Fig 2: Cc2d1a or Cc2d1b deficient intestine does not display alterations in goblet cell number, proliferating cells nor Notch signalling.(A) Immunoblotting of protein lysates from liver and intestine isolated from Villin-Cre tg/+,Cc2d1a 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 Villin-Cre tg/+,Cc2d1a 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 Hes1 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.

Fig 3: 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.

Fig 4: 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.

Fig 5: Disruption of Cc2d1a in the nervous system results in perinatal death.(A) PCR genotyping of different tissues (tb: tail biopsy, br: brain, lu: lung) of Nestin-Cre mediated wild type and heterozygous Cc2d1a mutants using primers P1,P3 and P5. DNA from heterozygous and homozygous Cc2d1a deficient mice was used as control. Cre mediated recombination of Cc2d1a is only detected in the brain of mutant mice. (B) Immunoblotting of protein lysates from abdominal organs (ao) and brain (br) isolated from Nestin-Cre tg/+;Cc2d1a 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.