{"CAPTION FIG1.png": "'\\nFig. 1: Clathrin-coated plaques are mechanosensitive structures. **a** HeLa cells were seeded on collagen-coated glass or polyacrylamide gels of indicated stiffness and fixed 24 h later before being stained for \u03b1-adaptin. Scale bar: 15 \u03bcm. Higher magnifications of boxed regions are shown. **b** Kymographs showing CCS dynamics in genome-edited HeLa cells expressing endogenous GFP-tagged \u03bcM2-adaptin seeded on the indicated collagen-coated substrate and imaged by spinning disk microscopy every 5 s for 5 min. **c** Quantification of the dynamics of CCSs observed as in **b** (\u201c+P < 0.001, as compared to 0.1 kPa condition, one-way analysis of variance\u2014ANOVA = 3). **d** HeLa cells seeded on collagen-coated glass were treated with Bleblithian or Cytokthalasian D as indicated for 30 min before being fixed and stained for \u03b1-adaptin. Scale bar: 10 \u03bcm. Higher magnifications of boxed regions are shown. **e** Quantification of the dynamics of CCSs observed as in **d** (\u201cP < 0.01, \u201cP < 0.05, ANOVA = 3). **f** EM micrographs of unrooted HeLa cells that were cultured on glass and treated for 30 min with blebbistatin before being fixed and processed. Clathrin-coated plaques are highlighted in green. Scale bar: 200 nm. All results are expressed as mean \u00b1 SD'", "CAPTION FIG2.png": "'\\nFig. 2: avlv5 integrin localizes to plaques and is required for their assembly. **a** HeLa cells were seeded on collagen-coated glass and fixed 24 h later before being stained for \u03b1-adaptin and avl/b5-integrin. Scale bar: 10 \u03bcm. Higher magnifications of boxed regions are shown. Arrows point to clathrin-coated plaques arrowheads point to CCPs. **b** Quantification of avl/b5 enrichment at plaques versus CCPs (*P < 0.005, two tailed Student\u2019s t-test. _n = 3_; 100 structures per experiment were counted). **c** Quantification of colocalization (Pearson\u2019s coefficient) between \u03b1/b5 and \u03b1-adaptin in cells cultured on the indicated collagen-coated substrate (*P < 0.005; *P < 0.001, one-way analysis of variance\u2013ANOVA _n = 3_). **d** HeLa cells treated with control (upper panel) or b5-specific (lower panel) siRNA were seeded on collagen-coated glass and fixed 24 h later before being stained for \u03b1-adaptin. Scale bar: 15 \u03bcm. **c** Kymgtaphs showing CCS dynamics in genome-edited HeLa cells treated with the indicated siRNA, seeded on collagen-coated glass, and imaged by spinning disk microscopy every 5 s for 5 min. **f** Quantification of the dynamics of CCSs observed as **in****e** and treated with the indicated siRNA (**_n_ < 0.001, ANOVA _n = 3_). **g** HeLa cells treated with b5-specific siRNAs were transfected with a siRNA-resistant \u03b2 5-GFP encoding construct and then fixed 24 h later before being stained for \u03b1-adaptin. Scale bar: 10 \u03bcm. The star marks a cell that is not transfected by b5-GFP. Higher magnifications of boxed regions are shown. All results are expressed as mean \u00b1 SD'", "CAPTION FIG3.png": "'\\nFig. 3: Clathrin-coated plaques assemble as a consequence of frustrated endocytosis. **a** Genome-edited HeLa cells expressing endogenous mCherry-tagged \\\\(\\\\mu^{2}\\\\)-adaptiv, seeded on collagen-coated glass, were treated with trypsin and imaged by spinning disk microscopy every 5 s. Time after trypsin addition is indicated in seconds. Arrowheads point to dot-like structure emanating from the disassembling plaques. Scale bar: 1 \u03bcm. **b** Kymographs showing plaque disassembly dynamics and concomitant GFP-auxillin bursts in HeLa cell treated and imaged as in **a**. Note that loss of \\\\(\\\\mu^{2}\\\\)-adaptiv-associated fluorescence is correlated with auxillin flashes. **c** Quantification of the number of auxillin flashes per plaque and per minute in HeLa cells before and after incubation with trypsin. Results are expressed as mean \\\\(\\\\pm\\\\) SD (\\\\(\\\\text{P}\\\\)\\\\(\\\\text{P}\\\\)\\\\(\\\\text{<}\\\\)\\\\(\\\\text{0.001}\\\\), Mann-Whitney rank sum test. **d** Total of 90 structures from three independent experiments was quantified). **d** Genome-edited HeLa cells expressing endogenous mCherry-tagged \\\\(\\\\mu^{2}\\\\)-adaptiv, seeded on collagen-coated glass, were treated with. Clingentide and imaged by spinning disk microscopy every 5 s. Time after Clingentide addition is indicated in seconds. Arrowheads point to dot-like structure emanating from the disassembling plaques. Scale bar: 1 \u03bcm. **e** Genome-edited HeLa cells treated with \\\\(\\\\text{P}\\\\)5-specific siRNA and transfected with a construct encoding mCherry-tagged TIR were seeded on anti-mCherry antibodies-coated glass and imaged 24 h later by spinning disk microscopy every 5 s. Scale bar: 10 \u03bcm. **f** Kymograph showing CCS dynamics in the region corresponding to the boxed area in **e**. Note that the cell on the left is not transfected by the TIR-mCherry construct and only display dynamic CCSs. **g** Quantification of the dynamics of CCSs observed as in **e** in genome-edited HeLa cells expressing the TIR-mCherry construct and treated as indicated (\\\\(\\\\text{P}\\\\)\\\\(\\\\text{P}\\\\)\\\\(\\\\text{<}\\\\)\\\\(\\\\text{0.001}\\\\), one-way analysis of variance--ANOVA. \\\\(n\\\\)\\\\(\\\\text{=}\\\\)\\\\(\\\\text{3}\\\\)). Results are expressed as mean \\\\(\\\\pm\\\\) SD'", "CAPTION FIG4.png": "'\\nFig. 4: Clathrin-coated plaques regulate stiffness-dependent Erk signaling. **a** Western-bit analysis of phospho-Erk (P-Erk) levels in HeLa cells growing on collagen-coated glass and treated with control or \\\\(\\\\beta\\\\)S-specific siRNAs as indicated (representative image of four independent experiments). Total-Erk was used as a leading control. **b** Densitometry analysis of bands obtained in western-blots as in **a** Results are expressed as mean \\\\(\\\\pm 5\\\\)D from four independent experiments (\u201cP < 0.05, one-way analysis of variance\u2014ANOVA). **c** Western-bit analysis of phospho-Erk (P-Erk) levels in HeLa cells growing on collagen-coated, 0.1 kPa polyacrylamide gels and treated with control or integrin \\\\(\\\\beta\\\\)S-specific siRNAs, as indicated. Total-Erk was used as a loading control (representative image of three independent experiments) **d** Densitometry analysis of bands obtained in western-blots as in **e** Results are expressed as mean \\\\(\\\\pm 5\\\\)D from three independent experiments. **e** GP-Erk-expressing HeLa cells treated or not with a \\\\(\\\\beta\\\\)S-specific siRNA and transfected or not with TRIzolCherry, as indicated, were seeded on anti-mCherry antibodies-coated glass and imaged 24 h later. Scale bar: 10 \u03bcm. A color-coded scale for low and high signal intensity is shown. **f** Quantification of GP-Erk nuclear enrichment index in cells as in **e** and cultured on control antibody- or anti-mCherry antibody-coated glass, as indicated, and treated or not with indicated siRNAs. Results are expressed as mean \\\\(\\\\pm 5\\\\)D (\u201cP < 0.01, \u201cP < 0.001, ANOVA, ControlAb-siCtr: 144 cells from \\\\(n=5\\\\) independent experiments; mCherryAb-siCtr: 19 cells from \\\\(n=5\\\\) independent experiments; ControlAb-siCtr: 146 cells from \\\\(n=5\\\\) independent experiments; mCherryAb-siCtr: 120 cells from \\\\(n=5\\\\) independent experiments; ControlAb-siCtr: 1- siRNA-2: 69 cells from \\\\(n=3\\\\) independent experiments; mCherryAb-siCtr: 1- siRNA-2: 68 cells from \\\\(n=3\\\\) independent experiments; ControlAb-siCtr: 1- siRNA-2: 67 cells from \\\\(n=3\\\\) independent experiments)'", "CAPTION FIG5.png": "'\\nFig. 5: Clathrin-coated plaques: totally regulate receptor-dependent Ek: signaling. **a** HeLa cells seeded on collagen-coated glass were starved for 4 h and then treated or not with 10 ng/ml EGF for 5 min alone or added after 30 min preincubation with 10 \u03bcM Gefitinib. Cells were then fixed and stained for a-adaptin and phosphotyrosine. The arrowhead points to one CCP and the star marks a plaque. Scale bar: 2 \u03bcM. **b** Quantification of phosphotyrosine accumulation at plaques or CCPs in the indicated conditions. Results are expressed as mean +- SOD (*_P_ < 0.05, *_P_ < 0.001, one-way analysis of variance\u2013ANOVA. \\\\(n\\\\) = 3. Number of structures analyzed per condition: Starved 160 plaques; EGF: 452 plaques, EGF + Gefitinib: 301 plaques, CCPs/EGF: 200 pits). **c** Western-blot analysis of phospho-Ek (P-Ek) levels in starved HeLa cells treated with control or b5-specific siRNA as indicated, and stimulated for the indicated time with 10 ng/ml EGF. Total-Ek was used as a leading control (representative image of three independent experiments. **d** Derstimometry analysis of bands obtained in western-blots as in **e**. Results are expressed as mean +- SOD (*_P_ < 0.05, ANOVA. \\\\(n\\\\) = 3). **e** HeLa cells treated with b5-specific siRNA were transfected with plasmids encoding for TIR-mCherry and EGFR-GFP and seeded on anti-mCherry antibodies-coated glass. Cells were serum-starved for 4 h and then treated with 10 ng/ml EGF for 470 s. Scale bar: 0.5 \u03bcM. Arrowheads point to plaques positive for EGFR-GFP. **f** HeLa cells treated with b5-specific siRNA were transfected with plasmids encoding for TIR-mCherry and HGFR-GFP and seeded on anti-mCherry antibodies-coated glass. Cells were serum-starved for 4 h and then treated with 50 ng/ml HGF for 570 s. Scale bar: 0.5 \u03bcM. Arrowheads point to plaques positive for HGFR-GFP. **g** HeLa cells treated with b5-specific siRNA were transfected with plasmids encoding for TIR-mCherry and seeded on anti-mCherry antibodies-coated glass. Cells were serum-starved for 4 h and then treated with 10 ng/ml EGF for 5 min prior to fixation and staining for phosphotyrosine. Scale bar: 1.5 \u03bcM. Arrowheads point to plaques positive for P-Tyr'", "CAPTION FIG6.png": "'\\nFig. 6: Signaling at plaques is contractility-independent and regulate cell proliferation. **a** HeLa cells on collagen-coated glass were treated for 1 h with 10 \u03bcM Blebbistatin or 10 \u03bcM Cytochalasin D prior to be fixed and stained for \u03b1-adaptin (red) and phosphotyrosines (P-Tyr, green). Scale bar: 3 \u03bcM. **b**, Quantification of phosphotyrosines accumulation at plaques in cells treated as in a, as indicated (*P < 0.05, one-way analysis of variance\u2014ANOVA). Control: 402 plaques from \\\\(n=3\\\\) independent experiments; Blebbistatin: 303 plaques from \\\\(n=3\\\\) independent experiments; Cytochalasin: 302 plaques from \\\\(n=3\\\\) independent experiments. **c** Western-blot analysis of phospho-Frk (P-Frk) levels in HeLa cells cultured on collagen- or vitronectin-coated glass or 0.1 \u03bcM kPa polyvinylamide gels, as indicated. Total-Erk was used as a loading control (representing image of three independent experiments). **d** Densimetry analysis of bands obtained in western-blots as in **e** (*P < 0.05, ANOVA. \\\\(n=3\\\\)). **e** Western-blot analysis of phospho-Frk (P-Erk) levels in HeLa cells growing on collagen-coated glass and treated with control or integrin b5-specific siRNAs and incubated or not with 10 \u03bcM Blebbistatin for 1 h, as indicated. Total-Erk was used as a loading control (representative image of three independent experiments). **f** Densitometry analysis of bands obtained in western-blots as in **e** (*P < 0.05, *P < 0.01, ANOVA. \\\\(n=5\\\\)). **g** Equal numbers of HeLa cells were plated on non-coated glass (open squares), or on collagen-coated (purple open circles) or vitronectin-coated glass (black open circles), as indicated. Cells were harvested and counted 24 and 48 h after plating (***P < 0.001, ANOVA. \\\\(n=3\\\\)). **h** HeLa cells treated with control (circles), b5-specific (triangles and crosses), or any-specific siRNAs (diamonds and hexagons) for 48 h were seeded on vitronectin-coated glass in equal numbers. 24 and 48 h later, cells were harvested and counted (*P < 0.05, ANOVA. \\\\(n=3\\\\)). **i** HeLa cells treated with b5-specific siRNAs were transfected with a plasmid encoding TIR-mCherry and seeded in equal numbers on glass coated with either anti-mCherry (black circles) or control antibodies (open circles). 24 and 48 h later, cells were harvested and counted (*P < 0.05, ANOVA. \\\\(n=3\\\\)). All results are expressed as mean \u00b1 SD'"}