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Fluorescence Lifetime Imaging of Interactions between Golgi Tethering Factors and Small GTPases in Plants
Authors:Anne Osterrieder  Claudine M Carvalho  Maita Latijnhouwers  Jorunn Nergaard Johansen  Christopher Stubbs  Stanley Botchway  Chris Hawes
Institution:School of Life Sciences, Oxford Brookes University, Headington, Oxford, OX3 0BP, UK; Current address: Department of Plant Pathology, Federal University of Viçosa, 36570000 Viçosa-MG, Brazil; Current address: Department of Mathematics and Natural Science, University of Stavanger, N-4036 Stavanger, Norway; Plant Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK; Current address: Institute for Cancer Research, Department of Immunology, Rikshospitalet -Radiumhospitalet Medical Centre, Montebello, 0310 Oslo, Norway; Central Laser Facility, STFC Harwell Science Innovation Campus, Didcot, Oxon. OX11 0QX, UK
Abstract:Peripheral tethering factors bind to small GTPases in order to obtain their correct location within the Golgi apparatus. Using fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) we visualized interactions between Arabidopsis homologues of tethering factors and small GTPases at the Golgi stacks in planta . Co-expression of the coiled-coil proteins AtGRIP and golgin candidate 5 (GC5) TATA element modulatory factor (TMF)] and the putative post-Golgi tethering factor AtVPS52 fused to green fluorescent protein (GFP) with mRFP (monomeric red fluorescent protein) fusions to the small GTPases AtRab-H1b, AtRab-H1c and AtARL1 resulted in reduced GFP lifetimes compared to the control proteins. Interestingly, we observed differences in GFP quenching between the different protein combinations as well as selective quenching of GFP-AtVPS52-labelled structures. The data presented here indicate that the FRET-FLIM technique should prove invaluable in assessing protein interactions in living plant cells at the organelle level.
Keywords:Flim                        FRET                        GFP                        Golgi                        small GTPase                        tethering factor
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