Spatiotemporal organization of RabGTPases in stimulated exocytosis in endothelial cells (Doctoral thesis)

Γκέκα, Δέσποινα

Weibel Palade Bodies (WPBs) are elongated secretory organelles of endothelial cells that contain von Willebrand factor (vWF) and other cargo proteins that contribute to hemostatic plug formation, inflammation, angiogenesis, and tissue repair. Thus, WPB exocytosis plays important role in the pathophysiology of blood vessels. To accomplish secretion of their cargo molecules, WPBs undergo maturation, transport and fusion with the plasma membrane. Rab GTPases are crucial factors in controlling the specific targeting of vesicles to their destination organelles. In previous studies we identified 5 Rabs (Rab3a, 15, 27a, 33a and 37) specifically localized at the membrane of WPBs (WPB-Rabs). In this Thesis, in order to get insights into the possible role of the five WPB-Rabs in the mechanisms of formation, transport and exocytosis of WPBs, we aimed in studying their spatio-temporal organization at WPBs. In particular, we studied the order and timing of recruitment of these Rabs at WPBs, from the time they are generated at the Golgi, until they mature and fuse with the plasma membrane. We found that the above 5 Rabs are recruited to the membrane of WPBs at least 1 hour after these organelles are produced from the Golgi compartment. The first Rabs that appear at WPBs, almost at the same time, are Rab27a and Rab33a; Rab37 is recruited soon after them, followed by the sequential recruitment of Rab3a and Rab15. Based on FRAP experiments, where we studied the fluorescence recovery time of WPBs after photobleaching of these 5 GFP-Rabs, we found that the recycling rate of Rabs, between membrane and cytoplasm, follow the order Rab3a>Rab15Rab33>Rab37Rab27. This order is indicative of the relative (comparatively between these five Rabs) stability of the complexes formed by these Rabs on the membrane, as well as of the rate of GTP hydrolysis and of their solubilization towards the cytoplasm. Finally, since Rab27a is the first recruited Rab at WPBs and, based on previous studies, the most important for their maturation and secretion, we studied the possible role of Rab27a in the membrane recruitment of the other four Rabs, by employing siRNA-based silencing experiments. These experiments showed that lack of Rab27a from the newly formed WPBs reduces the time of recruitment of Rab3a, Rab15, and Rab37, while it augments the time required for the recruitment of Rab33a. All in all, the above data provide the first to date information on the spatial-temporal recruitment and dynamics of WPB-Rabs, thus contributing to the understanding of the possible role of these Rabs on WPB maturation and exocytosis.
Institution and School/Department of submitter: Πανεπιστήμιο Ιωαννίνων. Σχολή Επιστημών Υγείας. Τμήμα Ιατρικής
Subject classification: Κύτταρα
Keywords: Εκκριτικά οργανίδια των ενδοθηλιακών κυττάρων,Παράγοντας von Willebrand (vWF),Εξωκυττάρωση,Σειρά στρατολόγησης Rab GT,Μικροσκοπία πραγματικού χρόνου,Μέθοδος ανάκτησης φθορισμού μετά από φωτοσκίαση,Weibel Palade Bodies (WPBs),von Willebrand factor (vWF),Εxocytosis,Οrder of Rab recruitment,Video Microscopy,FRAP
Appears in Collections:Διδακτορικές Διατριβές

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