Book Description

Professional secretory cells are responsible for regulated secretion of biologically active molecules to maintain homeostasis. These molecules, which include neuropeptides, digestive enzymes, and hormones, are stored in long-lasting secretory granules (SGs) that undergo exocytosis in response to external stimuli. The molecular mechanisms of SG biogenesis are poorly understood. Using Drosophila larval salivary glands as a model system, our laboratory has previously discovered that the type II phosphatidylinositol 4 kinase (PI4KII) is required for normal SG biogenesis. PI4KII is responsible for the generation of phosphatidylinositol 4-phosphate (PI4P) from phosphatidylinositol (PI) at the trans-Golgi network (TGN) and endosomes. To examine how PI4KII and its product PI4P regulate SG biogenesis, I employed genetic approaches and cell biology techniques to identify and characterize potential factors acting in this pathway. I discovered that Past1, syntaxin-16 (Syx16) and oxysterol binding protein (Osbp) are regulators of PI4KII and PI4KII-mediated SG maturation. I conducted a candidate RNA interference (RNAi) screen that identified many endocytic genes needed for proper SG maturation, suggesting that SG maturation requires endocytic contribution. A secondary screen suggested that Past1 and Syx16 are potential factors acting in the same pathway as PI4KII to facilitate SG maturation. Further characterization demonstrated that both PI4KII-dependent early endosomal sorting and accumulation of PI4P on SG membranes are associated with proper SG maturation. In addition, Past1 and Syx16 are needed for formation of PI4KII-positive endosomal tubules involved in this process. I also examined Osbp, which is a PI4P effector that regulates non-vesicular transport of PI4P and cholesterol across membrane contact sites (MCS). Loss of Osbp resulted in lowered levels of PI4P on SG membranes and an absence of PI4KII-positive tubules, similar to Past1 null mutants. Moreover, a cholesterol probe, D4H, showed that these tubules are enriched in cholesterol. My results indicate that Osbp is required for cholesterol accumulation at endosomal membranes to enable formation of PIKII-positive and cholesterol-rich membrane tubules. Overall, my results establish Past1, Syx16 and Osbp as regulators of PI4KII and describe a novel PI4KII-mediated early endosome retrograde trafficking pathway important for cellular PI4P distribution and SG maturation.