Control of vesicular flows by golgins – VesicleFiltering

Golgins are very long coiled-coil proteins that are anchored through their C-ter at the Golgi apparatus where they form a matrix of flexible strings whose N-ter end projects towards the cytosol. Golgins act as tethers between transport vesicles and Golgi cisternae and control the traffic of multiple cargoes within the cell. The function of some golgins is exacerbated in differentiated cells where golgin deletion leads to severe phenotypes. However, our understanding of the mechanisms and functions of golgins remains fragmental. The Antonny lab has previously shown that the golgin GMAP-210 selectively captures vesicles by a mechanism that departs from classical protein-protein or protein-lipid interactions. GMAP-210 harbors an amphipathic N-terminal ALPS motif, which binds preferentially to highly curved membranes enriched in unsaturated lipids. This observation suggests that GMAP-210 recognizes transport vesicles on the mere basis of their physicochemical features (high curvature and abundance of monounsaturated lipids). More recently, the Hivroz laboratory has uncovered a key role for GMAP-210 in the transport of the transmembrane adaptor protein LAT (linker for activation of T cells) between the Golgi apparatus and the immune synapse in T cells. Interestingly, two features of this transport echo back to the mechanism of membrane recognition by the ALPS motif. First, LAT vesicles are very small. Second, expressing the ALPS motif of GMAP-210 prevents the transport of LAT in a heterologous system, the primary cilium, which shows analogies with the immune synapse and which is very sensitive to changes in the lipid saturation/unsaturation ratio.

In this project, the Antonny, Hivroz and Perez groups team up to perform an in-depth study of the links between vesicle selection by golgins, membrane physicochemical features, and cellular functions. Our main questions are: do other golgins recognize transport vesicles according to their membrane features? Does the mechanism of vesicle capture by the ALPS motif of GMAP-210 explain the role of this golgin for the formation of the immune synapse? To address these questions, we will use approaches ranging from reconstitution experiments with purified proteins and liposomes of defined composition, to real-time cellular assays to follow cargo transport under conditions of modified lipid homeostasis in physiologically relevant cell systems. Our project includes four tasks. Task 1 (Antonny/Perez) is a comprehensive analysis of the binding of the N-terminus of golgins to lipid membranes of defined lipid composition and curvature. In task 2 (Perez/Antonny), we will determine the sensitivity of the transport of model cargoes at the Golgi to changes in the golgin repertoire and in membrane lipid composition. For this, the Perez lab will use and further develop a cellular system (RUSH) that allows for the synchronisation of the transport of fluorescent cargos, hence leading to quantitative and time-resolved measurements of their flow along the secretory pathway. In task 3 (Hivroz/Antonny), we will determine the mechanism of selective capture of LAT vesicles by GMAP-210 for the formation of the immune synapse. In task 4 (Hivroz/Perez/Antonny), we will use the primary cilium as a model structure to test the importance of membrane physicochemical features for the selective cargo transport by golgins. By covering a large range of analysis, from molecular interactions to physiological functions, our research should lead to a better understanding of the links between vesicular traffic, golgins, lipid metabolism, and the contrasting physicochemical properties of cellular membranes.