title: Visualization of PtdIns3P dynamics in living plant cells.
abstract: To investigate PtdIns3P localization and function in plants, a fluorescent PtdIns3P-specific biosensor (YFP-2xFYVE) was created. On lipid dot blots it bound specifically and with high affinity to PtdIns3P. Transient expression in cowpea protoplasts labelled vacuolar membranes and highly motile structures undergoing fusion and fission. Stable expression in tobacco BY-2 cells labelled similar motile structures, but labelled vacuolar membranes hardly at all. YFP-2xFYVE fluorescence strongly co-localized with the pre-vacuolar marker AtRABF2b, partially co-localized with the endosomal tracer FM4-64, but showed no overlap with the Golgi marker STtmd-CFP. Treatment of cells with wortmannin, a PI3 kinase inhibitor, caused the YFP-2xFYVE fluorescence to redistribute into the cytosol and nucleus within 15 min. BY-2 cells expressing YFP-2xFYVE contained twice as much PtdIns3P as YFP-transformed cells, but this had no effect on cell-growth or stress-induced phospholipid signalling responses. Upon treatment with wortmannin, PtdIns3P levels were reduced by approximately 40% within 15 min in both cell lines. Stable expression of YFP-2xFYVE in Arabidopsis plants labelled different subcellular structures in root compared with shoot tissues. In addition labelling the motile structures common to all cells, YFP-2xFYVE strongly labelled the vacuolar membrane in leaf epidermal and guard cells, suggesting that cell differentiation alters the distribution of PtdIns3P. In dividing BY-2 cells, YFP-2xFYVE-labelled vesicles surrounded the newly formed cell plate, suggesting a role for PtdIns3P in cytokinesis. Together, these data show that YFP-2xFYVE may be used as a biosensor to specifically visualize PtdIns3P in living plant cells.
authors.affiliation: Section of Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 316, Amsterdam, The Netherlands.
authors.forename: Joop E M
authors.initials: JE
authors.lastname: Vermeer
authors.forename: Wessel
authors.initials: W
authors.lastname: van Leeuwen
authors.forename: Rafa
authors.initials: R
authors.lastname: Tobeña-Santamaria
authors.forename: Ana M
authors.initials: AM
authors.lastname: Laxalt
authors.forename: David R
authors.initials: DR
authors.lastname: Jones
authors.forename: Nullin
authors.initials: N
authors.lastname: Divecha
authors.forename: Theodorus W J
authors.initials: TW
authors.lastname: Gadella
authors.forename: Teun
authors.initials: T
authors.lastname: Munnik
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D017360:Arabidopsis / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure; D048749:Cytokinesis / Q000502:physiology; D022162:Cytoplasmic Vesicles / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D003600:Cytosol / Q000378:metabolism / Q000648:ultrastructure; D056827:Endosomal Sorting Complexes Required for Transport; D007887:Fabaceae / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000032:analysis; D051379:Mice; D018613:Microscopy, Confocal; D018129:Phosphatidylinositol Phosphates / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics; D030821:Plants, Genetically Modified / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011523:Protoplasts / Q000378:metabolism / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry; D014026:Nicotiana / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D056827:Endosomal Sorting Complexes Required for Transport; D008164:Luminescent Proteins; D018129:Phosphatidylinositol Phosphates; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; C096164:hepatocyte growth factor-regulated tyrosine kinase substrate; C055525:phosphatidylinositol 3-phosphate; D000077191:Wortmannin