Imaging phosphatidylinositol 4-phosphate dynamics in living plant cells

Plant J. 2009 Jan;57(2):356-72. doi: 10.1111/j.1365-313X.2008.03679.x. Epub 2008 Oct 25.

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 (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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / cytology
  • Arabidopsis / metabolism
  • Biosensing Techniques
  • Cell Membrane / metabolism
  • Cells, Cultured
  • Image Processing, Computer-Assisted / methods*
  • Luminescent Proteins / analysis
  • Medicago truncatula / cytology
  • Medicago truncatula / metabolism
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Phosphatidylinositol Phosphates / analysis*
  • Plant Roots / cytology*
  • Plant Roots / metabolism
  • Plants, Genetically Modified / cytology
  • Plants, Genetically Modified / metabolism
  • Protoplasts / cytology
  • Protoplasts / metabolism
  • Seedlings / cytology
  • Seedlings / metabolism
  • Tobacco / cytology
  • Tobacco / metabolism

Substances

  • Luminescent Proteins
  • Phosphatidylinositol Phosphates
  • phosphatidylinositol 4-phosphate