The Mammalian Orthologs of <i>Drosophila</i> Lgd, CC2D1A and CC2D1B, Function in the Endocytic Pathway, but Their Individual Loss of Function Does Not Affect Notch Signalling

<div><p>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 <i>Drosophila melanogaster</i> 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 <i>Cc2d1a</i> and <i>Cc2d1b</i> conditional knockout mice. While <i>Cc2d1b</i> deficient mice displayed no obvious phenotype, we found that <i>Cc2d1a</i> 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 <i>Cc2d1a</i> deficient MEF cells. To analyse an influence on Notch signalling, we generated intestine specific <i>Cc2d1a</i> 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 <i>Cc2d1a</i> mutant cells, but not <i>Cc2d1b</i> 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 <i>D</i>. <i>melanogaster</i>, 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.</p></div

<i>Cc2d1a</i> or <i>Cc2d1b</i> deficient intestine does not display alterations in goblet cell number, proliferating cells nor Notch signalling.

<p>(A) Immunoblotting of protein lysates from liver and intestine isolated from <i>Villin-Cre</i>^tg/+,<i>Cc2d1a</i>^flox/flox and control mice. CC2D1A is expressed in the liver of all analysed animals and in the intestine of heterozygous and wild type mice, but not in homozygous animals. (B) Alcian blue staining of paraffin embedded intestinal sections revealed no alterations in goblet cell number between <i>Villin-Cre</i>^tg/+,<i>Cc2d1a</i>^flox/flox, Cc2d1b^-/- and wild type animals. (C, D) Immunohistochemical staining was performed on cryosections with antibodies directed against the proliferation marker Ki67 (C) and GFP (D). (E) Relative <i>Hes1</i> mRNA levels in intestine of wild type and mutant animals were monitored by qRT-PCR. No differences between mutant and control animals were detected (n = 3). Scale bars are 50 μm.</p

Ultra-structural characterisation of endo/lysosomal compartments in wild type, <i>Cc2d1a</i>^-/-, <i>Cc2d1b</i>^-/- and <i>Vps4a</i>^+/-,<i>Vps4b</i>^+/- MEFs.

<p>(A-A”) wild type, (B-B”) <i>Cc2d1a</i>^-/- mutant, (C-C”) <i>Cc2d1b</i>^-/- mutant and (D-D”) <i>Vps4a</i>^+/-,<i>Vps4b</i>^+/- double heterozygous MEFs. The comparison reveals that <i>Cc2d1a</i>^-/- and <i>Cc2d1b</i>^-/- 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 <i>Vps4a</i>^+/-,<i>Vps4b</i>^+/- 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 <i>Cc2d1a</i>^-/- and <i>Vps4a</i>^+/-,<i>Vps4b</i>^+/- 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 (<i>A-D</i>) and 0.5 μm (<i>A</i>`-<i>D</i>`, <i>A</i>”-<i>D</i>”).</p

CC2D1A appears not to regulate centrosome function.

<p>(A) Immunocytochemical staining of wild type, <i>Cc2d1a</i>^+/- and <i>Cc2d1a</i>^-/- MEF cells with an affinity purified anti-guinea-pig-CC2D1A antibody. No staining could be detected in <i>Cc2d1a</i> 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 <i>Cc2d1a</i> deficient MEF cells compared to wild type MEF cells. Two to three different passages were analysed per genotype. Scale bars are 20 μm.</p

Generation of a conditional knockout mouse of the <i>Cc2d1b</i> gene locus.

<p>(A) Scheme for generating animals carrying the conditional knockout allele (<i>Cc2d1b</i>^flox) and the recombined allele (<i>Cc2d1b</i>^<i>-</i>). LoxP sites are depicted as yellow triangles, FRT sites as orange triangles. The DM14 and C2 protein domains are marked in green and red, respectively. Primers for genotyping are depicted as black arrows. (B) PCR genotyping of wild-type and heterozygous and homozygous <i>Cc2d1b</i>^<i>-</i> mice using primers 1F, 3R and 3bR. (C) RT-PCR analysis of total RNA extracts of brain of newborn animals with exon specific primer pairs that confirm lack of expression of the deleted segment. (D) Immunoblotting of protein lysates from MEF (mouse embryonic fibroblast) cells isolated from <i>Cc2d1b</i>^-/- and control mice. The 130kDa CC2D1B band is lacking in the lysates of homozygous <i>Cc2d1b</i>^-/- mice.</p

Subcellular localisation of CC2D1A and CHMP4B in wild type cells and <i>Vps4a</i>^+/-,<i>Vps4b</i>^+/- cells.

<p>(A) Immunocytochemical staining was performed on wild type and <i>Cc2d1a</i> 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 <i>Vps4a</i>^+/-,<i>Vps4b</i>^+/- MEFs with antibodies directed against CC2D1A, CHMP4B and the late endosomal/lysosomal marker LAMP1. In <i>Vps4a</i>^+/-,<i>Vps4b</i>^+/- 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.</p

Disruption of <i>Cc2d1a</i> in the nervous system results in perinatal death.

<p>(A) PCR genotyping of different tissues (tb: tail biopsy, br: brain, lu: lung) of Nestin-Cre mediated wild type and heterozygous <i>Cc2d1a</i> mutants using primers P1,P3 and P5. DNA from heterozygous and homozygous <i>Cc2d1a</i> deficient mice was used as control. Cre mediated recombination of <i>Cc2d1a</i> is only detected in the brain of mutant mice. (B) Immunoblotting of protein lysates from abdominal organs (ao) and brain (br) isolated from <i>Nestin-Cre</i>^tg/+;<i>Cc2d1a</i>^flox/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.</p

Endogenous CC2D1A and CHMP4B interact in wild type MEFs.

<p>PLA (Proximity Ligation Assay) was performed on wild type and <i>Cc2d1a</i> 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.</p

Human CC2D1A and CC2D1B can replace the function of Lgd in <i>D</i>. <i>melanogaster</i>.

<p>(A) Expression of the Notch target gene <i>wingless</i> (Wg) in a wild type wing imaginal disc. (B) Expression of Wg in an <i>lgd</i> null mutant disc (lgd^d7). The expression domain has broadened dramatically, indicating an expansion of Notch activity. In addition, the disc is much larger due to over-proliferation of the disc cells. Expression of one copy of CC2D1B (C) or CC2D1A (D) normalises the expression of Wg. (E) Magnification of the wing area of the disc shown in (D). It reveals that a few cells still express Wg ectopically indication that the rescue of <i>lgd</i> mutants by CC2D1A is incomplete. (F) The ectopic expression of Wg is completely suppressed if two copies of CC2D1A are present in the genome. (G) The phenotype of <i>lgd</i> mutant discs rescued with one copy of CC2D1A worsened dramatically upon loss of one copy of <i>shrb</i>. (H) The wing imaginal disc of an <i>lgd</i> mutant, <i>shrub</i> heterozygous animal. The animals die during the early third larval instar stage and contain very small imaginal discs with Wg primarily located in dramatically enlarged endosomes. Arrows point to the expression domain of Wg along the dorsoventral boundary, which is controlled by the activity of the Notch pathway. Scale bars are 100μm.</p

Generation of a conditional knockout mouse of the <i>Cc2d1a</i> gene locus.

<p>(A) Scheme for generating animals carrying the conditional knockout allele (<i>Cc2d1a</i>^flox) and the recombined allele (<i>Cc2d1a</i> ^-). LoxP sites are depicted as yellow triangles, FRT sites as orange triangles. The DM14 and C2 protein domains are marked in green and red, respectively. Primers for genotyping are numbered P1-P5 and are depicted as black arrows. (B) PCR genotyping of wild type and heterozygous and homozygous <i>Cc2d1a</i>^- mice using primers P1, P3 and P5. (C) RT-PCR analysis of total RNA extracts of brain of newborn animals with exon specific primer pairs that confirm lack of expression of the deleted segment. (D) Immunoblotting of protein lysates from MEF cells isolated from <i>Cc2d1a</i>^<i>-/-</i> and control mice. The 130kDa CC2D1A band is lacking in the lysates of homozygous <i>Cc2d1a</i>^-/- mice.</p