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

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.

Structural Basis for Regulation of ESCRT-III Complexes by Lgd

“…Moreover, a single interaction-competent DM14 repeat is sufficient for rescue of the lgd null phenotype in vivo . The binding mode between Shrub and Lgd appears to be conserved in humans, as CC2D1A/Lgd2 and CC2D1B/Lgd1 can rescue lgd mutants in a shrb dependent manner (Drusenheimer et al, 2015) and charge reversal of the acidic face of CHMP4B, the mammalian homolog of Shrub, also disrupts interaction with human Lgd2/CC2D1A (Martinelli et al, 2012). In yeast, where the ESCRT pathway has been extensively defined, an Lgd ortholog appears to be lacking, suggesting that Lgd serves a regulatory role, as opposed to an obligate function in polymer assembly and ESCRT activity.…”

“…In animals, proper ESCRT-III function requires modulation of Shrub activity by the large multi-domain protein called Lethal (2) giant discs (Lgd) in flies, or Coiled-Coil and C2 Domain Containing proteins 1A and 1B (CC2D1A and B) in mammals (Drusenheimer et al, 2015; Troost et al, 2012). Loss of Lgd in flies results in aberrant sorting and signalling of membrane proteins such as Notch (Jaekel and Klein, 2006; Klein, 2003; Schneider et al, 2013), and produces defects in cell division (Matias et al, 2015; Morawa et al, 2015).…”

“…Loss of Lgd in flies results in aberrant sorting and signalling of membrane proteins such as Notch (Jaekel and Klein, 2006; Klein, 2003; Schneider et al, 2013), and produces defects in cell division (Matias et al, 2015; Morawa et al, 2015). In mice, loss of CC2D1A results in postnatal lethality from respiratory distress caused by central nervous system defects (Drusenheimer et al, 2015; Zhao et al, 2011). In humans, CC2D1A mutation is associated with a spectrum of neurological disorders, including intellectual disability, autism spectrum disorder, and seizures (Basel-Vanagaite et al, 2006; Manzini et al, 2014).…”

Structures, Functions, and Dynamics of ESCRT-III/Vps4 Membrane Remodeling and Fission Complexes

“…These helical hairpins again pack side to side, recapitulating the intermolecular hairpin stacking interactions seen in the open CHMP1B strand. Importantly, CHMP4 filament formation can be blocked by Lgd (CC2D1A/B), a negative regulator of the multivesicular body (MVB) pathway (Drusenheimer et al 2015, Martinelli et al 2012, Troost et al 2012, Usami et al 2012), and a recent crystal structure has shown how the third DM14 domain of Lgd binds the isolated Shrub hairpin to occlude polymerization sterically (McMillan et al 2017). Thus, the similarity of open subunit structures and interactions seen in the different ESCRT-III filaments, as well as the conserved mechanisms of the Lgd (CCD1) inhibitors (Martinelli et al 2012, McMillan et al 2017), indicate broad evolutionary conservation of the opening, polymerization, and regulatory mechanisms (McCullough et al 2015, McMillan et al 2016, Tang et al 2015).…”

Loss of the Intellectual Disability and Autism Gene Cc2d1a and Its Homolog Cc2d1b Differentially Affect Spatial Memory, Anxiety, and Hyperactivity

“…In Drosophila , where only one CC2D1 homolog, lethal giant discs lgd , is present, removal of lgd is lethal during the larval stage ( Gallagher and Knoblich, 2006 ; Jaekel and Klein, 2006 ). Expression of either human CC2D1A or CC2D1B can rescue the phenotypes observed in Drosophila ( Drusenheimer et al, 2015 ), suggesting that CC2D1A and CC2D1B act redundantly. Despite wide expression of CC2D1A and its binding to multiple proteins involved in the immune response ( Chang et al, 2011 ; Chen et al, 2012 ), CC2D1A LOF in humans appears to only affect the brain, leading to a spectrum of behavioral deficits.…”