First name
Caterina
Last name
Lucano
Year of Study
Thesis Title
A novel role of the endocytic adaptor proteins Eps15 and Eps15L1 in the regulation of Notch signaling
Thesis Abstract
Notch signaling is an evolutionary conserved signaling pathway that regulates multiple aspects of development and cell renewal. Endocytosis plays a critical role in Notch signaling regulation, both in the signal-sending and in the signal-receiving cell. Eps15 and Eps15L1 are two endocytic adaptors, involved in clathrin-dependent and independent endocytosis of tyrosine kinase receptors. Double Knockout (DKO) mice for Eps15/15L1 die between 9.5 and 11.5 dpc and show a Notch loss of function phenotype, mirrored by downregulation of Notch target genes. Based on these observations we decided to test whether Eps15 and Eps15L1 can regulate Notch signaling.
Using an in vitro coculture/transactivation assay we observed no reduction in Notch activity after knockdown (KD) of Eps15 or Eps15L1 in the signal-receiving cell. However, after KD of Eps15 or Eps15L1 in the signal-sending cell a 40-50% reduction in Notch activity was observed, even though no further decrease of the signal was observed after the combined Eps15/L1 KD. This observation was true for the four Notch ligands Dll1, Dll4, Jag1 and Jag2.
To understand the molecular mechanism underlying this regulation, we set up a FACS based Dll1 internalization assay. While we observed a strong reduction in Dll1 endocytic constant following KD of known endocytic regulators of Notch ligands, no reduction was observed after KD of Eps15, Eps15L1 or both. Moreover, we assessed that localization of Dll1 in detergent resistant membrane was not altered following Eps15/L1 KD. At the moment we are setting up a quantitative Dll1 recycling assay and a structure/function rescue with Eps15 mutants to understand other possible mechanisms through which Eps15/L1 can regulate Notch ligands activity.
Using an in vitro coculture/transactivation assay we observed no reduction in Notch activity after knockdown (KD) of Eps15 or Eps15L1 in the signal-receiving cell. However, after KD of Eps15 or Eps15L1 in the signal-sending cell a 40-50% reduction in Notch activity was observed, even though no further decrease of the signal was observed after the combined Eps15/L1 KD. This observation was true for the four Notch ligands Dll1, Dll4, Jag1 and Jag2.
To understand the molecular mechanism underlying this regulation, we set up a FACS based Dll1 internalization assay. While we observed a strong reduction in Dll1 endocytic constant following KD of known endocytic regulators of Notch ligands, no reduction was observed after KD of Eps15, Eps15L1 or both. Moreover, we assessed that localization of Dll1 in detergent resistant membrane was not altered following Eps15/L1 KD. At the moment we are setting up a quantitative Dll1 recycling assay and a structure/function rescue with Eps15 mutants to understand other possible mechanisms through which Eps15/L1 can regulate Notch ligands activity.
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