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- Title
Imaging phosphatidylinositol 4-phosphate dynamics in living plant cells.
- Authors
Vermeer, Joop E.M.; Thole, Julie M.; Goedhart, Joachim; Nielsen, Erik; Munnik, Teun; Gadella Jr., Theodorus W.J.
- Abstract
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 (PtdIns4 P) is the most abundant polyphosphoinositide. 32Pi-labelling studies have shown that the turnover of PtdIns4 P 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 PtdIns4 P dynamics in living plant cells. The biosensor consists of a fusion between a fluorescent protein and a lipid-binding domain that specifically binds PtdIns4 P, i.e. the pleckstrin homology domain of the human protein phosphatidylinositol-4-phosphate adaptor protein-1 (FAPP1). YFP–PHFAPP1 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–PHFAPP1 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 Ptdins3 P biosensor YFP–2 × FYVE revealed totally different localization patterns. During cell division, YFP–PHFAPP1 showed strong labelling of the cell plate, but PtdIns3 P was completely absent from the newly formed cell membrane. In root hairs of M. truncatula and A. thaliana, a clear PtdIns4 P 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 PtdIns4 P in tip growth.
- Subjects
LABELING-machines; CELL membranes; DETECTORS; HYDROGEN-ion concentration; MEDICAL equipment; CELLS; ROOT hairs (Botany); ARABIDOPSIS; CELL division; CELL proliferation
- Publication
Plant Journal, 2009, Vol 57, Issue 2, p356
- ISSN
0960-7412
- Publication type
Article
- DOI
10.1111/j.1365-313X.2008.03679.x