Examining phospho-modulation of regulated exocytosis and the release-ready state of native secretory vesicles

Abstract

Regulated exocytosis enables numerous critical cellular functions such as wound healing, fertilisation, and neurotransmission. In most eukaryotic cells, proteins and other compounds that need to be secreted into the extracellular space are sorted into secretory vesicles, which then translocate to the plasma membrane where they dock and undergo priming reactions. Upon elevation of intracellular calcium levels (caused by a physiological stimulus), release-ready secretory vesicles fuse with the plasma membrane and release their contents. The late, calcium regulated steps of exocytosis (including docking, priming, and fusion) have been studied using a variety of model systems; in particular, studies utilising sea urchin eggs, cell surface complexes (large sheets of plasma membrane with endogenous, docked vesicles) and isolated cortical vesicles have provided numerous original insights into fundamental, conserved molecular mechanisms (Reviewed in Chapter One, and methods pertaining to the use of the model are detailed in Chapter Two). In Chapter Three, using intact eggs and cell surface complexes, I test the hypothesis that the fully primed, release-ready state is one of stable hemifusion (i.e. with the contacting monolayers of the vesicle and plasma membrane having already merged). Studies utilising fluorescent dyes with well-defined membrane properties and membrane permeable cationic amphiphiles capable of disrupting hemifusion intermediates indicate that, contrary to current opinion, the release-ready state is dynamic, and capable of transiently transitioning between hemifusion and close bilayer apposition. The phosphorylation of proteins and lipids has been implicated in modulating all stages of regulated exocytosis, and in Chapter Four I use cortical vesicles and minimised docking and membrane fusion assays to screen a range of well characterised small molecule modulators of phosphorylation. Data from the screen implicate phosphatases and casein kinase 2 in modulating the calcium sensitivity of fusion, and sphingosine kinase in modulating the ability to fuse. As detailed in Chapter Five, combined molecular and functional analysis of cell surface complexes and cortical vesicles with altered membrane sphingolipid levels confirmed that a critical balance of sphingolipids and sphingolipid metabolites is needed to maintain efficient docking, calcium sensitivity, and capacity to fuse.

Date of Award	2016
Original language	English