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Title: Functions and mechanisms of Rab46 in endothelial cells
Author: Pedicini, Lucia
ISNI:       0000 0004 8506 1645
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2019
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Endothelial cells maintain vascular integrity by regulating a number of physiological and pathophysiological processes, including haemostasis, thrombosis and inflammation. A pivotal contribution to these processes is the exocytosis of cargo from specialized endothelial storage organelles, namely Weibel-Palade bodies (WPBs). WPBs provide an intracellular storage pool of pro-thrombotic and pro-inflammatory mediators which can be differentially released in response to different stimuli. Ca2+ raising agonists such as thrombin and histamine, respectively released following vascular injury or an immunogenic insult, evoke WPB exocytosis. However, inappropriate and untimely exocytosis of WPBs can promote the pro-thrombotic and pro-inflammatory environment evident in cardiovascular diseases. The mechanisms underlying differential cargo release in order to produce physiologically distinct responses are poorly understood. Some Rab GTPase family members, have been reported to be implicated in regulating the exocytosis of WPBs. Here, we describe a novel Rab GTPase (Rab46) in endothelial cells that is located on WPBs. Super-resolution microscopy confirmed that Rab46 is juxtaposed to von Willebrand Factor (vWF), on the cytosolic side of individual WPBs, whilst quantitative imaging analysis suggested that Rab46 may regulate a subpopulation of WPBs. Interestingly, Rab46 was necessary for acute histamine, but not thrombin, WPB trafficking towards the perinuclear area identified as the Microtubule Organizing Centre (MTOC). Biochemical analysis and mass spectrometry was used to investigate the molecular mechanisms underlying Rab46-dependent retrograde trafficking and the dynein heavy chain was identified as a candidate effector protein. Further biochemical experiments suggested a direct interaction between endogenous Rab46 and the dynein motor complex. Taken together, these results suggest that after acute histamine stimulation, dynein-bound Rab46 mediates retrograde transport of a subset of WPBs along microtubules to the MTOC. These observations indicate Rab46 as a key regulator of differential WPB cargo secretion, allowing an appropriate acute pro-inflammatory response whilst avoiding release of excessive pro-thrombotic mediators. Characterization of in vivo model of Rab46 represents the beginning of understanding the physiological contribution of Rab46 as well as its response to pathological conditions. Understanding the Rab46/WPB signalling axis, both in vitro and in vivo, could be important for achieving better appreciation of how the endothelial cell fine-tunes it's secretory response and thereby providing novel therapeutic targets for the prevention of endothelial dysfunction, which is often the trigger for cardiovascular diseases.
Supervisor: Mckeown, Lynn ; Beech, David ; Jayasinghe, Izzy Sponsor: University of Leeds ; MRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available