Molecular mechanisms of secretory granule membrane endocytosis in neuroendocrine cells: functional interplay between lipids, actin cytoskeleton and endocytic molecules – granule-endo

All mammalian cells exhibit complex and dynamic patterns of intracellular membrane traffic which are required not only for the survival of individual cells, but also allow their assembly into complex multicellular structures. Although much has been learned concerning the mechanisms of transport vesicle formation and vesicle fusion at donor and acceptor compartments, relatively little attention has been paid to understanding how cells maintain a balance of vesicular traffic between any two organelles. In secretory cells for example, endocytosis and exocytosis must be inexorably linked, but have generally been studied as separate processes. Recently, studies of dense-core granule exocytosis in neuroendocrine cells have suggested that granule release is tightly coupled to endocytosis both temporally and spatially. Hence, integrity of the granule membrane must be preserved at the exocytotic site after release of vesicular contents and the granule membrane subsequently retrieved per se. However, the molecular mechanisms underlying secretory granule endocytosis are largely unexplored today. Moreover, in contrast to the extensively studied receptor-mediated endocytosis process, the signal triggering granule membrane internalisation after exocytosis is currently unknown. The major goal of this project is to determine how granule endocytosis is coupled to exocytosis in neuroendocrine cells. To approach this challenging task, we will address these specific questions: i) how are granule membrane components maintained together at the plasma membrane during and after exocytosis? ii) What is the molecular pathway controlling subsequent retrieval of the granule membrane? iii) What is the signal that triggers secretory vesicle internalisation in such specialized secretory cells? Physical properties of individual lipids, as well as the cytoskeleton play fundamental roles in membrane trafficking by acting as scaffolding system to maintain specific machinery at restricted sites of the plasma membrane. Phosphatidylserine along with its carrier enzyme, scramblase, as well as actin filament dynamics regulated by the Rho GTPases represent attractive candidates to fulfill this function during exo-endocytosis events in neuroendocrine cells. With regards to the molecular mechanisms that control compensatory endocytosis in neuroendocrine cells, the involvement of clathrin coat and dynamin need to be firmly established. Finally, in view of their important implication in both exo- and endocytotic processes, bi-functional molecules like intersectin-1L and synaptotagmin may be potential signals triggering granule endocytosis. We propose here to investigate the endocytotic process and underlying regulatory mechanisms governing granule membrane retrieval in neuroendocrine cells using an innovative multidisciplinary approach which combines new assays to follow granule internalisation, electron microscopy and Total Internal Reflection Fluorescent Microscopy (TIR-FM) experiments with novel computational analysis method. This will allow us to visualize, measure and quantify regulated exocytosis and endocytosis simultaneously and with sufficient resolution to test the roles of actin and phospholipid dynamics and other potential mediators, like intersectin and synaptotagmin in the spatial restriction of these two events. This synergistic strategy will not only provide new insights into exo- and endocytic coupling, but will create general novel tools and approaches for understanding the complexity of membrane trafficking.