Spatiotemporal coordination and mechanisms of communication between endocytosis and regulated exocytosis upon VEGFR2 signaling in endothelial cells (Doctoral thesis)

Γούλα, Ευαγγελή

So far, endocytosis induced signaling as well as signaling induced exocytosis have been studied separately. Thus, it remains unknown whether endocytosis and exocytosis are interdependent processes and which are the basic molecular mechanisms of this relationship? In the present study, the inhibitors dyngo-4a (for clathrin-dependent endocytosis) and EIPA (for macropinocytosis) were used to block the different internalization routes of VEGFR2 and the consequences of this inhibition on WPBs exocytosis were tested. The results demonstrate that clathrin-dependent endocytosis of VEGFR2 causes a minor inhibition on the percentage of secreted vWF, as compared to macropinocytosis, which has the predominant inhibitory role. The inhibitory role of macropinocytosis on VEGF-induced WPBs exocytosis was also tested by siRNAs treatments of common regulatory proteins of the macropinocytosis route (Rabankyrin-5, CDC42), followed by assessment of the secreted vWF by an ELISA-based method. Indeed, siRNAs treatment of the above proteins, individually or in combination, increased the VEGF-induced WPBs exocytosis. In addition, time-lapse confocal video microscopy in HUVECs suggests that upon VEGF stimulation, VEGFR2 positive endosomes are in limited contact with WPBs. In order to better understanding of the molecular mechanisms responsible for VEGF-induced exocytosis of WPBs, we employed a proteomic analysis of the secreted proteins in activated endothelial cells. Among the secreted proteins which were analyzed by high resolution mass spectrometry (nLC-MS/MS) was the galectin-1 protein (Gal-1). In the present study we found that galectin-1 is targeted to the WPBs of endothelial cells, since confocal microscopy (LSCM/CLSM) with/or STED demonstrates that galectin-1 co-localizes with vWF, the main cargo molecule of WPBs. This observation was also confirmed by its co-localization with other bona fide markers of WPBs (P-selectin, Rab27a, Rab3a, Rab3d). Surprisingly, galectin-1 was found only in a subset of WPBs and in a “rare” subpopulation of HUVECs. The results show that, upon VEGF/bFGF/ATP induction galectin-1 is localized in WPBs that recapture the extracellular anti-vWF antibodies, thus undergo "kiss-and-run" exocytosis. In parallel, internalization assays of recombinant galectin-1 proteins added extracellularly in culture medium, in control and siRNA treated for galectin-1 HUVECs, show that recombinant galectin-1 is localized in a limited number of WPBs. In addition, removal of extracellular galectin-1 from the culture medium reduces the number of galectin-1 positive WPBs significantly. The above data provide novel insights into the mechanism of unconventional secretion of galectin-1, implying that its secretion is partly, due to “kiss-and-run” exocytosis of WPBs.
Institution and School/Department of submitter: Πανεπιστήμιο Ιωαννίνων. Σχολή Επιστημών Υγείας. Τμήμα Ιατρικής
Subject classification: Κύτταρα
Keywords: Ενδοκυττάρωση,Μακροπινοκυττάρωση,Σηματοδότηση του VEGFR2,UPS (Μη συμβατική πρωτεϊνική έκκριση),Ενδοθηλιακά κύτταρα,Endocytosis,Macropinocytosis,VEGFR2 signaling
Appears in Collections:Διδακτορικές Διατριβές

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