This grant focuses on the basic cell biological process of membrane trafficking through the secretory and endocytic pathways in mammalian cells. Secretion and endocytosis play fundamental biological roles at the level of both single cells and multi-cellular organisms to control a wide array of physiological processes including nutrient uptake, secretion of hormones and digestive enzymes, and defense against foreign pathogens. Secretion and endocytosis involve the transport of cargo between intracellular organelles of these pathways, 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, and atherosclerosis. In mammalian cells, membrane tubules (60-80 nm in diameter and to many microns in length) emanate from various organelles of the secretory and endocytic pathways, and these tubules have been implicated in many intracellular trafficking steps, including retrograde trafficking from the Golgi complex to the ER and recycling from receptors from endosomes to the cell surface. Recent evidence from my laboratory shows that cytoplasmic phospholipase A2 (PLA2) and lysophospholipid acyltransferase (LPAT) enzymes, with opposing enzymatic activities, may 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 PLA2 and LPAT enzymes with respect to secretion and endocytosis in mammalian cells. The goals of this work are encompassed in three Specific Aims: 1) identify and characterize the PLA2 enzymes and accessory proteins involved in stimulating tubule formation; 2) identify and characterize the LPATs involved in mediating tubule formation and/or coated vesicle fission; and, 3) identify and characterize protein and lipid kinases that regulate Golgi membrane tubule formation and function. These goals will be achieved through a combination of molecular, cellular, and genetic approaches. The in vivo roles of candidate PLA2 and LPAT enzymes in secretory and endocytic trafficking will be investigated by conducting over-expression and siRNA knockdown experiments. These experiments will focus on Golgi-to-ER retrograde trafficking, export from endocytic compartments, 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 PLA2 activity on membrane shape. These studies will reveal novel biological roles for PLA2 and LPAT enzymes in mediating intracellular trafficking events in the secretory and endocytic pathways.
This proposal is to conduct basic research into the structure and function of mammalian cells, with a focus on the processes of secretion and endocytosis. Both secretion and endocytosis are vital for human and animal health because they are 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 and endocytic pathways.
| Bechler, Marie E; Brown, William J (2014) G?1?2 activates phospholipase A2-dependent Golgi membrane tubule formation. Front Cell Dev Biol 2:0004 |
| Murugesan, Sricharan; Goldberg, Elysa B; Dou, Eda et al. (2013) Identification of diverse lipid droplet targeting motifs in the PNPLA family of triglyceride lipases. PLoS One 8:e64950 |
| 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 |
| Yang, Jia-Shu; Valente, Carmen; Polishchuk, Roman S et al. (2011) COPI acts in both vesicular and tubular transport. Nat Cell Biol 13:996-1003 |
| Bechler, Marie E; Doody, Anne M; Ha, Kevin D et al. (2011) The phospholipase A? enzyme complex PAFAH Ib mediates endosomal membrane tubule formation and trafficking. Mol Biol Cell 22:2348-59 |
| Schmidt, John A; Kalkofen, Danielle N; Donovan, Kirk W et al. (2010) A role for phospholipase A2 activity in membrane tubule formation and TGN trafficking. Traffic 11:1530-6 |
| Schmidt, John A; Yvone, Griselda Metta; Brown, William J (2010) Membrane topology of human AGPAT3 (LPAAT3). Biochem Biophys Res Commun 397:661-7 |
| Bechler, Marie E; Doody, Anne M; Racoosin, Esther et al. (2010) The phospholipase complex PAFAH Ib regulates the functional organization of the Golgi complex. J Cell Biol 190:45-53 |
| Judson, Bret L; Brown, William J (2009) Assembly of an intact Golgi complex requires phospholipase A2 (PLA2) activity, membrane tubules, and dynein-mediated microtubule transport. Biochem Biophys Res Commun 389:473-7 |
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