Imaging phosphatidylinositol 4-phosphate dynamics in living plant cells |
| |
Authors: | Vermeer Joop E M Thole Julie M Goedhart Joachim Nielsen Erik Munnik Teun Gadella Theodorus W J |
| |
Affiliation: | Department of Molecular Cytology, Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands;, Department of Plant Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands;, Department of Biology, Washington University, One Brookings Drive, St Louis, MO 63130, USA;, and Department of Molecular, Cellular &Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA |
| |
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. |
| |
Keywords: | phosphoinositides GFP membrane trafficking microscopy lipid binding domain |
本文献已被 PubMed 等数据库收录! |
|