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First published online December 11, 2006
doi: 10.1242/10.1242/jcs.000133

Journal of Cell Science 119, 5160-5168 (2006)
Published by The Company of Biologists 2006
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Localization of agonist-sensitive PtdIns(3,4,5)P3 reveals a nuclear pool that is insensitive to PTEN expression

Yvonne Lindsay1, David McCoull1, Lindsay Davidson1, Nick R. Leslie1, Alison Fairservice1, Alex Gray1, John Lucocq2 and C. Peter Downes1,*

1 Division of Molecular Physiology, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK
2 Division of Cell Biology and Immunology, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK


Figure 1
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  		Fig. 1. Analysis of GST-Grp1-PH binding by time-resolved FRET. (A) FRET saturation-binding isotherms. Protein (20 nM) was incubated with increasing concentrations of biotinylated PtdIns(3,4,5)P3. (B) IC50s of binding were measured by incubating FRET complexes containing 80 nM GST-Grp1-PH with increasing concentrations of the indicated unlabeled ligands. Data are presented as a mean of three independent experiments ± s.e.m. Values measured from A and B were used to calculate Ki values (see Materials and Methods) shown in C.

 

Figure 2
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  		Fig. 2. GST-Grp1-PH labeling on ultrathin thawed cryosections of Swiss 3T3 cells. Ultrathin cryosections of cells stimulated with 50 ng/ml PDGF were labeled with GST-Grp1-PH as described in the Materials and Methods. GST-Grp1-PH immunogold labeling, indicated by arrows, is visualised on the plasma membrane (PM) and endoplasmic reticulum (ER) of cells stimulated with PDGF for 5 minutes (A) and unstimulated cells (B). (C) GST-Grp1-PH labeling is located on a lamellipodium structure and plasma membrane (PM) of cells stimulated with PDGF for 2 minutes. Nuclear labeling in non-stimulated and stimulated (PDGF for 5 minutes) cells is shown in D and E, respectively. Arrows in F, G and H indicate gold labeling on internal vesicles of a MVB, Golgi stack and caveolae-like structures, respectively. Bars, 200 nm.

 

Figure 3
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  		Fig. 3. Quantification of GST-Grp1-PH labeling over cellular membranes after PDGF stimulation. Ultrathin cryosections of PDGF-stimulated Swiss 3T3 cells were labeled with GST-Grp1-PH and the labeling over cellular membranes was quantified as described previously (Watt et al., 2002Go). The proportions of GST-Grp1-PH labeling over individual compartments by on-section labeling are shown in A as percentages of the total gold particles counted, estimated by scanning cell profiles and assigning gold particles to compartments. The results are the means for three independent experiments, in which 1350-2100 gold particles were counted per experiment. The labeling densities over membrane compartments are shown in B, in which the number of gold particles were related to the number of intersections a scanning line makes with membranes from each compartment, as described by Watt et al. (Watt, et al., 2002Go). The results shown are one representative data set from three individual experiments, in which 839-1643 intersections and 1598-2643 gold particles were counted per experiment. Nuclear densities from a representative experiment, expressed as the number of gold particles related to area, are shown in the inset. Data are the means from an experiment in which 1342 gold particles were counted. Asterisks over the MVB and MLE columns indicate that the data were derived using intersections through the outer membrane and adjusted appropriately, as described (Watt et al., 2004Go). Error bars in A and B represent s.e.m. CYT, cytosol; ER, endoplasmic reticulum; GOL ST, Golgi stack; GOL VES, Golgi vesicles; LAM, lamellipodia; LYS, lysosome; MIT IN, mitochondria inner membrane; MLE, multilamellar body; MVB, multivesicular body; MIT OUT, mitochondria outer membrane; NUC, nucleus; NUC ENV, nuclear envelope; PM, plasma membrane; UN, unassigned (gold particles associated with indistinct structures).

 

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  		Fig. 4. GST-Grp1-PH [WT] and GST-Grp1-PH mutant [K273A] labeling in thawed cryosections of U87MG and Swiss 3T3 cells. U87MG cells (A) and Swiss 3T3 cells (B) were either treated with PDGF 50 ng/ml for 10 minutes or left unstimulated in the presence or absence of wortmannin, as indicated. The labeling densities for GST-Grp1-PH [WT] or the mutant PH domain [K273A] over the plasma membrane (PM), endoplasmic reticulum (ER) and nuclear area (nucleus) are shown. Labeling densities were calculated as golds per intersection (PM, ER) or golds per test point (nucleus), determined as described in the Materials and Methods. Data shown are expressed as fold-stimulation relative to unstimulated control cells. Results are the means of three experiments. Error bars represent s.e.m.

 

Figure 5
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  		Fig. 5. Effect of exogenous PTEN expression on GST-Grp1-PH labeling density after PDGF stimulation in U87MG cells. Ultrathin cryosections of both untransfected and GFP-PTEN-transfected U87MG cells, stimulated with 50 ng/ml PDGF for 10 minutes, were labeled with GST-Grp1-PH. (A) The labeling density over the plasma membrane and endoplasmic recticulum (ER) compartments in cells expressing PTEN [WT] is shown (densities obtained as described in Fig. 2 legend). (B) Nuclear GST-Grp1-PH labeling of cells transfected with PTEN [WT] is expressed as the number of gold particles related to area. Results are the means from a single experiment in which 616 intersections (PM and ER) or 1614 gold particles (nucleus) were counted. Error bars in A and B represent s.e.m. (C) The GST-Grp1-PH labeling for plasma membrane, ER and nuclear compartments in cells transfected with various PTEN constructs (WT; C124S phosphatase dead; or PTEN nls, containing an engineered nuclear localization signal). Data are normalized labeling densities from a representative data set of two similar experiments. (D) The subcellular localization of GFP-PTEN and GFP-NLS-PTEN in U87MG cells. Whereas GFP-PTEN is observed throughout the cytoplasm and nucleus, GFP-NLS-PTEN appears to be exclusively localized to the nucleus. (E) The effects of increasing expression of GFP-PTEN and GFP-NLS-PTEN on pS473-PKB phosphorylation in U87MG cells. The expression of each construct was titrated by adding increasing volumes of baculovirus supernatant to cells (0-1.0 ml in 10 ml of medium). After 24 hours, cells were lysed and the PTEN expression and pS473-PKB phosphorylation analysed by western blotting. The blots shown are representative of three independent experiments.

 

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