Final Report Summary - RETROMER (The mechanism of retrograde trafficking by retromer.) PROJECT-IGalectin-3 drives glycosphingolipid-dependent biogenesis of clathrin-independent carriers for retrograde trafficking. (Published in Nature Cell Biology) SUMMARYSeveral cell surface molecules including signaling receptors are internalized by clathrin-independent endocytosis. How this process is initiated without a cytosolic coat structure, how cargo proteins are sorted and membranes are bent to build endocytic pits has therefore been one of the challenging questions in the field of endocytosis. Here, we found that a carbohydrate-binding protein, galectin-3 (Gal3), triggered the glycosphingolipid (GSL)-dependent biogenesis of a morphologically distinct class of endocytic structures, termed clathrin-independent carriers (CLICs). Super-resolution and reconstitution studies showed that Gal3 required GSLs for clustering and membrane bending. Gal3 interacted with a defined set of cargo proteins. Cellular uptake of the CLIC cargo CD44 was dependent on Gal3, GSLs and branched N-glycosylation. Endocytosis of beta1-integrin was also reliant on Gal3. Analysis of different galectins revealed a distinct profile of cargoes and uptake structures, suggesting the existence of different CLIC populations using the same mechanism. Based on our results, a novel endocytic mechanism emerged in which galectins function like an endocytic adaptor by linking carbohydrate specificity on cargo proteins and GSLs into compositionally defined nanoenvironments at the plasma membrane in order to drive clathrin-independent plasma membrane bending as a first step of CLIC biogenesis. The mechanistic discovery of endocytosis of endogenous lectins will bath the way to design synthetic lectins, which are targeted to specific tumor cells with modified carbohydrate surface pattern. Those synthetic lectins might be used as probe to detect tumor cells by clinical imaging techniques or as carrier for specific drug delivery. BACKGROUNDMechanistically, endocytic events can be subdivided into clathrin-dependent and clathrin-independent processes1-6. Cargoes that are internalized in the absence of clathrin include endogenous surface molecules such as glycosylphosphatidylinositol (GPI)-anchored proteins, CD44, major histocompatibility complex (MHC) class I molecules, interleukin 2 (IL-2) receptor, and exogenous ligands such as the bacterial Shiga and cholera toxins and simian virus 40. A large fraction of fluid phase is also internalized by clathrin-independent endocytosis. CD44, cholera toxin and fluid-phase markers were mapped to early internalization structures with a distinct morphology, termed clathrin-independent carriers (CLICs). CLICs arise directly from the plasma membrane, mature into the GPI-enriched early endosomal compartments, and subsequently merge with early endosomes. Highly organized electron dense coat structures could not be detected at sites of membrane invagination in clathrin- and caveolin- independent uptake processes. How cargo proteins are sorted and membranes are bent to build endocytic pits in these cases has therefore been one of the challenging questions in the field of endocytosis. In mammals, the 14 members of the galectin family of N-glycanbinding proteins have functions in cancer, immunity, inflammation and development. Within this family, galectin-3 (Gal3) is unique in that it combines a carbohydrate recognition domain in its carboxy terminus with an amino-terminal non-lectin domain that favors the formation of Gal3 oligomers. Branched N-acetylglucosamine saccharides that result from the activity of the Golgi-localized beta-1,6-N-acetylglycosaminyltransferase V (Mgat5) are preferred binding determinants for Gal3 in the formation of an extracellular galectin-glycoprotein lattice, which regulates receptor tyrosine kinase signaling, cell migration and fibronectin fibril formation. The availability of Gal3 in tissues is controlled through expression and atypical secretion. Gal3 expression is deregulated in human cancers, and it has been suggested that Gal3 could be a tumourmarker. Binding to GSLs has been demonstrated for galectin-4 and galectin-9, and suggested for Gal3. The biological functions of these GSL interactions have remained elusive. GSLs are present in all mammalian cell types, throughout the animal kingdom, in bacteria, fungi and plants. Apart from being cellular receptors for pathogens and pathogenic molecules they are critical for cell adhesion, migration and signalling. Identifying mechanism(s) by which GSLs regulate these cellular functions is one of the major challenges in membrane biology research.PROJECT-IIGal-3 requires glycosphingolipids for cell migration and fibrillogenesis. (Revised manuscript according to comments of the referees will be submitted to PLOS ONE)This work showed the physiological relevance of PROJECT-I. SUMMARYWe demonstrated a physiological link between Gal3 and GSLs in cell migration and fibrillogenesis. GSLs were essential for Gal3 mediated regulation of cell migration and fibronectin remodeling. DIRECTION AND ANIMATION MANAGEMENTProject-I and -II were supported by a research engineer, who works under the supervision of the fellow. Project-I was done together with a PhD student (Ramya Lakshminarayan) and a post-doc (Ulrike Becken) of our group. Both contributed equally to the work published in Nature Cell Biology. The team leader, Ludger Johannes and the grant holder were responsible for guidance of both contributors.The manuscript for Project-II is in its final stage and a revised version (incl. rebuttle) will be submitted to PLOS One. The grant holder is corresponding author.In order to continue Project-I on a more mechanistical basis a physicist was hired. He is supporting the biophysical and future superresolution experiments. The superresolution project will be further supported by a newly hired chemist (post-doc). He was trained in DNA-nanotechnology, which will be used to generate cargo molecules for single particle tracking. ANIMATIONCollaboration – several collaborations supported each projectProject-I:• 3D EM-tomography expertise of Rob Parton’s group, (Queensland, Australia)• Super resolution microscopy of Katharina Gaus’s team (Sydney, Australia)• Mass spectrometry service of Damarys Loew’s group (Institute Curie, Paris)• Lipidomics by Andrej Shevchenko (Max-Plack Institute CBG, Dresden, Germany)Project-II:• Fibrillogenesis expertise of Ivan R. Nabi’s team (Vanvouver, Canada)• Cell migration expertise of Phillipe Chavrier’s team (Institute Curie, Paris) • The superesolution team of Melike Lakadamyali for STORM imaging (Barcelona, Spain)TeachingThe grant holder was successfully integrated into the teaching system of the Curie Institute and presented seminars and practical courses for Master students, PhD-students and Postdocs at the Pasteur Institute and Institut Jacques Monod.• Supervisor of the practical course “Molecular Biology of the cell” Bacterial toxins and retrograde trafficking in health and disease Pasteur Institute, Paris, France 2011- 2014• Advanced Course in Cellular Dynamics, Master-2, PhD-students Talk: Retrograde trafficking – Mechanism, Function, Application Institut Jacques Monod, Paris, France 2011-2014Furthermore, the grant holder disseminated his knowledge about in vitro reconstitution of protein complex on giant unilamellar vesicles within an INSERM workshop:• Supervisor of the INSERM Workshop “Membrane domains: Translating compositional complexity into biological functions“ 11/2013Advisory panels Evaluation expert for ERA: FP7-PEOPLE, FP7-CIG, Horizon-2020-People 2012-2014 External evaluation expert for LIGUE CONTRE LE CANCER Finally, the grant holder started the process of habilitation (HDR) at University Paris VII.
