Role and regulation of 3-phosphorylated phosphoinositides in neuroexocytosis

In mammalian cells, membrane phospholipids not only serve to maintain structural integrity but play a dynamic and active role in a variety of essential biological functions including cell signalling and membrane trafficking. One type of phospholipid called phosphatidylinositol, contains a myo-inositol headgroup that can be phosphorylated at three distinct locations to give rise to seven different phosphoinositide species. Phosphoinositides have pleiotropic cellular roles ranging from signalling and membrane trafficking to modulation of ion channels and cell survival. The central theme of the research undertaken in this PhD was to investigate the role of phosphoinositides in neuroexocytosis. Previous work from our group had shown a critical role for class II phosphatidylinositol 3-kinase C2α (PI3K-C2α) in the priming of exocytosis. This raised the question of whether a pool of phosphatidylinoitol-3-phosphate (PtdIns3P) was produced by PI3K-C2α on large dense core vesicles (LDCVs) of neurosecretory cells (Chromaffin cells and Pheochromocytoma (PC12) cells). In Chapter 2, this question was directly addressed by examining the localization and regulation of PtdIns3P pools produced by PI3K-C2α in neurosecretory cells. The results showed that in PC12 cells there exist two pools of PtdIns3P, an endosomal pool and a vesicular pool, that are differentially sensitive to a pan-PI3-kinase inhibitor, wortmannin. The PtdIn3P pool located on LDCVs was found to be sensitive to PI3K-C2α knockdown and transiently upregulated upon stimulation of exocytosis. Importantly the activity of PI3K-C2α was tightly regulated by Ca2+, providing the first evidence for Ca2+ regulation of a PI3K family member. Further analysis by total internal reflection fluorescence microscopy showed that PtdIns3P-positive vesicles, labelled with the FYVE domain from EEA1 (FYVEEEA1) could undergo translocation to the plasma membrane upon stimulation of exocytosis but failed to fuse with the plasma membrane, suggesting that FYVEEEA1 acted as a dominant negative for vesicle fusion. This data demonstrated that PtdIns3P is not only involved in endosomal trafficking but also serves as an essential signal in Ca2+-regulated exocytosis. The finding that PtdIns3P could be produced on LDCVs raised an immediate question as to how PtdIns3P levels could be regulated upon stimulation of exocytosis. In Chapter 3, the role of PIKfyve, a phosphoinositide 5-kinase that predominantly produces PtdIns(3,5)P2 using PtdIns3P as a substrate, in modulating PtdIns3P levels on LDCVs was examined. Using a variety of approaches including overexpression, siRNA knockdown and a PIKfyve specific inhibitor YM201636, it was found that PIKfyve could negatively regulate exocytosis in neurosecretory cells. In PC12 cells expressing PIKfyve-GFP, a cytosolic pool of PIKfyve was transiently recruited onto LDCVs upon stimulation of exocytosis. This suggested that PIKfyve could be recruited to secretory vesicles by binding to PtdIns3P, presumably via its FYVE domain, which would both allow access to its substrates and negatively regulate exocytosis. Furthermore, the recruitment of PIKfyve onto LDCVs upon stimulation of exocytosis was found to be dependent of PI3K-C2α expression, suggesting PIKfyve is acting downstream of PI3K-C2α. These data strongly suggested that PIKfyve negatively regulates exocytosis by converting PtdIns3P pools produced by PI3K-C2α on LDCVs to PtdIns(3,5)P2. In the last chapter the role of Class I PI3-kinase δ (PI3Kδ) in the regulation of PtdIns(4,5)P2 levels was investigated during stimulated exocytosis in chromaffin cells. Inhibition of PI3Kδ activity using a PI3Kδ–specific inhibitor IC87114 lead to a transient increase in PtdIns(4,5)P2 levels on the plasma membrane that is attributed to the activation of a PtdIns(3,4,5)P3 3-phosphatase, phosphatase and tensin homolog deleted on chromosome ten (PTEN). By exploiting this pathway, it was found that a PtdIns(4,5)P2 signal is sufficient, in the absence of any stimulus, to promote an actin-mediated vesicular translocation to the plasma membrane. This report represents the first functional link between PtdIns(4,5)P2 signalling and actin-mediated vesicular translocation. Collectively, the results from these studies provide strong evidence for a regulatory role for 3-phosphorylated phosphoinositides in exocytosis and importantly, show that phosphoinositides act as both site-specific and temporally regulated signals in the dynamic events underpinning neuroexocytosis. This understanding of the role and regulation of phosphoinositides could open new avenues of research to help overcome the growing lists of phosphoinositide-related pathologies such as Charcot-Marie-Tooth disease, Francois-Neetens Mouchetee fleck corneal dystrophy, various cancers and Alzheimer’s disease