Visualization of PtdIns3P dynamics in living plant cells

被引:171
作者
Vermeer, Joop E. M.
van Leeuwen, Wessel
Tobena-Santamaria, Rafa
Laxalt, Ana M.
Jones, David R.
Divecha, Nullin
Gadella, Theodorus W. J., Jr.
Munnik, Teun
机构
[1] Univ Amsterdam, Swammerdam Inst Life Sci, Sect Mol Cytol, Amsterdam, Netherlands
[2] Univ Amsterdam, Swammerdam Inst Life Sci, Sect Plant Physiol, Amsterdam, Netherlands
[3] Univ Amsterdam, Swammerdam Inst Life Sci, Ctr Adv Microscopy, Amsterdam, Netherlands
[4] Univ Nacl Mar del Plate, Fac Ciencias Exactas & Nat, Inst Invest Biol, Mar Del Plata, Argentina
[5] Netherlands Canc Inst, Div Cellular Biochem, NL-1066 CX Amsterdam, Netherlands
关键词
yellow fluorescent protein; FYVE domain; lipid signalling; confocal laser scanning microscopy; vesicle trafficking;
D O I
10.1111/j.1365-313X.2006.02830.x
中图分类号
Q94 [植物学];
学科分类号
071001 ;
摘要
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.
引用
收藏
页码:687 / 700
页数:14
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