Proximal interaction of ICAM-1 with EBP50/NHERF1/SLC9A3R1 into nano-scale microvillar domains
(A). The ICAM-1 BioID interactome reveals the proximal interaction of ICAM-1 with EBP50. Parental polarized HepG2 cells (WT) and HepG2 cells stably expressing ICAM-1-BirA* were incubated with 50 μM biotin for 16 h, lysed and subjected to a pull-down (PD) assay with neutravidin-agarose. Western blots show ICAM-1 and EBP50 biotinylation. Exo70 and ERK are shown as negative controls. ICAM-1 and ICAM-1-BirA* were detected with anti-ICAM-1 antibody. (B) Polarized hepatic epithelial cells were fixed and stained for ICAM-1 and EBP50. BC, bile canaliculi. The Manders’ analyses for the indicated pairs of staining show the remarkable co-localization between ICAM-1 and EBP50. Scale bar, 5 μm. (C) EBP50 is localized in murine hepatic sinusoids (S) and in the liver bile canaliculi (red arrows). Scale bar, 50 μm. (D) Triple staining of EBP50, F-actin and pMLC in BCs. Central panel. Relative distribution of the staining intensity for the indicated proteins, starting from the distal parts of BCs, which are enriched in actomyosin, towards the centers of the BCs, which contain the microvillar tips. Right panel. The Manders’ analyses for the indicated pairs of staining show the remarkable co-localization of EBP50 with F-actin, but not with pMLC. Scale bars, 3 μm. (E) Hepatic epithelial cells were transfected with the indicated expression vectors coding for FLAG-tagged full-length EBP50 or its N-termina PDZ domains. 24 post-transfection, cells were lysed, subjected to immunoprecipitation with anti-FLAG antibodies and the immunoprecipitates and the lysates were analyzed by western blot for the indicated proteins. (F,G) Polarized hepatic epithelial cells were stained for the indicated proteins and analyzed by STED super-resolution microscopy. Only two different fluorophores could be subjected to simultaneous STED. (F) STED analysis of ICAM-1 and EBP50. Central and right images are a 2.5-fold enlargement of the boxed area in the left image, which corresponds to BCs. Bottom images are a 2-fold enlargement of the boxed area in top central and right images, which corresponds to a canalicular microvilli-rich area. (G) STED analysis of F-actin and EBP50 in a BC. Bottom images are a 2-fold enlargement of the boxed area in the top image, which corresponds to a canalicular microvilli-rich area. Scale bars, 5 μm. (H) STED analyses of non-polarized epithelial cells exposed to anti-ICAM-1 for 30 min in the cold and then incubated at 37°C for the indicated times. ICAM-1 and EBP50 did not overlap at t=0 in these non-canalicular regions. However, 90 min at 37°C with anti-ICAM-1 antibody induced a redistribution of EBP50 into ring-shaped macroclusters that overlapped with ICAM-1 aggregates. Scale bars, 5 μm. (I) Manders’ analyses for the indicated pairs of staining from (F) (apical) and (H) (non-polarized). (J) Polarized WT and ICAM-1_KO cells were fixed, stained and the intensity of EBP50 at the apical BCs was quantified by confocal microscopy (right). (K) Polarized WT and ICAM-1_KO cells were fixed, stained and analyzed by STED confocal microscopy. Intensity and nearest neighbor distance (nnd) of detected spots at BCs and eBCs were calculated.