Figure 4.
The β3 integrin dependent contractility is associated with the defect of β3 integrin endocytosis. (A) The β3 integrin uptake was measured using β3 integrin specific antibody (LucA.5), coupled with pH-Rhodo. The volume of the endocytotic vesicles per cell volume was measured at 37 and 4°C. Note the decrease of β3 integrin endocytosis in cell lines deleted in ICAP-1. The quantification was performed after 45 min incubation. Error bars represent SD. N ≥ 127 cells. ns, adjusted P value > 0.05; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001; ****, P value ≤ 0.0001. (B) Representative images of PLA performed with antibodies against ICAP-1 and β3 integrin. Red dots denote regions of signal amplification indicating the close proximity between ICAP-1 and β3 integrin. PLA performed on ICAP-1 deficient cell line is used as control. Nuclei are stained in blue with DAPI. (C) Representative images of proximity ligation assays performed with antibodies against ICAP-1 and AP2 or Dynamin 2. Red dots denote regions of signal amplification indicating that ICAP-1 belongs to clathrin endocytosis machinery. PLA performed on ICAP-1 deficient cell line is used as control. Nuclei are stained in blue with DAPI. Scale bar, 20 µm. (D) PLA performed with antibodies against AP2 and β1 integrin or β3 integrin and quantification of the number of PLA spots per cell shows that deficiency in ICAP-1 leads to increase of the association of β1 and β3 integrins with AP2. N ≥ 25 cells/condition from three independent experiments. Scale bars, 20 µm. Error bars represent SD. ns, adjusted P value > 0.05; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001; ****, P value ≤ 0.0001. (E) Immunofluorescence staining of the phospho-myosin (ppMLC antibody, red) and β3 integrin (LucA.5 antibody, green). Representative micrographs of the four osteoblast cell lines treated with clathrin siRNA (si Clat, right) or scramble siRNA (si Scr, left). Inhibition of clathrin expression in β1 integrin−/−/icap-1+/+ cell line rescues cell spreading through β3 integrin-mediated FA and development of acto-myosin cytoskeleton (see also graphs F–H). Scale bar, 20 µm. (F) Quantification of cell spreading area of the four osteoblast cell lines treated with clathrin siRNA (si Clathrin, light colors) or scramble siRNA (si Scr, dark colors). Error bars represent SD. N ≥ 27 cells. ns, adjusted P value > 0.05; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001; ****, P value ≤ 0.0001. (G) Quantification of area of β3 integrin containing FAs normalized to the cellular surface area of the four osteoblast cell lines treated with clathrin siRNA (si Clathrin, light colors) or scramble siRNA (si Scr, dark colors). Error bars represent SD. N ≥ 27 cells. ns, adjusted P value > 0.05; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001; ****, P value ≤ 0.0001. (H) Quantification of phospho-myosin staining area normalized to the cellular surface area of the four osteoblast cell lines treated with clathrin siRNA (si Clathrin, light colors) or scramble siRNA (si Scr, dark colors). Error bars represent SD. N ≥ 27 cells. ns, adjusted P value > 0.05; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001; ****, P value ≤ 0.0001.

The β3 integrin dependent contractility is associated with the defect of β3 integrin endocytosis. (A) The β3 integrin uptake was measured using β3 integrin specific antibody (LucA.5), coupled with pH-Rhodo. The volume of the endocytotic vesicles per cell volume was measured at 37 and 4°C. Note the decrease of β3 integrin endocytosis in cell lines deleted in ICAP-1. The quantification was performed after 45 min incubation. Error bars represent SD. N ≥ 127 cells. ns, adjusted P value > 0.05; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001; ****, P value ≤ 0.0001. (B) Representative images of PLA performed with antibodies against ICAP-1 and β3 integrin. Red dots denote regions of signal amplification indicating the close proximity between ICAP-1 and β3 integrin. PLA performed on ICAP-1 deficient cell line is used as control. Nuclei are stained in blue with DAPI. (C) Representative images of proximity ligation assays performed with antibodies against ICAP-1 and AP2 or Dynamin 2. Red dots denote regions of signal amplification indicating that ICAP-1 belongs to clathrin endocytosis machinery. PLA performed on ICAP-1 deficient cell line is used as control. Nuclei are stained in blue with DAPI. Scale bar, 20 µm. (D) PLA performed with antibodies against AP2 and β1 integrin or β3 integrin and quantification of the number of PLA spots per cell shows that deficiency in ICAP-1 leads to increase of the association of β1 and β3 integrins with AP2. N ≥ 25 cells/condition from three independent experiments. Scale bars, 20 µm. Error bars represent SD. ns, adjusted P value > 0.05; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001; ****, P value ≤ 0.0001. (E) Immunofluorescence staining of the phospho-myosin (ppMLC antibody, red) and β3 integrin (LucA.5 antibody, green). Representative micrographs of the four osteoblast cell lines treated with clathrin siRNA (si Clat, right) or scramble siRNA (si Scr, left). Inhibition of clathrin expression in β1 integrin−/−/icap-1+/+ cell line rescues cell spreading through β3 integrin-mediated FA and development of acto-myosin cytoskeleton (see also graphs F–H). Scale bar, 20 µm. (F) Quantification of cell spreading area of the four osteoblast cell lines treated with clathrin siRNA (si Clathrin, light colors) or scramble siRNA (si Scr, dark colors). Error bars represent SD. N ≥ 27 cells. ns, adjusted P value > 0.05; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001; ****, P value ≤ 0.0001. (G) Quantification of area of β3 integrin containing FAs normalized to the cellular surface area of the four osteoblast cell lines treated with clathrin siRNA (si Clathrin, light colors) or scramble siRNA (si Scr, dark colors). Error bars represent SD. N ≥ 27 cells. ns, adjusted P value > 0.05; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001; ****, P value ≤ 0.0001. (H) Quantification of phospho-myosin staining area normalized to the cellular surface area of the four osteoblast cell lines treated with clathrin siRNA (si Clathrin, light colors) or scramble siRNA (si Scr, dark colors). Error bars represent SD. N ≥ 27 cells. ns, adjusted P value > 0.05; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001; ****, P value ≤ 0.0001.

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