Thompson, Nicola; (2001) An investigation into the regulated secretory pathway of granulocytes. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In recent years great strides have been made in our understanding of the secretory pathway. Much of the new information regarding regulated exocytosis has come from biochemical studies of neuronal cells and tissues. The aim of the work described in this thesis was to determine if cells of the haematopoietic lineage use the same granule docking and fusion machinery as that used for neurotransmitter release and to investigate the function of candidate genes involved in granulocyte degranulation. Western blotting across eosinophil subfractions failed to identify known components of the synaptic vesicle fusion machinery. cDNA library screening resulted in the isolation of VAMP2 and cellubrevin cDNA clones from eosinophil and RBL-2H3 cDNA libraries. Tagged VAMP2 or cellubrevin cDNA constructs transiently expressed in RBL-2H3 cells localised to the endocytic pathway. To determine if key gene products thought to play a role in regulated exocytosis affected RBL-2H3 cell degranulation, a functional assay utilising flow cytometry was developed. This enabled the effect of overexpression of GFP-tagged ARF and PLD 1b on RBL-2H3 degranulation to be determined. Using this approach the GDP-bound mutant of ARF6 inhibited ionomycin stimulated degranulation while overexpression of wild-type PLD 1b increased granule fusion and localised to RBL-2H3 granules. Therefore PLD1b and ARF6 are implicated in granulocyte degranulation.

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