Figure 5.
ICAP-1 is required for NME recruitment in clathrin-coated pits and for keeping the proximity between integrin and NME. (A) Western blot analysis showing the efficiency of SiNME1/2 in osteoblast cells. (B) TIRF/FRAP analysis shows that deletion of NME1/2 complex by siRNAs (siNME1/2, light colors) or scramble siRNA (si Scr, dark colors) impedes the turnover of eGFP-β3 integrins at the plasma membrane in β1 integrin−/−/icap-1+/+ cell line. Six FAs per cell were bleached for each experiment. eGFP-β3 integrin recovery was monitored for 5 min. 19 cells ≤ N ≤ 107 cells. 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. (C and D) Representative images of PLAs (C) and PLA assay quantification (D) of the number of PLA spots per cell performed with antibodies against NME and ICAP-1 or AP2 or Dynamin 2. Red dots denote regions of signal amplification consistent with NME/ICAP-1 interaction, NME/AP2 interaction, and NME/dynamin 2 interaction in β1 integrin+/+- icap-1+/+ osteoblast cells (left). PLA performed on ICAP-1 deficient cell line is used as control. The deletion of ICAP-1 induces a decrease of red dots in all three cases indicating the crucial role of ICAP-1 for keeping NME in clathrin endocytosis machinery (right). Nuclei are stained in blue with DAPI. Scale bar, 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 and F) Representative images (E) of PLAs with quantification (F) performed with antibodies against NME and β1 integrin or β3 integrin or transferrin receptor. Red dots denote regions of signal amplification consistent with NME/integrin proximity in β1 integrin+/+- icap-1+/+ osteoblast cells (left). The deletion of ICAP-1 induces a decrease of the number of red dots indicating the crucial role of ICAP-1 for keeping NME and integrin vicinity (right). PLA performed on ICAP-1 deficient cell line is used as control. Nuclei are stained in blue with DAPI. Scale bar, 20 µm. Error bars represent SD. N ≥ 25 cells/condition from three independent experiments. ns, adjusted P value > 0.05; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001; ****, P value ≤ 0.0001. Source data are available for this figure: SourceData F5.

ICAP-1 is required for NME recruitment in clathrin-coated pits and for keeping the proximity between integrin and NME. (A) Western blot analysis showing the efficiency of SiNME1/2 in osteoblast cells. (B) TIRF/FRAP analysis shows that deletion of NME1/2 complex by siRNAs (siNME1/2, light colors) or scramble siRNA (si Scr, dark colors) impedes the turnover of eGFP-β3 integrins at the plasma membrane in β1 integrin−/−/icap-1+/+ cell line. Six FAs per cell were bleached for each experiment. eGFP-β3 integrin recovery was monitored for 5 min. 19 cells ≤ N ≤ 107 cells. 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. (C and D) Representative images of PLAs (C) and PLA assay quantification (D) of the number of PLA spots per cell performed with antibodies against NME and ICAP-1 or AP2 or Dynamin 2. Red dots denote regions of signal amplification consistent with NME/ICAP-1 interaction, NME/AP2 interaction, and NME/dynamin 2 interaction in β1 integrin+/+- icap-1+/+ osteoblast cells (left). PLA performed on ICAP-1 deficient cell line is used as control. The deletion of ICAP-1 induces a decrease of red dots in all three cases indicating the crucial role of ICAP-1 for keeping NME in clathrin endocytosis machinery (right). Nuclei are stained in blue with DAPI. Scale bar, 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 and F) Representative images (E) of PLAs with quantification (F) performed with antibodies against NME and β1 integrin or β3 integrin or transferrin receptor. Red dots denote regions of signal amplification consistent with NME/integrin proximity in β1 integrin+/+- icap-1+/+ osteoblast cells (left). The deletion of ICAP-1 induces a decrease of the number of red dots indicating the crucial role of ICAP-1 for keeping NME and integrin vicinity (right). PLA performed on ICAP-1 deficient cell line is used as control. Nuclei are stained in blue with DAPI. Scale bar, 20 µm. Error bars represent SD. N ≥ 25 cells/condition from three independent experiments. ns, adjusted P value > 0.05; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001; ****, P value ≤ 0.0001. Source data are available for this figure: SourceData F5.

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