically changed in Vps4a+/-,Vps4b+/- double heterozygous cells. These cells contain massively enlarged MEs with a class E like phenotype. The MEs partially lose their normal round shape and contain many intraluminal vesicles or membrane layers (arrowheads). (E) Statistical analysis of the endo/lysosomal perimeter revealed that the organelles in Cc2d1a-/- and Vps4a+/-,Vps4b+/- MEFs are significantly enlarged compared to wild type cells. Data are mean ± SD values from 4 independent experiments (** p < 0.001). Arrows highlight individual endosomes. Scale bars are 5 μm (A-D) and 0.5 μm (A`-D`, A”-D”).
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Related In: Results  -  Collection

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

pgen.1005749.g009: Ultra-structural characterisation of endo/lysosomal compartments in wild type, Cc2d1a-/-, Cc2d1b-/- and Vps4a+/-,Vps4b+/- MEFs.(A-A”) wild type, (B-B”) Cc2d1a-/- mutant, (C-C”) Cc2d1b-/- mutant and (D-D”) Vps4a+/-,Vps4b+/- double heterozygous MEFs. The comparison reveals that Cc2d1a-/- and Cc2d1b-/- contain endo/lysosomal organelles that are similar to wild type endo/lysosomal organelles in appearance. In contrast, the appearance of the organelles is dramatically changed in Vps4a+/-,Vps4b+/- double heterozygous cells. These cells contain massively enlarged MEs with a class E like phenotype. The MEs partially lose their normal round shape and contain many intraluminal vesicles or membrane layers (arrowheads). (E) Statistical analysis of the endo/lysosomal perimeter revealed that the organelles in Cc2d1a-/- and Vps4a+/-,Vps4b+/- MEFs are significantly enlarged compared to wild type cells. Data are mean ± SD values from 4 independent experiments (** p < 0.001). Arrows highlight individual endosomes. Scale bars are 5 μm (A-D) and 0.5 μm (A`-D`, A”-D”).
Mentions: To further elucidate a function of CC2D1 proteins in endosome maturation we analysed the endo/lysosomal compartments of wild type and mutant MEFs with the transmission electron microscope (TEM) (Fig 9). Endo/lysosomal organelles were identified by their ultrastructural characteristics proposed by others [48–50]. Exemplary pictures of the analysed endo/lysosomal organelles are depicted in S7A–S7D Fig. Cc2d1b-/- cells show no differences in size or morphology of the endo/lysosomal compartments compared to wild type cells (Figs 9A–9A”, 9C–9C”, 9E and S7E). In contrast and in accordance with the fluorescence microscopy data, Vps4a+/-,Vps4b+/- cells had enlarged endo/lysosomal compartments. We detected large multi-membrane structures that resembled the class E compartment described for loss of ESCRT function in yeast and mammalian cells [51,52]. We did not observe these structures in Cc2d1a-/- cells, but found that the average size of their endo/lysosomal organelles was significantly increased (Figs 9B–9B”, 9E and S7E). The endo/lysosomal phenotype of Cc2d1a-/- cells resembled that observed in lgd mutant cells of D. melanogaster [53]. When we allocated the endo/lysosomal organelles to different size classes, we found classes in Cc2d1a-/- and Vps4a+/-,Vps4b+/- MEFs that were not present in Cc2d1b-/- or wild type MEFs (S7E Fig). Thus, the loss of Cc2d1a function results in a moderate enlargement of the endo/lysosomal compartment, indicating that it is involved in endosomal maturation. Moreover, already the double heterozygosity of Vps4a and Vps4b results in a dramatic morphological defect of the endo/lysosomal phenotype, which appears to have no direct consequences for the viability of mice in captivity.

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