Figure 4. 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.