Figure 1. Energetic requirement for... | Rockefeller University Press

Figure 1.

$Figure 1. Energetic requirement for cargo sorting, but not clathrin assembly. (A) Representative images of CCP assembly induced by cytosol on stripped membrane sheets. CCPs were visualized by Clathrin-AF555 using TIRF microscopy. (B) Representative images of CCP assembly induced by cytosol on unstripped membrane sheets. CCPs were immunostained with an anti-clathrin heavy chain (X22) antibody. (C) Normalized intensity of Clathrin-AF555 during CCP real-time assembly induced by cytosol or by cytosol with 0.5 µM latrunculin A. (D) Representative images of membranes expressing TfR-pHluorin and CCPs after incubation with cytosol. (E) Normalized intensity of CCP real-time assembly and corresponding TfR-pHluorin puncta. (F) Representative images of membranes expressing TfR-pHluorin and CCPs after incubation with coat fraction. (G) Density of CCPs assembled with cytosol (left) or coat fraction (right) in the presence or absence of ATP. (H) Representative images of membranes expressing TfR-pHluorin and CCPs after AP fraction incubation. (I) Normalized intensity of CCP real-time assembly induced by AP fraction. Each profile of C, E, and I is the average intensity of ROI from >12 membrane sheets. Error bars indicate SD. Scale bars, 2 µm.$

Energetic requirement for cargo sorting, but not clathrin assembly. (A) Representative images of CCP assembly induced by cytosol on stripped membrane sheets. CCPs were visualized by Clathrin-AF555 using TIRF microscopy. (B) Representative images of CCP assembly induced by cytosol on unstripped membrane sheets. CCPs were immunostained with an anti-clathrin heavy chain (X22) antibody. (C) Normalized intensity of Clathrin-AF555 during CCP real-time assembly induced by cytosol or by cytosol with 0.5 µM latrunculin A. (D) Representative images of membranes expressing TfR-pHluorin and CCPs after incubation with cytosol. (E) Normalized intensity of CCP real-time assembly and corresponding TfR-pHluorin puncta. (F) Representative images of membranes expressing TfR-pHluorin and CCPs after incubation with coat fraction. (G) Density of CCPs assembled with cytosol (left) or coat fraction (right) in the presence or absence of ATP. (H) Representative images of membranes expressing TfR-pHluorin and CCPs after AP fraction incubation. (I) Normalized intensity of CCP real-time assembly induced by AP fraction. Each profile of C, E, and I is the average intensity of ROI from >12 membrane sheets. Error bars indicate SD. Scale bars, 2 µm.