This grant focuses on the basic cell biological process of membrane trafficking through the secretory pathway in mammalian cells. Secretion plays fundamental biological roles at the level of both single cells and multi-cellular organisms t control a wide array of physiological processes including release of hormones and digestive enzymes, neurotransmission, and defense against foreign pathogens. Secretion involves the transport of cargo between intracellular organelles, e.g., between the Golgi complex and plasma membrane. Defects in the transport of proteins through these pathways are associated with a host of diseases including cancer, cystic fibrosis, neurodegeneration, and atherosclerosis. In mammalian cells, membrane tubules (60-80 nm in diameter and up to many microns in length) emanate from various organelles of the secretory pathway, and these tubules have been implicated in many intracellular trafficking steps, e.g., retrograde trafficking from the Golgi complex to the ER and anterograde movement through the Golgi stack. Recent evidence from my laboratory indicates that cytoplasmic phospholipase A (PLA) and integral membrane lysophospholipid acyltransferase (LPAT) enzymes, with opposing enzymatic activities, work in concert to mediate membrane tubule formation by directly altering membrane phospholipid composition and consequently membrane shape. The long-term objectives of this grant are to elucidate the roles of membrane tubules in intracellular trafficking, and to determine the biological functions of specific PLA and LPAT enzymes with respect to secretion in mammalian cells. The goals of this work are encompassed in three Specific Aims, with a focus on the mammalian Golgi complex: 1) identify and characterize PLA and LPAT enzymes involved in intracellular membrane trafficking;2) determine the contribution of PLA2 and LPAT enzymes to the production of Golgi coated vesicles and membrane tubules;and 3) determine the molecular mechanisms by which PLA2 and LPATs contribute to membrane tubule formation. These goals will be achieved through a combination of molecular, cellular, and genetic approaches. The in vivo roles of candidate PLA and LPAT enzymes in secretory trafficking will be investigated by conducting over expression and siRNA knockdown experiments. These experiments will focus on membrane trafficking to, through, and from the Golgi complex, and vesicle fission from membrane-bound organelles. In vitro assays that reconstitute tubule or vesicle formation will also be conducted to elucidate the mechanisms underlying the effects of PLA and LPAT activity on membrane shape. These studies will reveal novel biological roles for PLA and LPAT enzymes in mediating intracellular trafficking events in the secretory pathway and advance our understanding of membrane tubule-mediated transport.
This proposal is to conduct basic research into the structure and function of mammalian cells, with a focus on the process of secretion and the function of the mammalian Golgi complex. Secretion is vital for human and animal health because it is required for many important processes including digestion, neurotransmission, and hormone signaling. Many human diseases, such as cancer, atherosclerosis, and neurodegeneration, are caused by mutations that disrupt normal secretory trafficking.
|Kalkofen, Danielle N; de Figueiredo, Paul; Brown, William J (2015) Methods for analyzing the role of phospholipase Aâ‚‚ enzymes in endosome membrane tubule formation. Methods Cell Biol 130:157-80|
|Apodaca, Gerard; Brown, William J (2014) Membrane traffic research: challenges for the next decade. Front Cell Dev Biol 2:52|
|Bechler, Marie E; Brown, William J (2014) GÎ²1Î³2 activates phospholipase A2-dependent Golgi membrane tubule formation. Front Cell Dev Biol 2:0004|
|Bechler, Marie E; Brown, William J (2013) PAFAH Ib phospholipase A2 subunits have distinct roles in maintaining Golgi structure and function. Biochim Biophys Acta 1831:595-601|
|Bechler, Marie E; de Figueiredo, Paul; Brown, William J (2012) A PLA1-2 punch regulates the Golgi complex. Trends Cell Biol 22:116-24|
|Ha, Kevin D; Clarke, Benjamin A; Brown, William J (2012) Regulation of the Golgi complex by phospholipid remodeling enzymes. Biochim Biophys Acta 1821:1078-88|