Bilateral regulation of EGFR activity and local PI(4,5)P2 dynamics in mammalian cells observed with superresolution microscopy
- Cellular Informatics Laboratory, RIKEN Cluster for Pioneering Research, Japan
- Molecular and Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Japan
- Laboratory of Single Molecule Biology, Graduate School of Frontier Biosciences, Osaka University, Japan
- Laboratoire de Bioimagerie et Pathologies, UMR 7021 CNRS, Université de Strasbourg, Faculté de Pharmacie, France
Figures
Figure 1 with 2 supplements
Epidermal growth factor receptor (EGFR), PI(4,5)P2, and phosphatidylserine (PS) are distributed nonrandomly in the plasma membrane.
(A) Images of EGFR–rsKame (cyan), PAmCherry–PI(4,5)P2 (magenta), and HMSiR–PS (yellow) before epidermal growth factor (EGF) stimulation. PAmCherry–PI(4,5)P2 and Halo–evt2–PH (Halo–PS) were transiently expressed in a cell line stably expressing EGFR–rsKame. Cells were incubated in serum-free medium in the presence of the HMSiR–Halo ligand overnight and treated with paraformaldehyde and glutaraldehyde. Left, a typical image of 15×15 µm. Right, enlarged image of the square region surrounded by a bold line in the left image. Enlarged images of other square regions surrounded by thin lines in the left image are shown in Figure 1—figure supplement 2C. (B) Distribution of the densities of EGFR (left) and PI(4,5)P2 cluster areas (right). Cells were incubated in serum-free medium overnight, stimulated with or without 20 nM EGF for 1 min, and treated with paraformaldehyde and glutaraldehyde. From the single-molecule localization microscopy (SMLM) images, the cluster areas and the number of clusters were measured. After the cluster number was normalized to the cell area (density), the cluster density was plotted as a function of the cluster area. Inset, enlarged graphs for the cluster areas of <0.03 µm2. Blue and red indicate before and after EGF stimulation, respectively. Data are means ± SEM of at least eight cells. (C) Univariate H(R) values of EGFR–rsKame (left), PAmCherry–PI(4,5)P2 (middle), and HMSiR–PS (right) in cells incubated in the absence (blue) or presence (red) of 20 nM EGF for 1 min. Ripley’s univariate H-function was calculated from the SMLM images. Data are means ± SEM of nine (EGFR–rsKame and PAmCherry–PI(4,5)P2) or 10 (HMSiR–PS) cells.
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Figure 1—source data 1
Original image files displayed in Figure 1A.
- https://cdn.elifesciences.org/articles/101652/elife-101652-fig1-data1-v1.zip
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Figure 1—source data 2
Raw data files displayed in Figure 1B and C.
- https://cdn.elifesciences.org/articles/101652/elife-101652-fig1-data2-v1.zip
Figure 1—figure supplement 1
Multicolor single-molecule localization microscopy (SMLM) analysis workflow.
(A) Typical images of 512×512 pixels (34.3×34.3 µm with a pixel size of 67 nm). Bright spots around the cells indicate 100 nm TetraSpeck Microspheres (beads), and those in the cells indicate fluorescence-labeled epidermal growth factor receptor (EGFR), PI(4,5)P2, or phosphatidylserine (PS) (samples). (B) Single-molecule tracking (SMT) analysis followed by alignment using affine transformation. An affine transformation matrix was calculated from the XY coordinates of the same beads in the nth frame (or color channel) and a target frame (or channel) using least-squares regression. Example trajectories of the same beads before (white) and after (yellow) drift correction during 1000 frames are shown in the left panel. A histogram of the X coordinates of the beads after affine transformation is shown under the trajectory. The difference in the XY coordinates of the same beads between two color channels, before (white) and after (yellow) affine transformation, is shown in the right panel. (C) Reconstituted images. After the alignment of localization with affine transformation, images were reconstituted by convolution with a Gaussian kernel around each localization of the sample. The 5×5 µm area is merged and expanded in the right panel. (D) Definition and calculation flow of Ripley’s K-function variants. Npi(r) is the number of points within distance r around the ith point. In the calculation of the univariate K-function, the number of points belonging to the same channel was counted. In the bivariate K-function calculation, the number of points belonging to the other channel was counted. Kpi(r) is a local K-function around the ith point, where λ is the mean density in the region of interest (ROI). K(r) is Ripley’s K-function, which is the ensemble average of all Kpi(r) for n points in the ROI. K(r) is πr2 if the spatial distribution of the n points in the ROI is random. Because intuitive comparison with πr2 is difficult, variants L(r) and H(r) are often used; L(r) and H(r) become r and 0 if random, respectively. G(r) is the difference function of K(r), where the number of points in the donut area between circles of radii rm+1 and rm is centered at each point. G0(r) is normalized G(r), where μ and σ are the mean and SD of G(r) in a random distribution. Unlike H(r), G(r) does not involve nonlinear computations, and therefore is suitable for the following spatial mapping analysis. (E) Automated multidistance spatial cluster analysis. After alignment with affine transformation, the image area was divided into ROIs with two rectangles (top panels). K(r) and its variants for the sample and for random simulation data were computed for the center points within the red region. In the calculation, the target points within both the red and blue areas were considered, to reduce the edge effect. After the calculations for all ROIs, ROIs containing cellular regions were detected with tiling images of the mean density of each ROI (lower left panel). The Otsu method was used to binarize the cellular and noncellular regions. The lower right panel shows an example of the mean H(r) for the cellular region. Data are shown as means ± SEM of N ROIs within the cellular regions. (F) G-function spatial map (G-SMAP) analysis. In the computation process in (E), the local K-function and its variants, including G0 pi(r), were calculated for all the sample and random simulation data points. G-SMAP images were generated by integrating the G0 pi(r) kernel around each point. Example G-SMAP images for sample and random simulation data in the same ROI are shown in pseudo rainbow colors (upper left panel). A pixel-intensity histogram of the G-SMAP images is shown in the upper right panel. Pixels above a threshold value (e.g. four SD of the random distribution) were defined as cluster regions, and closed cluster regions were numbered as individual clusters (pseudo rainbow colors in bottom panels).
Figure 1—figure supplement 2
Construction and evaluation of multicolor single-molecule localization microscopy (SMLM).
(A) Amounts of phosphorylated-Tyr1068 epidermal growth factor receptor (EGFR) in the cell line. Top: Western blotting analysis of phosphorylated-Tyr1068 EGFR and total EGFR. Parental HeLa cells and EGFR-KO cells stably expressing EGFR–rsKame fusion protein were incubated in a serum-free medium overnight and stimulated with 20 nM epidermal growth factor (EGF) for 1 min. Phospho-EGFR and total EGFR were detected with anti-pY1068 EGFR and EGFR, respectively. Bottom: Ratio of phosphorylated-Tyr1068 EGFR/total EGFR. The ratio was normalized to the mean value of parental HeLa cells after EGF stimulation. Data are means ± SD of three experiments. (B) Amounts of phosphorylated extracellular signal-regulated kinase (ERK) in the cell line. Top: Western blotting analysis of phosphorylated ERK and total ERK. Cells were prepared as described in (A). Phospho-ERK and total ERK were detected with anti-pERK and ERK, respectively. Bottom: Ratio of phosphorylated-ERK/total ERK. The ratio was normalized to the mean value of parental HeLa cells after EGF stimulation. Data are means ± SD of three experiments. (C) Enlarged images of square regions surrounded by thin lines in Figure 1A. (D) Full-width half-maximum (FWHM) of the positional distributions for single immobile fluorescent beads. Data are means ± SEM of 10 beads. (E) Position accuracy of the dual-color immobile fluorescent beads. The accuracy of the superimposition of two images was calculated after affine transformation. Data are means ± SEM of 10 beads. NS (not significant) on Welch’s t-test.
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Figure 1—figure supplement 2—source data 1
PDF file for western blotting analysis displayed in Figure 1—figure supplement 2A and B.
- https://cdn.elifesciences.org/articles/101652/elife-101652-fig1-figsupp2-data1-v1.zip
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Figure 1—figure supplement 2—source data 2
Original files for western blotting analysis displayed in Figure 1—figure supplement 2A and B.
- https://cdn.elifesciences.org/articles/101652/elife-101652-fig1-figsupp2-data2-v1.zip
Figure 2
Bivariate H(r) calculated from SMLM images reveals that lateral coaggregation of epidermal growth factor receptor (EGFR) and PI(4,5)P2 decreases after epidermal growth factor (EGF) stimulation.
(A) Bivariate H(r) value of EGFR–rsKame and HMSiR–PS, PAmCherry–PI(4,5)P2 and HMSiR–PS, and EGFR–rsKame and PAmCherry–PI(4,5)P2 before (blue) and after incubation with 20 nM EGF for 1 min (red). Cells were prepared as in Figure 1. Ripley’s bivariate H-function was calculated from the single-molecule localization microscopy (SMLM) images. Data are means ± SEM of eight cells. (B) Bivariate H(r) value of EGFR–rsKame and HMSiR–PI(4,5)P2 before (blue) and after incubation with 20 nM EGF for 0.5 min (green) or 2 min (red). Halo–PI(4,5)P2 was transiently expressed in a cell line stably expressing EGFR–rsKame. Cells were incubated in serum-free medium in the presence of the HMSiR–Halo ligand overnight, stimulated with or without 20 nM EGF for the indicated times, and treated with paraformaldehyde and glutaraldehyde. Data are means ± SEM of 17 (without EGF stimulation), 10 (with EGF stimulation for 0.5 min), or 20 cells (with EGF stimulation for 2 min). (C) Bivariate H(r) value of EGFR–rsKame and HMSiR–TubbyC before (blue) and after incubation with 20 nM EGF for 2 min (red). Halo–TubbyC was transiently expressed in a cell line stably expressing EGFR–rsKame. Data are means ± SEM of 8 (without EGF stimulation) or 13 cells (with EGF stimulation for 2 min). (D) Images of EGFR–rsKame (green) and HMSiR–GRB2 (magenta) after EGF stimulation for 2 min (right). Halo–GRB2 was transiently expressed in a cell line stably expressing EGFR–rsKame. Right, enlarged image of the square region surrounded by a bold line in the left image. Arrows indicate EGFR and GRB2 overlaps in the images. (E) Bivariate H(r) value of EGFR–rsKame and HMSiR–GRB2 before (blue) and after incubation with 20 nM EGF for 2 min (red). Halo–GRB2 was transiently expressed in a cell line stably expressing EGFR–rsKame. Data are means ± SEM of 10 cells. (F) Images of EGFR–rsKame (green) and PAmCherry–PI(3,4,5)P3 (magenta) before (left) and after EGF stimulation for 2 min (right). PAmCherry1–GRP1-PH (PAmCherry–PI(3,4,5)P3) was transiently expressed in a cell line stably expressing EGFR–rsKame. (G) Bivariate H(r) value of EGFR–rsKame, and PAmCherry–PI(3,4,5)P3 before (blue) and after incubation with 20 nM EGF for 2 min (red). Data are means ± SEM of 10 (before EGF stimulation) or nine cells (after 20 nM EGF stimulation).
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Figure 2—source data 1
Raw data files displayed in Figure 2A, B, C, E and G.
- https://cdn.elifesciences.org/articles/101652/elife-101652-fig2-data1-v1.zip
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Figure 2—source data 2
Original image files displayed in Figure 2D and F.
- https://cdn.elifesciences.org/articles/101652/elife-101652-fig2-data2-v1.zip
Figure 3 with 2 supplements
Single-molecule tracking (SMT) analysis reveals that partial and transient overlap between epidermal growth factor receptor (EGFR) and PI(4,5)P2 decreases after epidermal growth factor (EGF) stimulation.
(A) Trajectories of EGFR–GFP (left) and JF549–PI(4,5)P2 (right) over 6 s. EGFR–GFP and Halo–PLCδ–PH (Halo–PI(4,5)P2) were transiently expressed in EGFR-KO HeLa cells. After the cells were incubated in a serum-free medium overnight, Halo–PI(4,5)P2 was stained with JF549–Halo ligand. EGFR–GFP and JF549–PI(4,5)P2 were observed in 30 cells at a time resolution of 30 ms for 6 s before EGF stimulation. After stimulation with 20 nM EGF, EGFR–GFP and JF549–PI(4,5)P2 were observed in the same cells between 1 and 5 min after the addition of EGF. SMT analysis was fractionated into the immobile (blue), slow-mobile (yellow), and fast-mobile (green) fractions. (B) Proportions of immobile (blue), slow-mobile (yellow), and fast-mobile (green) fractions. (C) Mean square displacement per unit time (MSD-Δt) plots of the trajectories of EGFR–EGFP and JF549–PI(4,5)P2. (D) Trajectories of EGFR–GFP (green), JF549–PI(4,5)P2 (magenta), and colocalization (white) during 6 s before (left) and after incubation with 20 nM EGF (right) in the same cell. (E) Relative colocalization rates of EGFR–GFP and JF549–PI(4,5)P2 before and after incubation with 20 nM EGF. To consider the differences in expression levels among cells, the colocalization rate was divided by the densities of EGFR–GFP and JF549–PI(4,5)P2 and normalized to the mean value obtained before EGF stimulation. Violin plots show the mean value and distribution of 30 cells. ***p<0.001 on Welch’s t-test.
Figure 3—figure supplement 1
Single-molecule tracking (SMT) analysis of epidermal growth factor receptor (EGFR)–EGFP and JF549–PI(4,5)P2.
(A) Amounts of phosphorylated-Tyr1068 EGFR in the cell for SMT analysis. Left: Western blotting analysis of phosphorylated-Tyr1068 EGFR and total EGFR. Parental HeLa cells and EGFR-knockout (KO) cells transiently expressing EGFR–GFP at low levels were incubated in a serum-free medium overnight and stimulated with 20 nM epidermal growth factor (EGF) for 1 min. Phospho-EGFR and total EGFR were detected with anti-pY1068 EGFR and EGFR, respectively. Right: Ratio of phosphorylated-Tyr1068 EGFR/total EGFR. The ratio was normalized to the mean value of parental HeLa cells after EGF stimulation. Data are means ± SD of three experiments. (B) Diffusion coefficients of EGFR–EGFP and JF549–PI(4,5)P2. Cells were prepared as described in Figure 3A. (C) Confinement lengths of EGFR–EGFP and JF549–PI(4,5)P2. (D) Relative colocalization rate of EGFR–EGFP and JF549–PI(4,5)P2 in the immobile (blue), slow-mobile (yellow), and fast-mobile (green) fractions before and after incubation with 20 nM EGF. To consider the differences in expression levels among cells, the colocalization rate of each fraction was divided by the densities of EGFR–GFP and JF549–PI(4,5)P2 of the total fractions and normalized to the mean value obtained before EGF stimulation. Violin plots show the mean value and distribution of 30 cells. (E) Trajectories of EGFR–GFP (green), JF549–TubbyC (magenta), and colocalization (white) for 5 s before (left) and after incubation with 20 nM EGF (right) in the same cell. EGFR–GFP and Halo–TubbyC were transiently expressed in EGFR-KO HeLa cells. After overnight incubation in a serum-free medium, Halo–TubbyC was stained with JF549–Halo ligand. EGFR–GFP and JF549–TubbyC were observed at a time resolution of 30ms for 5 s before EGF stimulation. After stimulation with 20 nM EGF, EGFR–GFP, and JF549–TubbyC were observed in the same cells between 1 and 5 min after the addition of EGF. (F) Relative colocalization rate of EGFR and biosensors of PI(4,5)P2 before and after incubation with 20 nM EGF. To consider the differences in expression levels among cells, the colocalization rate was divided by the densities of EGFR–GFP and biosensors of PI(4,5)P2, and normalized to the mean value of EGFR–GFP and JF549–PLCδ–PH (JF549–PI(4,5)P2) obtained before EGF stimulation. Violin plots show the mean value and distribution of 36 cells. ***p<0.001, NS (not significant) on Welch’s t-test.
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Figure 3—figure supplement 1—source data 1
PDF file for western blotting analysis displayed in Figure 3—figure supplement 1A.
- https://cdn.elifesciences.org/articles/101652/elife-101652-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
Original files for western blotting analysis displayed in Figure 3—figure supplement 1A.
- https://cdn.elifesciences.org/articles/101652/elife-101652-fig3-figsupp1-data2-v1.zip
Figure 3—figure supplement 2
Time-dependent colocalization of epidermal growth factor receptor (EGFR)-PI(4,5)P2 and EGFR-growth factor receptor-bound protein 2 (GRB2).
(A) Relative colocalization rate of EGFR–green fluorescent protein (GFP) and JF549–PI(4,5)P2 before and after incubation with 20 nM EGF. EGFR–GFP and Halo–PI(4,5)P2 were transiently expressed in EGFR-knockout (KO) HeLa cells. After the cells were incubated in a serum-free medium overnight, Halo–PI(4,5)P2 was stained with JF549–Halo ligand. EGFR–GFP and JF549–PI(4,5)P2 were observed in different cells from 0 to 5 min after epidermal growth factor (EGF) stimulation. To consider the differences in expression levels among cells, the colocalization rate was divided by the densities of EGFR–GFP and JF549–PI(4,5)P2 and normalized to the mean value obtained before EGF stimulation. Violin plots show the mean value and distribution of the 18–20 cells. ***p<0.001, NS (not significant) on Tukey’s multiple comparison test. (B) Relative colocalization rate of EGFR–GFP and JF549–GRB2 before and after incubation with 20 nM EGF. EGFR–GFP and Halo–GRB2 were transiently expressed in EGFR-KO HeLa cells. Similar to EGFR–GFP and Halo–PI(4,5)P2 in (A), EGFR–GFP and JF549–GRB2 were observed in different cells from 0 to 5 min after EGF stimulation. To consider the differences in expression levels among cells, the colocalization rate was divided by the densities of EGFR–GFP and JF549–GRB2 and normalized to the mean value obtained before EGF stimulation. Violin plots show the mean value and distribution of the 17–20 cells. **p<0.01, NS (not significant) on Welch’s t-test.
Figure 4 with 2 supplements
Single-molecule tracking (SMT) analysis shows that PI(4,5)P2 is important for stabilizing epidermal growth factor receptor (EGFR) dimers.
(A) SMT analysis of EGFR–SF650 in control and synaptojanin (SYNJ)-expressing cells. Only EGFR–Halo or both EGFR-Halo and green fluorescent protein (GFP)–SYNJ were transiently expressed in Chinese Hamster Ovary (CHO)-K1 cells. After cells were incubated in a serum-free medium overnight, EGFR–Halo was stained with the SF650–Halo ligand. EGFR–SF650 was observed in control (n=40) and SYNJ-expressing cells (n=40) at a time resolution of 30 ms before epidermal growth factor (EGF) stimulation. Following stimulation with 100 nM EGF, EGFR–SF650 was observed in the same cells between 1 and 6 min after the addition of EGF. (B) SMT analysis of EGFR(WT)–SF650 and EGFR(3RN)–SF650. EGFR–Halo or EGFR(3RN)–Halo was transiently expressed in CHO-K1 cells. After the cells were incubated in a serum-free medium overnight, EGFR–Halo was stained with the SF650–Halo ligand. EGFR(WT)–SF650 (n=40) and EGFR(3RN)–SF650 (n=45) were observed as described in (A). Data are means ± SEM.
Figure 4—figure supplement 1
Single-molecule tracking (SMT) analysis of epidermal growth factor receptor (EGFR) under PI(4,5)P2-depleted condition.
(A) SMT analysis of EGFR–SF650 in control and synaptojanin (SYNJ)-expressing HeLa cells. EGFR-Halo was transiently expressed in EGFR-KO HeLa cells. After cells were incubated in a serum-free medium overnight, EGFR–Halo was stained with the SF650–Halo ligand. EGFR–SF650 was observed in control (n=40) and SYNJ-expressing cells (n=40) at a time resolution of 30 ms before epidermal growth factor (EGF) stimulation. Following stimulation with 100 nM EGF, EGFR–SF650 was observed in the same cells between 1 and 6 min after the addition of EGF. (B) Distribution of the total fluorescence intensity of EGFR–SF650 in control (magenta) and SYNJ-expressing cells (green) after stimulation with 100 nM EGF in immobile (top), slow-mobile (middle), and fast-mobile fractions (bottom). EGFR–SF650 in control (n=40) or SYNJ-expressing cells (n=40) was observed as described in Figure 4A. Data are means ± SEM. (C) Distribution of the fluorescence intensity of EGFR–SF650 in control (magenta) and SYNJ-expressing cells (green) after stimulation with 100 nM EGF in the immobile fraction. The putative oligomer size was estimated from the total fluorescence intensity (bold line) with Gaussian functions (thin lines). The estimates are shown in Figure 4A. (D) The mean density of EGFR–SF650 in control (n=40) and SYNJ-expressing cells (n=40) before EGF stimulation. (E) The mean intensity of EGFR–SF650 in control (n=40) and SYNJ-expressing cells (n=40) before EGF stimulation. The intensities of EGFR–SF650 were measured at the first frame of the observation and normalized with the area. Violin plots show the mean value and distribution of 40 cells. NS (not significant) on Welch’s t-test.
Figure 4—figure supplement 2
Single-molecule tracking (SMT) analysis of epidermal growth factor receptor (EGFR) under PI(4,5)P2-interaction-depleted condition.
(A) Distribution of the total fluorescence intensity of EGFR(WT)–SF650 (magenta) and EGFR(3RN)–SF650 (green) after stimulation with 100 nM epidermal growth factor (EGF) in immobile (top), slow-mobile (middle), and fast-mobile fractions (bottom). EGFR(WT)–SF650 (n=40) and EGFR(3RN)–SF650 (n=45) were observed as described in Figure 4B. Data are means ± SEM. (B) Distribution of the fluorescence intensities of EGFR(WT)–SF650 (magenta) and EGFR(3RN)–SF650 (green) after stimulation with 100 nM EGF in the immobile fraction. The putative oligomer size was estimated from the total fluorescence intensity (bold line) with Gaussian functions (thin lines). The estimates are shown in Figure 4B. (C) Images of EGFR (top) and Cy5-labeled EGF (bottom). EGFR(WT), EGFR(3RN), or EGFRvIII lacking amino acid residues involved in the ligand binding in the extracellular region was expressed in EGFR-KO cells and labeled with Cy5-EGF for 10 min. (D) The relative intensity of Cy5–EGF bound to EGFR. To consider the difference in the expression level of EGFR, the fluorescent intensities of Cy5-EGF and EGFR–JF549 were measured, and the intensity of Cy5-EGF was normalized with that of EGFR–JF549. The ratio was normalized to the mean value of control cells. Violin plots show the mean value and distribution of 26 images. ***p<0.001, NS (not significant) on Tukey’s multiple comparison test.
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Figure 4—figure supplement 2—source data 1
Original image files are displayed in Figure 4—figure supplement 2C.
- https://cdn.elifesciences.org/articles/101652/elife-101652-fig4-figsupp2-data1-v1.zip
Figure 5
PI(4,5)P2 is important for not only the dimerization of epidermal growth factor receptor (EGFR) but also its subsequent phosphorylation.
(A) Western blotting analysis of crosslinked EGFR. After EGFR-knockout (KO) cells were transfected with only EGFR–Halo or both EGFR–Halo and green fluorescent protein (GFP)–synaptojanin (SYNJ), the cells were incubated in a serum-free medium overnight. The cells were then stimulated with 0, 0.2, or 20 nM epidermal growth factor (EGF), and treated with a crosslinker for 1 hr. EGFR–Halo was detected with an anti-EGFR antibody. (B) Amounts of EGFR–Halo dimer. Relative intensity was normalized to the mean intensity of lane #3 in (A). Data are means ± SD of four experiments. (C) Western blotting analysis of crosslinked EGFR(3RN). After EGFR-KO cells were transfected with only EGFR(3RN)–Halo or both EGFR(3RN)–Halo and GFP–SYNJ, the cells were incubated in a serum-free medium overnight. The cells were then stimulated with 0.2 or 20 nM EGF, and treated with a crosslinker for 1 hr. EGFR(3RN)–Halo was detected with an anti-EGFR antibody. (D) Amounts of EGFR(3RN)–Halo dimer. Relative intensity was normalized to the mean intensity of lane #1 in (C). Data are means ± SD of three experiments. (E) Western blotting analysis of phosphorylated EGFR and total EGFR. After EGFR-KO cells were transfected with EGFR(WT)–Halo, EGFR(WT)–Halo, and GFP–SYNJ, or EGFR(3RN)–Halo, the cells were incubated in serum-free medium overnight and stimulated with 0.2 nM or 20 nM EGF for 2 min. Phospho-EGFR and total EGFR were detected with anti-pY1068 EGFR and EGFR, respectively. (F) Ratio of phosphorylated-Tyr1068 EGFR/total EGFR. The ratio was normalized to the mean value of lane #4 in (E). Data are means ± SD of three experiments. *p<0.05, **p<0.01, NS (not significant) on Welch’s t-test.
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Figure 5—source data 1
PDF file for western blotting analysis are displayed in Figure 5A, C and E.
- https://cdn.elifesciences.org/articles/101652/elife-101652-fig5-data1-v1.zip
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Figure 5—source data 2
Original files for western blotting analysis are displayed in Figure 5A, C and E.
- https://cdn.elifesciences.org/articles/101652/elife-101652-fig5-data2-v1.zip
Figure 6 with 1 supplement
Phospholipase Cγ (PLCγ) is responsible for epidermal growth factor receptor (EGFR) dissociation from PI(4,5)P2 nanodomains after EGFR is phosphorylated.
(A) Effect of wortmannin on phosphorylation of AKT. A cell line stably expressing EGFR–rsKame was incubated in a serum-free medium overnight and treated with 10 µM wortmannin for 1 hr. The cells were then stimulated with or without 20 nM EGF for 1 min. Phosphorylated AKT was detected with an anti-phospho-AKT (Ser473) antibody in a western blotting analysis. Relative intensity was normalized to the mean value of the control cells without EGF stimulation. Data are means ± SD of three experiments. (B) Effect of DN PLCγ on diacylglycerol (DAG) production. DN PLCγ was transiently expressed in a cell line stably expressing EGFR–rsKame. Cells were incubated in a serum-free medium overnight and treated with or without 20 nM EGF for 1 min. Amounts of DAG were measured with a DAG assay kit. Relative intensity was normalized to the mean intensity of the control cells without EGF stimulation. Data are means ± SD of three experiments. (C) Bivariate H(r) value of EGFR–rsKame and PAmCherry–PI(4,5)P2 in control (red), wortmannin-treated (green), and DN-PLCγ-expressing cells (black) after incubation with 20 nM EGF for 1 min. In control and wortmannin-treated cells, PAmCherry1–PLCδ–PH was transiently expressed in a cell line stably expressing EGFR–rsKame. To co-express PAmCherry1–PLCδ–PH and DN PLCγ in the same cells, plasmid DEST40/PAmCherry1–PLCδ–PH–IRES–DN PLCγ was transfected to the cell line stably expressing EGFR–rsKame. Cells were incubated in a serum-free medium overnight and treated with (control and DN PLCγ) or without 10 µM wortmannin for 1 h (wortmannin). The cells were then stimulated with 20 nM EGF for 1 min and treated with paraformaldehyde and glutaraldehyde. Data are means ± SEM of seven (control and wortmannin) or eight cells (DN PLCγ). (D) Bivariate H(r) value of PAmCherry–PI(4,5)P2 and HMSiR–pY1068 in control (red), wortmannin-treated (green), and DN-PLCγ-expressing cells (black) after incubation with 20 nM EGF for 1 min. After cells were prepared as described in (E), phosphorylated EGFR was immunostained with anti-EGFR (pY1068) and HMSiR-labeled antibodies. Data are means ± SEM of six (control), seven (wortmannin), or eight cells (DN PLCγ). (E) Images of PAmCherry–PI(4,5)P2 (magenta) and HMSiR-labeled pY1068 EGFR (green) after stimulation with EGF for 1 min. Cells were prepared as described in (F). **p<0.01, ***p<0.001 on Welch’s t-test.
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Figure 6—source data 1
Raw data files are displayed in Figure 6C and D.
- https://cdn.elifesciences.org/articles/101652/elife-101652-fig6-data1-v1.zip
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Figure 6—source data 2
Original image files are displayed in Figure 6E.
- https://cdn.elifesciences.org/articles/101652/elife-101652-fig6-data2-v1.zip
Figure 6—figure supplement 1
Phosphoinositide 3-kinase (PI3K) is not responsible for epidermal growth factor receptor (EGFR) dissociation from PI(4,5)P2 nanodomains after EGFR is phosphorylated.
(A) Univariate H(r) value of PAmCherry–PI(4,5)P2 in control (red), wortmannin-treated (green), and DN phospholipase Cγ (PLCγ)-expressing cells (black) after incubation with 20 nM EGF for 1 min. Cells were prepared as described in Figure 6D. Data are means ± SEM of nine cells. (B) Univariate H(r) value of HMSiR–pY1068 in control (red), wortmannin-treated (green), and DN-PLCγ-expressing cells (black) after incubation with 20 nM epidermal growth factor (EGF) for 1 min. Cells were prepared as described in Figure 6D. Data are means ± SEM of nine (control) or 10 (wortmannin-treated and DN PLCγ-expressing) cells. (C) Western blotting analysis of phosphorylated AKT (top) and phosphorylated EGFR (bottom). After EGFR-KO and ERBB3-KO cells were transfected with EGFR–Halo, the cells were incubated in serum-free medium overnight and stimulated with 20 nM EGF for 2 min. Phosphorylated AKT and phosphorylated EGFR were detected with anti-phospho-AKT (Ser473) and anti-pY1173 EGFR antibodies, respectively. (D) Amount of phosphorylated AKT. Relative intensities were normalized to the mean of lane #3 in (C). Data are means ± SD of four experiments. (E) Trajectories of EGFR–GFP (cyan), PLCγ–TMR (magenta), SF650–PI3K (yellow), and colocalization (white) for 6 s after incubation with 20 nM EGF (right). EGFR–GFP, PLCγ (PLCG1)–SNAP, and Halo–PI3K (p85α) were transiently expressed in EGFR-KO HeLa cells. After overnight incubation in a serum-free medium, the cells were stained with TMR–SNAP and SF650–Halo ligands. EGFR–GFP, PLCγ–TMR, SF650–PI3K were observed at a time resolution of 30 ms for 5 s, between 1 and 5 min after the addition of EGF. (F) Relative colocalization of EGFR, PLCγ, and PI3K after incubation with 20 nM EGF. To consider the differences in expression levels among cells, the colocalization rate was divided by the density and normalized to the mean value of EGFR–GFP and PLCγ–TMR. Violin plots show the mean value and distribution of 35 cells. ***p<0.001 based on Tukey’s multiple comparison test.
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Figure 6—figure supplement 1—source data 1
Raw data files are displayed in Figure 6—figure supplement 1A and B.
- https://cdn.elifesciences.org/articles/101652/elife-101652-fig6-figsupp1-data1-v1.zip
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Figure 6—figure supplement 1—source data 2
PDF file for western blotting analysis are displayed in Figure 6—figure supplement 1C.
- https://cdn.elifesciences.org/articles/101652/elife-101652-fig6-figsupp1-data2-v1.zip
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Figure 6—figure supplement 1—source data 3
Original files for western blotting analysis displayed in Figure 6—figure supplement 1C.
- https://cdn.elifesciences.org/articles/101652/elife-101652-fig6-figsupp1-data3-v1.zip
Figure 7
Thr654 of epidermal growth factor receptor (EGFR) is phosphorylated in a phospholipase Cγ (PLCγ)-dependent manner.
(A) Effect of PLCγ on phosphorylation of EGFR. After EGFR-knockout (KO) cells were transfected with EGFR–Halo, EGFR–Halo and DN PLCγ, or EGFR–Halo and PLCG1 siRNA, the cells were incubated in serum-free medium overnight and stimulated with 10 nM EGF for 1 min or 5 min. DN PLCγ was transiently expressed in EGFR-KO cells expressing EGFR-Halo. Phosphorylated-Tyr1068 EGFR, phosphorylated-Thr654 EGFR, and total EGFR were detected with anti-EGFR pY1068, anti-EGFR pT654, and anti-EGFR antibodies, respectively. (B) Ratios of phosphorylated-Tyr1068 EGFR/total EGFR and phosphorylated-Thr654 EGFR/total EGFR. The ratios were normalized to the mean value of lane #7 in (A). Data are means ± SD of three experiments. (C) Effects of AG1478 and 4β-phorbol 12-myristate 13-acetate (PMA) on phosphorylation of EGFR. After EGFR-KO cells were transfected with EGFR–Halo or EGFR–Halo and PLCG1 siRNA, the cells were incubated in a serum-free medium overnight and stimulated with 10 nM EGF for 5 min in the presence or absence of 100 nM PMA. For inhibition of autophosphorylation of EGFR, cells were treated with 1 µM AG1478 for 30 min and stimulated with 10 nM EGF for 5 min. Phosphorylated-Tyr1068 EGFR, phosphorylated-Thr654 EGFR, and total EGFR were detected with anti-EGFR pY1068, anti-EGFR pT654, and anti-EGFR antibodies, respectively. (D) Ratios of phosphorylated-Tyr1068 EGFR/total EGFR and phosphorylated-Thr654 EGFR/total EGFR. The ratios were normalized to the mean value of lane #1 in (C). Data are means ± SD of three experiments. (E) Schematic model of the activation and signal transduction process for EGFR. Left, after EGF stimulation, PI(4,5)P2 (magenta) interacts with the arginine residues in the JM-A region (red) of EGFR, stabilizing the JM-A antiparallel dimer, and inducing the formation of asymmetric dimers of the kinase region (green) of EGFR. This dimerization results in the phosphorylation of several tyrosine residues in the tail region of EGFR. Right, Phosphorylated-Tyr1068 EGFR recruits PLCγ (yellow), degrading PI(4,5)P2 and producing DAG. DAG activates protein kinase C (cyan), which phosphorylates Thr654 of EGFR, deactivating EGFR. *p<0.05, **p<0.01, NS (not significant) on Welch’s t-test.
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Figure 7—source data 1
PDF file for western blotting analysis displayed in Figure 7A and C.
- https://cdn.elifesciences.org/articles/101652/elife-101652-fig7-data1-v1.zip
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Figure 7—source data 2
Original files for western blotting analysis are displayed in Figure 7A and C.
- https://cdn.elifesciences.org/articles/101652/elife-101652-fig7-data2-v1.zip
Videos
Video 1
Lateral colocalization rate of epidermal growth factor receptor (EGFR) and PI(4,5)P2 decreases after epidermal growth factor (EGF) stimulation.
EGFR–GFP (green) and Halo–PI(4,5)P2 (magenta) were transiently expressed in EGFR-KO HeLa cells. After the cells were incubated in a serum-free medium overnight, Halo–PI(4,5)P2 was stained with JF549–Halo ligand. EGFR–GFP and JF549–PI(4,5)P2 were observed at a time resolution of 30 ms for 6 s before EGF stimulation (left). After stimulation with 20 nM EGF, EGFR–GFP and JF549–PI(4,5)P2 were observed in the same cell 2 min after the addition of EGF (right).
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Bilateral regulation of EGFR activity and local PI(4,5)P2 dynamics in mammalian cells observed with superresolution microscopy
eLife 13:e101652.
https://doi.org/10.7554/eLife.101652
