Intersectin-1s (ITSN-1s), a protein containing five SH3 (A-E) domains, regulates via the SH3A the function of dynamin-2 (dyn2) at the endocytic site. ITSN-1s expression was modulated in mouse lung endothelium by liposome delivery of either a plasmid cDNA encoding myc-SH3A or a specific siRNA targeting ITSN-1 gene. The lung vasculature of SH3A-transduced and ITSN-1s- deficient mice was perfused with gold albumin (Au-BSA) to analyze by electron microscopy the morphological intermediates and pathways involved in transendothelial transport or with dinitrophenylated (DNP)-BSA to quantify by ELISA its transport. Acute modulation of ITSN-1s expression decreased the number of caveolae, impaired their transport, and opened the interendothelial junctions, while upregulating compensatory nonconventional endocytic/transcytotic structures. Chronic inhibition of ITSN-1s further increased the occurrence of nonconventional intermediates and partially restored the junctional integrity. These findings indicate that ITSN-1s expression is required for caveolae function and efficient transendothelial transport. Moreover, our results demonstrate that ECs are highly adapted to perform their transport function while maintaining lung homeostasis. 1. Introduction ITSN-1s is a multimodular protein, evolutionary conserved and widely expressed [1, 2]; it consists of two NH2-terminal EH domains, a central coiled-coil domain, and five consecutive COOH-terminal SH3 domains, SH3A-E, [3, 4]. Similarly to Dyn, ITSN-1s localizes to endocytic clathrin-coated pits and caveolae at the plasma membrane and associates preferentially with the neck region of caveolae [5, 6]. The simultaneous presence of multiple SH3 and EH domains, best known for their function in endocytosis, as well as the subcellular localization of ITSN-1s, led to the early assumption that ITSN-1s may function as an adaptor/scaffold of the general endocytic machinery [3, 5]. Subsequent studies have shown that ITSN-1s is capable of binding essential endocytic proteins, Epsin1/2, Eps15 , both the neuronal and ubiquitously expressed Dyn isoforms, [3, 5, 6, 8], stonin 2 [9, 10], and the signaling proteins RaIBP-associated Eps15-homology domain protein , mSos [12, 13], and 5-phosphatase SHIP2 . ITSN-1s binds several Dyn molecules simultaneously and clusters them at the endocytic sites, creating a high concentration of Dyn required for collar formation around the necks of endocytic vesicles [6, 8]. This is a crucial endocytic event since the GTPase activity of Dyn is allosterically dependent on Dyn protein concentration
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