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Visualization of PtdIns3 P dynamics in living plant cells
Author(s) -
Vermeer Joop E.M.,
van Leeuwen Wessel,
TobeñaSantamaria Rafa,
Laxalt Ana M.,
Jones David R.,
Divecha Nullin,
Gadella Theodorus W.J.,
Munnik Teun
Publication year - 2006
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/j.1365-313x.2006.02830.x
Subject(s) - yellow fluorescent protein , microbiology and biotechnology , wortmannin , biology , cytosol , guard cell , arabidopsis , cytoplasm , endosome , chemistry , intracellular , kinase , biochemistry , phosphatidylinositol , mutant , gene , enzyme
Summary To investigate PtdIns3 P localization and function in plants, a fluorescent PtdIns3 P ‐specific biosensor (YFP–2xFYVE) was created. On lipid dot blots it bound specifically and with high affinity to PtdIns3 P . 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 PtdIns3 P as YFP‐transformed cells, but this had no effect on cell‐growth or stress‐induced phospholipid signalling responses. Upon treatment with wortmannin, PtdIns3 P 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 PtdIns3 P . In dividing BY‐2 cells, YFP–2xFYVE‐labelled vesicles surrounded the newly formed cell plate, suggesting a role for PtdIns3 P in cytokinesis. Together, these data show that YFP–2xFYVE may be used as a biosensor to specifically visualize PtdIns3 P in living plant cells.