REDUNDANT AND NON-REDUNDANT ROLES OF THE ENDOCYTIC ADAPTOR PROTEINS EPS15 AND EPS15L1 IN MAMMALS
Creators: name: MILESI, CINZIA
Publisher: name: Universit� degli Studi di Milano
Year: 2017

description: Eps15 and Eps15L1 are two endocytic adaptor proteins involved in both clathrin-dependent and clathrin-independent endocytosis of receptor tyrosine kinases. Due to their homology, Eps15 and Eps15L1 are thought to be redundant in several cellular processes. Their redundancy, however, has never been demonstrated in in vivo model systems.
Our laboratory has generated genetically engineered mice to unravel the physiological functions of Eps15 and Eps15L1. We found that Eps15-KO (knockout) mice were healthy and fertile, while Eps15L1-KO mice died at birth because of neural defects, showing the specific function of Eps15L1 in neuronal development. Importantly, Eps15/Eps15L1-DKO (double knockout) mice had a more severe phenotype, dying at midgestation, suggesting redundancy in one or more fundamental developmental programs. 
The aim of this thesis project was to investigate redundant and non-redundant roles of Eps15 and Eps15L1, with the final goal to unmask the underlying causes of embryo lethality of Eps15/Eps15L1-DKO mice. 
Since Eps15/Eps15L1-DKO mice displayed a Notch loss-of-function phenotype, accompanied by a downregulation of Notch target genes, our initial hypothesis was that Eps15 and Eps15L1 were redundantly required in the regulation of the Notch signalling. To address this issue, we set-up a co-culture model system to recapitulate Notch signalling. In detail, we used MEFs (mouse embryonic fibroblasts) derived from Eps15- and Eps15L1-KO mice as a model system for the signal-sending cell and we co-cultured them with CHO (chinese hamster ovary) cells expressing the Notch receptor, as signal receiving cells. We found that Eps15L1, but not Eps15, played a non-redundant role in Notch signalling activation. This finding indicated that impaired Notch signalling was not responsible for the more severe phenotype observed in Eps15/Eps15L1-DKO mice compared to single KO mice. Whether Eps15 and Eps15L1 are required in signal-receiving cells will be addressed, using MEFs as a model for the signal-receiving cell. 
We found that angiogenesis was seriously compromised in Eps15/Eps15L1-DKO mice and, therefore, hypothesized that impaired vascular development might be the major cause of midgestation lethality of these mice. To address this issue, we generated cDKO (conditional Eps15/Eps15L1-DKO mice), which lack Eps15 and Eps15L1 in endothelial and hematopoietic cells. We found that cDKO mice displayed vascular defects but did not recapitulate the severe phenotype of constitutive DKO mice. This finding indicated that impaired angiogenesis was not the major lethality cause of constitutive DKO mice. However, by in vitro studies in endothelial cells, we found that Eps15 and Eps15L1 redundantly regulated VEGFR-2 turnover, thus indicating a possible cell-autonomous function of the proteins in vascular homeostasis, even if not sufficient to cause embryo lethality when functionally impaired.
Previous in vitro studies have demonstrated a role for Eps15 and Eps15L1 in the CDE (clathrin-dependent endocytosis) of EGFR (epidermal growth factor receptor) and TfR (transferrin receptor). To confirm this role in a clean background, we used MEFs as a model system. By using radioactive assays, we found that Eps15 and Eps15L1 redundantly regulate the CDE of TfR. Indeed, in DKO cells, the Ke (endocytic rate constant) of the TfR was reduced to ~50% and, as a consequence, surface levels of TfR were increased, while single KO cells showed only a minor, if any, defect. Moreover, in DKO cells, we found that the number of AP-2-positive structures (which label clathrin-structures that form during CDE) was increased, but the structures were significantly smaller in size. Whether the maturation of clathrin-coated structures is altered in DKO cells will be addressed by live imaging studies. 
Next, we asked whether the role of Eps15 and Eps15L1 in TfR internalization had a physiological relevance in vivo. In detail, since TfR is essential for the biology of erythroid cells, we investigated whether erythroid development was impaired in cDKO mice, lacking Eps15 and Eps15L1 in endothelial and hematopoietic cells. We found that these mice suffered from microcytic hypochromic anemia: RBCs (red blood cells) had reduced MCV (mean corpuscular volume) and high RDW (red blood cell distribution width, index of anisocytosis), and reticulocyte counts were higher. These findings suggest that Eps15 and Eps15L1 redundantly regulate erythroid development. However, since cDKO mice did not recapitulate the severe phenotype of constitutive DKO mice, altered erythroid development per se was not the only cause of the early lethality of constitutive DKO mice.
Further studies are required to investigate whether altered development of Eps15/Eps15L1-DKO erytroid cells correlates with increased surface levels of TfR and reduced iron uptake.
These findings, combined with previous data generated in our laboratory, highlight that Eps15 and Eps15L1 regulate several cellular processes, both in a redundant and in a non-redundant manner. Functional impairment of these processes, together with other unexplored processes, might addictively contribute to the DKO phenotype.