Paul Gleeson Professor - Department of Biochemistry and Molecular ...

Paul GleesonProfessorRoom 335Bio21 Molecular Science and Biotechnology Institute30 Flemington RoadTel +61 3 8344 2354pgleeson@unimelb.edu.auhttp://www.biochemistry.unimelb.edu.au/research/res_gleeson.htmlMembrane trafficking and protein sorting in model systems and specialized primary cellsMembrane trafficking underpins many physiological processes, including secretion, receptor signalling,endocytosis, antigen presentation, and neural networking. Membrane trafficking is also exploited byinfectious organisms and toxins to gain entry into the cell. Moreover, many diseases arise from defects inmembrane trafficking, including Alzheimer’s disease. Our aim is to understand the molecular basis ofmembrane and protein sorting in the secretory and endocytic pathways in a variety of physiologicalprocesses using cultured cells and differentiated primary cells.1. Defining molecular machinery and membrane organization of Golgi transport pathwaysThe trans‐Golgi network (TGN) is a major traffic hub. Our goal is to identify the molecular machinery thatregulates both the export and the import pathways to and from the TGN. We have identified novelcomponents of the trafficking machinery, including small G proteins and golgins, and have developed aninterference RNA‐based approach to silence these components in vivo andexplore their physiological function. This project will investigate the role oftwo novel Golgi small G proteins, Arl5b and Rab30. We have constitutivelyactive (GTP restricted) and dominant –negative (GDP‐restricted) mutants ofboth Arlb5b and Rab30. Using these mutants we will examine their preciselocalisation of these small G proteins and different cargos in the TGN usingnew methods of super resolution confocal microscopy to define membranesubdomains. In addition the role of these small G proteins in transportingdifferent cargos will be assessed using transport assays in a variety of celltypes.Cartoon of the secretory and endocyticpathways2. Regulated endocytosis and antigen uptake by immune cellsMacropinocytosis is a regulated form of endocytosis and is highly active in macrophages and dendritic cells(antigen presenting cells) where it is a major pathway for the capture of antigens. Despite the importanceof this pathway, the molecular basis for the formation and maturation of macropinosomes is poorlydefined. We have recently identified the molecule sortin nexin 5 (SNX5) as a key regulator ofmacropinocytosis in macrophages. By generating SNX5 deficient mice and cell lines, this project aims toinvestigate the role of macropinocytosis in antigen uptake and immune responses by macrophages anddendritic cells both in vitro and in vivo.3. Cell biology of membrane receptors regulating the serum half‐life of novel therapeutic proteinsThe neonatal Fc receptor (FcR) plays a critical role in regulating the half‐live of a range of serum proteins,including IgG, in the adult individual. The Fc receptor protects serum proteins from degradation by bindingto IgG in endosomes after internalization by cells and releasing the proteins back into the plasma. There isconsideration interest in exploiting this protective pathway to prolong the live time of engineeredtherapeutic proteins. In collaboration with CSL this project will define the membrane recycling pathway ofthe neonatal Fc receptor, information which is critical for the optimizing the life span of therapeuticproteins.Page 14

HeLa cell expressing a epitope taggedmembraneprotein, mutated in a Tyrbasedinternalization motif, resultingin the accumulation of the membraneprotein (red) on the cell surface oftransfected cells. 3D reconstruction ofconfocal images showing membraneprotein (red), Golgi marker (green) andnuclear DAPI staining (blue).4. Signals for endosomal sorting in development and diseaseA number of important membrane proteins recycle between the plasmamembrane and the Golgi, and this recycling is essential for their function.Perturbation of these pathways can result in diseases, including neurodegenerativediseases such as Alzheimer’s disease. Very little is known about the targetingsignals of cargo proteins that define this transport pathway. This project willdefine these targeting signals by the generation and analysis of mutants ofmembrane cargos using defined transport assays and by the silencing pathwayspecifictrafficking machinery.References/publications/recommended reading1. Kerr M.C., Lindsay M.R. Luetterforst R., Hamilton N., Simpson F., Parton R.G., Gleeson P.A., TeasdaleR.D. (2006). Visualisation of macropinosome maturation by the recruitment of sorting nexins. J. Cell Sci.119:3967‐80.2. Lieu, Z.Z., Lock, J.G., Hammond, L.A., La Gruta, N. L, Stow, J. L. and Gleeson, P.A. (2008). A trans‐Golginetwork golgin is required for the regulated secretion of TNFa in activated macrophages in vivo. Proc. Natl.Acad. Sci USA, 105:3351‐63. Houghton F, Chew PL, Lohedo S, Goud B and Gleeson PA (2009) The localization of the golgin, GCC185,is independent of Rab6 and Arl1. Cell 138: 787‐7944. Gleeson PA and Goud, B (2010) TGN golgins, Rabs and cytoskeleton: Regulating the Golgi trafficking highways. Trends Cell Biol 20(6):329‐36.5. Phey Lim, J. and Gleeson, P.A. (2011). Macropinocytosis: An endocytic pathway for internalising large gulps. Immunol Cell Biol 89:836‐8436. Chia, PZC, Gasnereau, I, Lieu, ZZ and Gleeson, PA (2011). Rab‐9 dependent retrograde transport and endosomal sorting of the endopeptidase,furin. J. Cell Sci. 124:2401‐2413Autoimmunity and molecular immunology –Collaborators Professor Ian van Driel and Dr Dorothee Bourges,Department of Biochemistry and Molecular BiologyFailure of immunological self‐tolerance often leads to the development of autoimmune diseases, whichafflict up to 5% of the population. The underlying causes of autoimmune diseases are still unknown,although infections are strong candidates for initiating autoimmunity by promoting the maturation ofdendritic cells (DCs) that present tissue self‐antigens. Research in the van Driel/Gleeson laboratory focuseson defining how autoimmune disease develops, with a particular emphasis on the role of dendritic cells andregulatory T cells. The lab works on a murine model of autoimmune gastritis, a highly define system inwhich the target autoantigen is naturally expressed in the stomach.This project will analyse the relationship between inflammatory stimuli, the maturation status of DCs thatpresent gastric self‐epitopes and the development of organ‐specific autoimmunity.We have established an experimental autoimmune disease of the stomach(autoimmune gastritis) as a powerful model of organ‐specific autoimmunityand have identified the gastric autoantigens that are recognized by the selfreactiveeffector T cells. CD4 + T cells that mediate the disease recognize thehighly abundant gastric H/K ATPase heterodimer. Immune tolerance to thesegastric self‐antigens occurs primarily in the periphery. Most importantly, wehave also identified a subpopulation of migratory DCs in the draining lymphnode of the stomach that presents the endogenous gastric H/K ATPaseantigen. Hence, we are now able to compare the status of DCs whichpresent the gastric antigen, from normal mice and mice with autoimmunegastritis, as a basis for understanding the shift from tolerance toautoimmunity. This project will involve the isolation of the migratorypopulation of DCs by standards methods in the lab, analysis of TLR expression of these migratory DCs andassessment of the impact of different TLR ligands on the maturation and functional status of these DCs byanalysis of cytokine profiles and activation of gastric‐specific effector T cell responses.Figure 2 Gastric cells express anautoantigen which is presented in thelocal draining lymph nodes by migratorydendritic cellReferences/publications/recommended reading.1. Read S, Hogan, T, Zwar, T, Gleeson, PA and van Driel, IR (2007). Prevention of autoimmune gastritis in mice requires extra‐thymic deletionand regulation of CD4+CD25+ T cells. Gastroenterology 133: 547‐552. Kim JM, Rasmussen, JP and Rudensky, AY. (2007) regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice. NatImmunol 8, 191‐197.3. Allen, S, Turner, SJ, Bourges, D, Gleeson, PA and van Driel, IR (2011). Shaping the T‐cell repertoire in the periphery. Immunol Cell Biol. 89 : 60‐69 (IF 4.2)Page 15