Impaired Caveolae Function and Upregulation of Alternative Endocytic Pathways Induced by Experimental Modulation of Intersectin-1s Expression in Mouse Lung Endothelium
Figure 2
Increased endothelial permeability, impaired membrane fission, and increased occurrence of nonconventional endocytic/transcytotic intermediates in the mouse lungs expressing the SH3A domain of ITSN-1s. (a) ELISA applied on mouse lung lysates of control, empty liposomes, and myc-SH3A-transduced mice perfused with 10 mg/mL BSA-DNP, for 10 min. Results were expressed in ng BSA-DNP/mg total protein/10 min. Bars ± SD. Results were obtained in 4-5 different experiments, with three mice per experimental condition. (b) Representative EM image showing the transendothelial transport of 8 nm Au-BSA in control mouse lung. Tracer particles were detected in the vessel lumen, (t), in vesicles open to the luminal side of ECs (v1), in vesicles within the endothelial cytoplasm, (v2), and in vesicles discharging their load in the perivascular space (pvs; v3). Tracer particles did not permeate the IEJ and generated filtration residues (arrowhead), in the luminal introit of the junction. Scale bar: 100 nm. (c) Representative electron micrographs showing transendothelial transport of 8 nm Au-BSA in SH3A-transduced mouse lung specimens as well as nonconventional endocytic/transcytotic intermediates in ECs expressing the SH3A domain of ITSN-1. Open IEJ permeated by 8 nm gold BSA complexes (c1) and heavy association of gold particles with the abluminal exit of an IEJ (c2). Caveolae (c3, c4, arrows) and clathrin-coated pits (c7, arrow) with very long necks, and caveolae with their narrow necks surrounded by staining-dense collars (c5, c6 arrowheads) were frequently observed. A clathrin-coated pit with elongated neck is shown (c8, boxed area, inset). Elongated elements and tubular membranous structures, labeled by gold particles (c1, c9, black boxed structures, c9.1, a9.2) and comprising caveolae and coated pits (c9, c10, white boxed structures) as well as large and uncharacteristic caveolae clusters (c10, arrow and inset). Bars: 200 nm—(c1), (c3), (c7), (c8), (c9.1); 100 nm—(c2), (c4), (c6), (c9), (c8) inset, (c10); 50 nm—(c5), (c9.2).
