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Identifier

000388614

Title

Ο υποκυτταρικός εντοπισμός του Delta, διαμεμβρανικού προσδέτη του υποδοχέα Notch, στη Drosophila melanogaster

Alternative Title

Subcellular localization of Delta, Notch transmembrane ligand, in Drosophila melanogaster

Author

Σαματά, Μαρία Μ.

Thesis advisor

Δελιδάκης, Χρήστος

Abstract

Notch is an evolutionary conserved signaling mechanism, very important in many developmental processes and disease. Notch, the trans-membrane receptor, has to engage with its ligands, Delta or Serrate/Jagged, in trans for the activation of the downstream pathway. It has been shown that the ligands have to be priori activated by two E3 ubiquitin ligases, Neuralized and Mindbomb, which induce clearance of the ligand from the plasma membrane and its localization in endocytic vesicles. As ligand endocytosis appears to be crucial for Notch activation, we decided to study further the endocytic route that Delta follows as a result of the ubiquitination signaling from the E3 ligases. For this analysis, well- characterized proteins that serve as markers for the different endocytic compartments, the Rab-GTPases were used. Using Delta variants, constructed by our laboratory, which distinctively do not interact with the E3 ligases, we examined the hypothesis that Delta is targeted to a different endocytic compartment as a result of its ubiquitination. Using confocal microscopy and subsequently performing a quantitative analysis, using a semi-autonomous algorithm (OBCOL), we measured the colocalisation rates between Delta and the different endosomal compartments. Of great interest were the results from the Rab4GTPase analysis, which is associated with the recycling endosomes. Delta was measured to be increasingly colocalized with Rab4 recycling endosomes. However, Dli1/2, the dominant negative variant showed statistically important decreased rates of colocalisation with Rab4 positive endosomes. Therefore, activation by the E3 ubiquitin ligases may be responsible for Delta targeting to Rab4- dependent recycling route which might be important for Delta – Notch signaling. For the validation of the above analysis, we decided, also, to perform a functional assay of Delta, in conditions of malfunctioned endocytosis. Using constitutively active or dominant negative forms of specific RabGTPases, using induced mosaic analysis, we wanted to examine if Delta would promote the expression of a Notch target gene, wingless, in the wing disc of 3rd instar larvae. In this work, we started optimizing the conditions of the analysis, but we weren’t able to acquire clones of cell with disrupted endocytic nodes, possibly due to cell lethality.

Language

Greek

Subject

Analysis

Colocalisation

E3 ligases

Endocytosis

Notch signaling

Rab-gtpases

Ανάλυση

Ε3 λιγάσες

Ενδοκυττάρωση