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{"CAPTION FIG1.png": "'\\n\\n**Fig. 1 EGF modifies the ultrastructure of clathrin at the plasma membrane.****a**_N_Montaged PREM image of an unroofed control HCS-EGFR-GFP cell and the mask created after segmentation of the full membrane outlined (yellow). Flat, dome, and sphere clathrin-coated structures (CCCs) are shown in green, blue, and magenta, respectively. **b** High-magnification image of the cropped PREM in (**a**); the different segmented CCSs are color-coded as in (**a**), with grayscale in magnified insets. **c**_Montaged PREM image of an unroofed HCS-EGFR-GFP cell treated with 50 ng/mL EGF for 15 min and the mask created after the segmentation. **d**_High-magnification of a representative region of the PREM in (**c**), the magnification insets are shown at the same scale and are outlined with dashed squares in each image. **e**_Representative clathrin masks of the EGF stimulation time course for 0, 2, 5, 15, 30, and 60 min. PREM images corresponding to the masks and cropped images are shown in Supplementary Fig. 1. **f-h**_Morphometric analysis of the percentage of plasma membrane (PM) area occupation for each CCS subtype. 1-_N_Morphometric analysis of the percentage of plasma maximum data point markers with a coefficient value of 1.5. **f-k**_Morphometric analysis of the size of flat, dome, and sphere CCSs during the EGF time course for three clathrin subtypes. Dot plots show every structure segmented, the bar is the median, \\\\(N\\\\) = 2 biologically independent experiments in (**a**-**a**); 0 min: \\\\(N_{\\\\text{Max}}\\\\) = 141, \\\\(N_{\\\\text{dena}}\\\\) = 46, \\\\(N_{\\\\text{dena}}\\\\) = 68, \\\\(N_{\\\\text{exta}}\\\\) = 4; 2 min: \\\\(N_{\\\\text{Int}}\\\\) = 164, \\\\(N_{\\\\text{dena}}\\\\) = 32, \\\\(N_{\\\\text{dena}}\\\\) = 30, \\\\(N_{\\\\text{exta}}\\\\) = 3; 5 min: \\\\(N_{\\\\text{Max}}\\\\) = 184, \\\\(N_{\\\\text{dena}}\\\\) = 26, \\\\(N_{\\\\text{dena}}\\\\) = 36, \\\\(N_{\\\\text{dena}}\\\\) = 4; 15 min: \\\\(N_{\\\\text{Int}}\\\\) = 559, \\\\(N_{\\\\text{dena}}\\\\) = 67, \\\\(N_{\\\\text{dena}}\\\\) = 207, \\\\(N_{\\\\text{exta}}\\\\) = 4; 30 min: \\\\(N_{\\\\text{Int}}\\\\) = 395, \\\\(N_{\\\\text{dena}}\\\\) = 149, \\\\(N_{\\\\text{dena}}\\\\) = 113, \\\\(N_{\\\\text{exta}}\\\\) = 3; 60 min: \\\\(N_{\\\\text{int}}\\\\) = 81, \\\\(N_{\\\\text{dena}}\\\\) = 57, \\\\(N_{\\\\text{exta}}\\\\) = 5 examined over the indicated independent experiments. Scale bars in (**a**) and (**c**) are 5 min. Scale bars in (**b**, **d**, **e**) are 1 min insets are 200 nm. EGF epidermal growth factor, EGFR epidermal growth factor receptor, PREM platinum replica electron microscopy.\\n\\n'", "CAPTION FIG2.png": "'\\n\\n**Fig. 2 Flat clathrin lattice formation requires EGFR.****a** Representative PREM images of control HSC3-EGFR-GFP cells (Ctrl), **b** cells treated either with ESO ng/mL EGF alone (EGF) for 15 min, or in presence of (**c**) 10 \u03bcM gefitinib (Gefi + EGF), and **d** EGFR siRNA (EGFR siRNA + EGF). EGFR siRNA validation is shown in Supplementary Fig. 3. The magnification insets are shown at the same scale and are outlined with dashed squares in each image. Flat, dome, and sphere clathrin-coated structures (CCSs) are shown in transparent green, blue, and magenta, respectively, with native grayscale in magnified insets. **e** Representative masks of segmented cells treated as in (**a-d**). PREM images corresponding to the masks and cropped images are shown in Supplementary Fig. 4. **f** Morphometric analysis of the percentage of plasma membrane (PM) area occupation for flat clathrin structures. 1-shaped box plots show median extended from 25th to 75th percentiles, and minimum and maximum data point whiskers with a coefficient value of 15. **g** Morphometric analysis of the size of flat structures of cells treated as indicated in (**a-d**). Dot plots show every structure segmented the bar indicates the median. Ctrl: _N_Hua = 14L \\\\(N_{\\\\text{cells}}\\\\) = 4; EGF: _N_Hua = 559, _N_cells = 4; Gefi + EGF: _N_Hua = 160, _N_cells = 4; EGFR siRNA + EGF: _N_Hua = 346, _N_cells = 4. Number of biologically independent experiments = 2. Scale bars in (**a-e**) are 1 \u03bcm; insets are 200 nm. Ctrl and EGF data were from Fig. 1 and shown for reference. EGF epidermal growth factor receptor, PREM platinum replica electron microscopy.\\n\\n'", "CAPTION FIG3.png": "'Fig. 3: **Flat clathrin lattice formation requires Src.****a** Representative PREM images of control HSC3-EGFR-GFP cells (CtrU), **b** cells treated either with 50 ng/mL EGF alone (EGF) for TS min, or in presence of **c**10 \u03bcM PP2 (PP2 + EGF), and **d** Src siRNA (Src siRNA + EGF). Src siRNA validation is shown in Supplementary Fig. 3. The magnification insets are shown at the same scale and are outlined with dashed squares in each image. Flat, dome, and sphere clathrin-coated structures (CCSs) are shown in green, blue, and magenta, respectively, with native grayscale in magnified insets. **e** Representative masks of segmented cells treated as in (**a\u2013d**). PREM images corresponding to the masks and cropped images are shown in Supplementary Fig. 4. **f** Morphometric analysis of the percentage of plasma membrane (PM) area occupation for flat clathrin structure. **i**-shaped box plots show median extended from 25th to 75th percentiles, and minimum and maximum data point whiskers with a coefficient value of 1.5. **g** Morphometric analysis of the size of flat structures of cells treated as indicated in (**a\u2013d**). Dot plots show every structure segmented the bar indicates the median. CtrU \\\\({}_{\\\\text{Mat}}\\\\) = 141, \\\\(N_{\\\\text{cells}}\\\\) = 4; EGF: \\\\(N_{\\\\text{Mat}}\\\\) = 559, \\\\(N_{\\\\text{cells}}\\\\) = 4; PP2 + EGF: \\\\(N_{\\\\text{Mat}}\\\\) = 267, \\\\(N_{\\\\text{cells}}\\\\) = 8; Src KD + EGF: \\\\(N_{\\\\text{Mat}}\\\\) = 71, \\\\(N_{\\\\text{cells}}\\\\) = 7. Number of biologically independent experiments - 2. Scale bars in (**a\u2013e**) are 1 \u03bcm; insets are 200 nm. CtrU and EGF data were from Fig. 1 and shown for reference. EGF epidermal growth factor, EGFR epidermal growth factor receptor, PREM platinum replica electron microscopy.\\n\\n'", "CAPTION FIG4.png": "'\\nFig. 4: **Flat clathrin lattice formation requires \\\\(\\\\beta\\\\)S-integrin.** A Representative PREMs of control HSC3-EGR-GFP cells (Ctrl), **b** treated either with 50 ng/mL EGF alone (EGF) for 15 min, or in presence of **e** 10 \u03bcM clengicide acid (CTA + EGF), and **d** \\\\(\\\\beta\\\\)S-integrin siRNA (Q5 siRNA + EGF). \\\\(\\\\beta\\\\)S-integrin siRNA isolation is shown in Supplementary Fig. 3. The magnification insets are shown at the same scale and are outlined with dashed squares in each image. Flat, _red_, and sphere clathrin-coated structures (CCSs) are shown in green, blue, and magenta, respectively, with native grayscale in magnified insets. **e** Representative masks of segmented cells treated as in (**a\u2013d**). PREM images corresponding to the masks and creoped images are shown in Supplementary Fig. 4. **f** Morphometric analysis of the percentage of plasma membrane (PM) area occupation for flat clathrin structures. 1-shaped box plots show median extended from 25th to 75th percentiles, and minimum and maximum data point whiskers with a coefficient value of 15. **g** Morphometric analysis of the size of flat structures of cells treated as indicated in (**a\u2013d**). Dot plots show every structure segmented the bar indicates the median. Ctrl: \\\\(N_{\\\\text{int}}\\\\) = 14U, \\\\(N_{\\\\text{cells}}\\\\) = 4; EGF: \\\\(N_{\\\\text{tot}}\\\\) = 559, \\\\(N_{\\\\text{cells}}\\\\) = 4; CTA + EGF: \\\\(N_{\\\\text{tot}}\\\\) = 244, \\\\(N_{\\\\text{cells}}\\\\) = 4; pS siRNA + EGF: \\\\(N_{\\\\text{tot}}\\\\) = 304, \\\\(N_{\\\\text{cells}}\\\\) = 7. Number of biologically independent experiments = 2. Scale bars in (**a\u2013e**) are 1 \u03bcm; insets are 200 nm. Ctrl and EGF data were from Fig. 1 and shown for reference. EGF epidermal growth factor receptor, PREM platinum replica electron microscopy.\\n\\n'", "CAPTION FIG5.png": "'\\n\\n**Fig. 5 Differential location of EGFR; Src, and (BS-integrin in clathrin-coated structures.****a** Representative two-calar TIRF images of genome-edited HSC3 expressing EGFR-GFP and transfected with mScarlet-CLCa or HSC3 WT cells co-transfected with mScarlet-CLCa + Src-GFP or DESIGN-GFP before (Ctrl) or after 50 ng/ml EGF stimulation for 15 mm. Scale bar is 10 mm; insets are 7.3 mm x 7.3 mm. **b** Automated correlation analysis between clathrin and EGFR, Src, and (BS-integrin. Dot box plots show median extended from 25th to 75th percentiles, mean (square), and minimum and maximum data paint whiskers with a coefficient value of 1.5. Significance was tested by a two-tailed _t_-test. EGFR, ***_P_ = 5.9 x 10-7. Src ***_P_ = 1.7 x 10-11; (BS-integrin. rnp = 0.358. \\\\(N\\\\) = 4 biologically independent experiments with consistent results. \\\\(N_{\\\\text{EGFR-Ca}} = 23\\\\) cells - 3728 spots, \\\\(N_{\\\\text{ECF-ECF}} = 22\\\\) cells - 2173 spots, \\\\(N_{\\\\text{exc-CH}} = 28\\\\) cells - 1394 spots, \\\\(N_{\\\\text{exc}}\\\\). \\\\(\\\\text{EGF} = 27\\\\) cells - 1407 spots, \\\\(N_{\\\\text{EG-CH}} = 21\\\\) cells - 1037 spots; \\\\(N_{\\\\text{EG-CH}} = 20\\\\) cells - 1011 spots examined over the indicated independent experiments. EGFR epidermal growth factor receptor, TIRF total internal reflection fluorescence, CLCa clathrin light chain a, WT wild type, EGF epidermal growth factor.\\n\\n'", "CAPTION FIG6.png": "'\\n\\n**Fig. 6****BS-integrin phosphorylation controls spatial correlation with clathrin.****a** Diagram of \\\\(\\\\beta\\\\)S-integrin and magnification of the cytoplasmic domain showing different mutants. Numbers indicate the residue positions, and letters identify the amino acid. The truncated line in the diagram indicates deletion of the sequence coding for amino acids 743-799. Other constructs are wild type (WT), carboxyl-truncated (\\\\(\\\\Delta\\\\)C), none-phosphorylatable (3Y-F), phosphomimetic (3Y-E), and PAK-targeted (2S-A). **b** In vitro phosphorylation assay using purified Src or PAK4 and peptides corresponding to the B5-integrin carboxyl domain (742-799) WT and mutants in (**a**). Significance was tested by a one-way ANOVA test: \\\\({}^{*}P\\\\) = 1.51 x 10-4, \\\\({}^{**}p\\\\) = 0.002, \\\\({}^{***}P\\\\) = 1.09 x 10-5, \\\\({}^{**}p\\\\) = 0.020S. \\\\(N\\\\) = 3 biologically independent experiments with consistent results. **c** Representative TIRF images of HSC3 WT cells can transfected with mScarlet-CLCa and B5-integrin-GFP WT or containing the different mutations shown in (**a**), either before (Ctrl) or after 50 ng/mL EGF stimulation for 15 min. Scale bars are 10 min; insets are 2.3 mm x 7.3 mm. **d** Automated two-color correlation analysis of (**a**). Dot box lists show median extended from 25th to 75th percentiles, mean (square), and minimum and maximum data point whiskers with a coefficient value of 1.5. Significance was tested by a two-tailed _t_-test, \\\\({}^{**}p_{\\\\text{E-WT}}\\\\) = 0.0811, \\\\({}^{***}p_{\\\\text{B5-AC}}\\\\) = 4.03 x 10-7, \\\\({}^{***}p_{\\\\text{E-WT}}\\\\) = 8.9 x 10-5, \\\\({}^{***}p_{\\\\text{E-WT},\\\\text{C}}\\\\) = 0.9149, \\\\({}^{**}p_{\\\\text{E-WT},\\\\text{C}}\\\\) = 0.5331. \\\\(N\\\\) = 4 biologically independent experiments with consistent results. \\\\({}^{**}p_{\\\\text{E-WT},\\\\text{C}}\\\\) = 193 cells - 1492 spots, \\\\({}^{**}p_{\\\\text{E-WT},\\\\text{C}}\\\\) = 16 cells - 872 spots, \\\\({}^{**}p_{\\\\text{E-WT},\\\\text{C}}\\\\) = 16 cells - 872 spots, \\\\({}^{**}p_{\\\\text{E-WT},\\\\text{C}}\\\\) = 16 cells - 872 spots, \\\\({}^{**}p_{\\\\text{E-WT},\\\\text{C}}\\\\) = 18 cells - 1099 spots, \\\\({}^{**}p_{\\\\text{E-WT},\\\\text{C}}\\\\) = 17 cells - 1245 spots, \\\\({}^{**}p_{\\\\text{E-WT},\\\\text{C}}\\\\) = 16 cells - 1122 spots,'", "CAPTION FIG7.png": "'\\n\\n**Fig. 7 Flat clathrin lattices partition sustained signals at the plasma membrane.****a** Representative TIRF images of control (Ctrl) unroofed genome-edited HSC3 cells expressing EGFR-GFP transfected with mScarlet-CLCa and immunelabeled with anti-phospho EGFR (P-EGFR) coupled to Alexa 647, treated with 50 ng/ml EGF alone (EGF) or in the presence of bSintegrin mRNA (05 siRNA + EGF). **b** Automated correlation analysis of (**a**). \\\\({}^{*}\\\\)P = 3.67 x 10-21, \\\\({}^{**}\\\\)P = 4.84 x 10-8, \\\\({}^{***}\\\\)P = 6.31 x 10-9. \\\\(N\\\\) = 4 biologically independent experiments with consistent results. _N_(c)(c)(c)(c)(c)(c) = 19 cells - 1770 spots, _N_(c)(c)(c)(c) = 19 cells - 1240 spots, _N_(c)(c)(c) = 20 cells - 1660 spots, _N_(c)(c)(c) = 19 cells - 1678 spots examined over the indicated independent experiments. **c** Fluorescence intensity measurements of the signal from membrane P-EGFR. \\\\({}^{*}\\\\)P = 4.71 x 10-23, \\\\({}^{**}\\\\)P = 2.6 x 10-5, \\\\({}^{**}\\\\)P = 0.052. _N_(c)(c)(c)(c)(c)(c)(c) = 30 cells, _N_(c)(c)(c)(c)(c) = 30 cells, _N_(c)(c)(c) = 30 cells, _N_(c)(c)(c) = 30 cells, _N_(c)(c)(c) = 30 cells. **d** Fluorescence intensity measurements of the signal from membrane total EGFR-GFP (T-EGFR). \\\\({}^{*}\\\\)P = 1.45 x 10-4, \\\\({}^{**}\\\\)P = 2.46 x 10-4, \\\\({}^{***}\\\\)P = 8.53 x 10-9. _N_(c)(c)(c)(c) = 30 cells, _N_(c)(c)(c) = 30 cells, _N_(c)(c)(c) = 30 cells, _N_(c)(c)(c) = 30 cells. **e** Representative TIRF images of HSC3 WT cells transfected with mScarlet-CLCa and immunelabeled with anti-Grb2 coupled to Alexa 647 before (Ctrl) and treated as in (**a**). **f** Automated correlation analysis of (**e**). \\\\({}^{*}\\\\)P = 3.44 x 10-7, \\\\({}^{**}\\\\)P = 3.54 x 10-5, \\\\({}^{***}\\\\)P = 4.47 x 10-3. \\\\(N\\\\) = 4 biologically independent experiments with consistent results. _N_(c)(c)(c)(c)(c) = 19 cells - 1965 spots, _N_(c)(c)(c)(c)(c) = 20 cells - 1532 spots, _N_(c)(c)(c)(c) = 19 cells - 2165 spots, _N_(c)(c)(c)(c) = 14 cells - 801 spots. **g** Fluorescence intensity measurements of the signal coming from immunelabeled Grb2. \\\\({}^{*}\\\\)P = 2.69 x 10-14, \\\\({}^{**}\\\\)P = 2.67 x 10-7, \\\\({}^{***}\\\\)P = 0.089 _N_(c)(c)(c)(c) = 30 cells, _N_(c)(c)(c)(c) = 30 cells, _N_(c)(c)(c) = 30 cells, _N_(c)(c)(c) = 30 cells, _N_(c)(c)(c) = 30 cells. Scale bars are 10 \\\\(\\\\mu\\\\)m\\\\(\\\\times\\\\) insets are 7.3 \\\\(\\\\mu\\\\)m\\\\(\\\\times\\\\) 7.3 \\\\(\\\\mu\\\\)m. Dot and box plots show median extended from 25th to 75th percentiles, mean (square) and minimum and maximum data point whiskers with a coefficient value of 1.5. Significance between groups was evaluated by a one-way ANOVA test. Number of independent experiments = 4 (**c**, **d**, **g**). AU fluorescence arbitrary units, EGFR epidermal growth factor receptor, CLCa clathrin light chain a, WT wild type, EGF epidermal growth factor, TIRF total internal reflection fluorescence.\\n\\n'", "CAPTION FIG8.png": "'\\n\\n**Fig. 8****Model of flat clathrin lattices expansion during growth factor response.** **a** Small flat clathrin lattices are in proximity to Src and are enriched with HS-integrin. **b** EGF triggers the dimerization, clustering, and cross-phosphorylation of EGFR at growing FCLs. This in parallel allows the biding of the downstream scaffold Grb2 and locally activates Src, which in turn phosphorylates BS-integrin cytoplasmic domain. The maintenance of the EGFR/Src/BS-integrin axis promotes flat clathrin lattice growth. A key implication of this model is that two different receptor systems are spatially organized at the nanoscale within flat clathrin lattices. EGF epidermal growth factor, EGFR epidermal growth factor receptor, FCLs flat clathrin lattices.\\n\\n'"} |
