Endophilin A2 controls scission in clathrin-independent endocytosis of Shiga toxin.

Endocytosis is an essential biological process by which cells internalize extracellular components and portions of their plasma membrane. Together with adaptor proteins, clathrin was first discovered as a key player for the formation of endocytic invaginations. However in the recent years, it has been discovered that many cargoes can be endocytosed in a clathrin-independent manner. Our lab showed that bacterial and viral lectins (Shiga toxin B-subunit = STxB, VP1 protein from SV40) are able to induce plasma membrane bending by clusterization of their glycosphingolipid receptors (1,2). The produced tubular invaginations undergo scission via a process involving actin polymerisation (3). In order to discover new actors involved this clathrin-independent endocytic mechanism, we screened cDNA libraries to identify proteins localized on STxB-induced membrane invaginations, or perturbing their formation. We focused especially on the BAR domain protein family of curvature sensors. Amongst the 20 BAR domain proteins analyzed, only Endophilin A2 (EA2) had a clear effect on STxB-induced membrane invaginations. Overexpression of EA2 induced a decrease of their length and density, while EA2 depletion had the opposite effect. EA2 depletion also inhibited retograde transport of STxB to the Golgi apparatus. Diverse microscopy techniques allowed us to show that STxB was internalized in EA2 positive structures. Release of EA2 sequestered at the mitochondria by the “knocksideways” technique (4) induced a decrease of STxB-invagination size. Purified EA2 will allow us to determine if the protein alone is able to favour scission of STxB-induced invaginations in a model membrane system (ongoing). Taken together, our data suggest a role for EA2 in the scission process of STxB-induced invaginations. This could be a direct effect via insertion of amphipathic helices in the membrane (5). We also propose that EA2 might be an adaptor for actin recruitment onto STxB-induced invaginations. (1) Römer W et al. 2007. Nature. 450(7170):670-5. (2) Ewers H et al. 2010. Nat Cell Biol. 12(1):11-8. (3) Römer W et al. 2010. Cell. 140(4):540-53. (4) Robinson MS et al. 2010. Dev Cell. 18(2):324-31. (5) Boucrot E et al. 2012. Cell. 149(1):12