Imaging phosphatidylinositol 4-phosphate dynamics in living plant cells.
Polyphosphoinositides represent a minor group of phospholipids, accounting for less than 1% of the total. Despite their low abundance, these molecules have been implicated in various signalling and membrane trafficking events. Phosphatidylinositol 4-phosphate (PtdIns4P) is the most abundant polyphosphoinositide. (32)Pi-labelling studies have shown that the turnover of PtdIns4P is rapid, but little is known about where in the cell or plant this occurs. Here, we describe the use of a lipid biosensor that monitors PtdIns4P dynamics in living plant cells. The biosensor consists of a fusion between a fluorescent protein and a lipid-binding domain that specifically binds PtdIns4P, i.e. the pleckstrin homology domain of the human protein phosphatidylinositol-4-phosphate adaptor protein-1 (FAPP1). YFP-PH(FAPP1) was expressed in four plant systems: transiently in cowpea protoplasts, and stably in tobacco BY-2 cells, Medicago truncatula roots and Arabidopsis thaliana seedlings. All systems allowed YFP-PH(FAPP1) expression without detrimental effects. Two distinct fluorescence patterns were observed: labelling of motile punctate structures and the plasma membrane. Co-expression studies with organelle markers revealed strong co-labelling with the Golgi marker STtmd-CFP, but not with the endocytic/pre-vacuolar marker GFP-AtRABF2b. Co-expression with the Ptdins3P biosensor YFP-2 x FYVE revealed totally different localization patterns. During cell division, YFP-PH(FAPP1) showed strong labelling of the cell plate, but PtdIns3P was completely absent from the newly formed cell membrane. In root hairs of M. truncatula and A. thaliana, a clear PtdIns4P gradient was apparent in the plasma membrane, with the highest concentration in the tip. This only occurred in growing root hairs, indicating a role for PtdIns4P in tip growth.
Vermeer
Joop E M
JE
Department of Molecular Cytology, Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands.
Thole
Julie M
JM
Goedhart
Joachim
J
Nielsen
Erik
E
Munnik
Teun
T
Gadella
Theodorus W J
TW
D017360:Arabidopsis / Q000166:cytology / Q000378:metabolism; D015374:Biosensing Techniques; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008164:Luminescent Proteins / Q000032:analysis; D046913:Medicago truncatula / Q000166:cytology / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D018129:Phosphatidylinositol Phosphates / Q000032:analysis*; D018517:Plant Roots / Q000166:cytology* / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000166:cytology / Q000378:metabolism; D011523:Protoplasts / Q000166:cytology / Q000378:metabolism; D036226:Seedlings / Q000166:cytology / Q000378:metabolism; D014026:Nicotiana / Q000166:cytology / Q000378:metabolism
D008164:Luminescent Proteins; D018129:Phosphatidylinositol Phosphates; C037178:phosphatidylinositol 4-phosphate