Visualization of PtdIns3P dynamics in living plant cells

Abstract

SummaryTo investigate PtdIns3P localization and function in plants, a fluorescent PtdIns3P-specific biosensor (YFP2xFYVE) 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 stressinduced 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, YFP2xFYVE-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.

ATG5 defines a phagophore domain connected to the endoplasmic reticulum during autophagosome formation in plants

“…1a-d). Both changes were abolished by the PtdIns(3)-kinase inhibitor wortmannin, which impedes autophagosome formation 39 ( Fig. 1e,f).…”

Vesicle Dynamics during Plant Cell Cytokinesis Reveals Distinct Developmental Phases

“…It is intriguing that the exocyst complexes move back and forth between the plasma membrane and the cytoplasm (Fendrych et al, 2010(Fendrych et al, , 2013. In addition, FM4-64 is observed at the cell plate as early as the IPA stage, and phosphatidylinositol-4-phosphate (originating from both the Golgi and the plasma membrane) is integrated into the growing cell plate (Vermeer et al, 2009), with the accumulation of YFP-2x-FYVE-positive endosomes at the periphery of the growing cell plate (Vermeer et al, 2006(Vermeer et al, , 2009.…”

Structural Basis for Interactions of thePhytophthora sojaeRxLR Effector Avh5 with Phosphatidylinositol 3-Phosphate and for Host Cell Entry

“…PtdIns(3)P represents 2-15% of the total monophosphate PIPs (38). Using a chimeric YFP fused with a tandem dimer of the PtdIns(3)P-binding FYVE domain from the growth factor-regulated tyrosine kinase substrate, it has been demonstrated that PtdIns(3)P localizes in both endosomal/pre-vacuolar vesicles and vacuolar membranes (119). Indeed, PtdIns(3)P has been shown to participate in vacuolar trafficking in Arabidopsis (22) and Nicotiana (120).…”