Characterization of the FRET biosensor for DAG and PI(4)P. (A) Schematic representation of the monitor for DAG (Digda). YFP and CFP denote a yellow-emitting mutant of GFP and cyan-emitting mutant of GFP, respectively. The C1 domain of PKCβII was used as the DAG-binding domain. In this biosensor design, the FRET level will increase when the biosensor recognizes DAG. (B) An NIH3T3 cell expressing Digda was stimulated with 50 μM of OAG, an analog of DAG, and the FRET/CFP image was processed as in Figure 1B. (C) The net intensities of CFP and FRET in each cell were measured and the averaged emission ratio (FRET/CFP) was calculated as in Figure 1C. Digda-expressing cells were stimulated with OAG at the time point 0 min as indicated by the arrow. The results were obtained from the wild-type (shown in red; n = 13) and a mutant (C67/132S; shown in blue; n = 3). Bar, SD. The asterisk indicates the significance of t test analysis: *p < 0.05 (vs. cells expressing the mutant biosensor stimulated for the same period). (D) An NIH3T3 cell expressing Digda were stimulated with PDGF (50 ng/ml). FRET images were processed as in Figure 1B. (E) The net intensities of CFP and FRET in each cell were measured and the averaged emission ratio (FRET/CFP) was calculated as in Figure 1C. Results were obtained from the wild type (shown in red; n = 17) and a mutant (C67/132S, shown in blue; n = 5). Digda-expressing cells were stimulated with PDGF. At the time point 0 min, PDGF was added to the cell as indicated by the arrow. Bar, SD. The asterisk indicates the significance of t test analysis: *p < 0.001 (vs. cells expressing the mutant biosensor stimulated for the same period). (F) Schematic representation of the FRET biosensor for PI(4)P. The PH domain of Pippi-PI(4,5)P₂ (shown in Figure 1A) was replaced by that of FAPP1. On binding to PI(4)P, CFP in the biosensor came into proximity to YFP, thereby causing FRET. The YFP (excitation 433 nm, emission 530 nm)/CFP (excitation 433 nm, emission 475 nm) ratio of Pippi-PI(4)P was used as a representation of the FRET efficiency. (G) MDCK cells were transfected with pPippi-PI(4)P, treated with the inhibitor of PAO at the concentration of 0.1 μM and processed as described in Figure 1B, except that phase images were obtained instead of DIC images. The numbers above are time points (in minutes). (H) MDCK cells were transfected with expression plasmids for Pippi-PI(4)P, mRFP-FKBP-5-ptase-dom, and ERed-NLS-LDR. Forty hours after transfection, cells were treated with 50 nM rapamycin; phase, RFP, CFP, and FRET images were obtained; and FRET/CFP images were processed as described in Figure 2G. The numbers above are time points after rapamycin treatment (in minutes). Bar, 20 μm. (I) MDCK cells were transfected with pPippi-PI(4,5)P₂- (top) and -PI(4)P (bottom)-HRas-CT, which carry the C-terminal region of H-Ras. Images were processed in a manner similar to that shown in Figure 2G. Bar, 20 μm.

Effect of inhibitors of PtdIns metabolism on the spatial distribution of PI(4,5)P₂ and PIP₃. (A–D) MDCK cells expressing the biosensors were treated with the inhibitors as indicated. Representative FRET/CFP ratio images are shown at the indicated time points (in minutes). The normalized FRET/CFP values from three cells are expressed in the graph (A, right). The bars indicate the SD (E and F). The boxed region in D was processed for kymographic analysis (E) and line scan analysis (F). The FRET/CFP value is represented in the pseudocolor mode with the range shown at the bottom of the figure (E) and in the line scan plots at different time points (F). Bars, 20 μm.

In silico analysis of the metabolism of PtdInss and DAG at the plasma membrane of migrating MDCK cells. (A) The metabolic pathway of PtdInss and DAG used in the model. DAG-K, DAG kinase. All reactions and parameters are listed in the supplemental information. (B) Schematic view of the one-dimensional model of PtdInss and DAG metabolism in a migrating cell. There are 256 elements along the axis of the standardized cell described in Figure 5. In each element the reactions were calculated while taking the lateral diffusion of lipids into consideration. (C and D) A predicted distribution of kinases, PLC, and lipid phosphatases. The concentrations of their molecules are normalized to the highest region. (E–H) Distribution of PtdInss and DAG in vivo (black plots) and in silico (red lines) in a migrating cell. (I and J) In silico simulation of the U73122-induced increase in PI(4,5)P₂ in a migrating cell. The time course of the PI(4,5)P₂ increase is shown in a kymograph (I) and a line scan plot (J).

Development of a FRET biosensor for PI(4,5)P₂. (A) Schematic representation of a monitor for PI(4,5)P₂ [Pippi-PI(4,5)P₂]. YFP and CFP denote a yellow-emitting mutant of GFP and cyan-emitting mutant of GFP, respectively. K-Ras4B-CT and PH indicate the C-terminal region of Ki-Ras4B and the pleckstrin homology domain of PLCδ, respectively. (B) NIH3T3 cells were transfected with pPippi-PI(4,5)P₂. Twenty-four hours later, cells were serum starved, stimulated with PDGF (50 ng/ml), and observed by fluorescent microscopy. CFP (excitation 433 nm/emission 475 nm) and FRET (excitation 433 nm/emission 530 nm) images were obtained every 1 min with a time-lapse epifluorescent microscope. After recording, the ratio FRET/CFP was calculated by MetaMorph software. At the same time, differential interference contrast (DIC) images were obtained. The images shown here are of a representative cell stimulated with PDGF for 0, 3, 10, 15, and 20 min. The calculated FRET efficiency was colored in intensity-modulated display (IMD) modes as shown at the right. The upper and lower limits of the ratio range are shown at the right of the panel. At least 20 similar images were obtained. Bar, 20 μm. (C) The net intensities of CFP and FRET in each cell were measured to calculate the averaged emission ratio (FRET/CFP). The FRET/CFP ratio was normalized by the average value before stimulation. The results obtained using the wild-type PH domain are shown in red (n = 6). The biosensor containing amino acid substitution in the PH domain (R37D/R38E) was used as a negative control (shown in blue, n = 3). Bars indicate the SD. The bar graph shows the averaged FRET/CFP values at 5 min after PDGF addition. The asterisk indicates the significance by t test analysis: *p < 0.05 (vs. cells expressing the mutant biosensor stimulated for the same period). (D) NIH3T3 cells were labeled with ³²P_i (0.4 mCi/ml) for 4 h and stimulated with PDGF (50 ng/ml) for the indicated periods. Lipids were extracted with CHCl₃:methanol:H₂O (1:1:1, vol/vol), separated by TLC (separating solvent: CHCl₃:methanol:ammonium:H₂O [45:35:6:4, vol/vol]), and quantified by BAS2000 (Fuji-Film). To mark the region for phosphatidylcholine and phosphatidylinositol (denoted as PC and PI at the left, respectively), the standard lipids were separated by the same TLC plate and chemically detected. (E) PIP₂, PC, and PI in D were quantitated and normalized to the basal level (time point 0 min; i.e., before stimulation). The results shown here are the averages from three independent experiments. Bar, SD. The asterisk indicates the significance of t test: *p < 0.01 (vs. that of before stimulation). (F) NIH3T3 cells were stimulated with PDGF and subjected to SDS-PAGE and Western blotting by using the antibodies against phospho-PLCγ1 (pY783, top, denoted as p-PLCγ1) and PLCγ1 (bottom). (G) The intensity of the phospho-PLCγ and total PLCγ1 was measured, calculated by phospho-PLC/PLC, and normalized by the value at the time point 5 min. Results are the average from three independent experiments. Bar, SD.

Distribution of PI(4,5)P₂, DAG, PIP₃, PI(3,4)P₂, and PI(4)P in migrating cells. MDCK cells were transfected with FRET probes for the lipids indicated at the left. Twenty-four hours after transfection, cells were observed by the fluorescent microscopy, and the FRET ratio images were processed in a manner similar to that described in Figure 2G. Results are shown for Pippi-PI(4,5)P₂, (available as a Supplemental Video) (A), Digda (B), Pippi-PI(3,4,5)P₃ (C), Pippi-PI(3,4)P₂ (D), and Pippi-PI(4)P (E).

Quantitative analysis of the distribution of PtdInss and DAG in migrating MDCK cells. MDCK cells were imaged as in Figure 3 (A, C, E, and G). Shown are representative phase contrast images (left) and FRET/CFP ratio images in pseudocolor mode (right). The rainbow bars show ratio ranges. White bars, 20 μm. Red and blue arrows indicate the front and back of migrating cells. The origin of the cell is set on the boundary between the cell body and the front of the cell (white circle). The ordinates of the front and back ends are set to 1.0 and -1.0 in the standardized cell size, respectively. FRET/CFP values are also normalized to that at the center and plotted along the arrows (B, D, F, and H). Each panel contains data for five different time points. The slope of the gradient in the front was calculated by the least-squares method.