One of the hallmarks of eukaryotic cells is their internal membrane compartmentalization, fundamental to normal cell function. Many human diseases result from deregulation of pathways linking membrane-bound organelles. The best-studied mechanism of communication between organelles is vesicle-mediated trafficking. However, recently we have begun to appreciate the significance of information transfer between intracellular organelles at membrane contact sites (MCS). MCS are zones where heterologous membranes, usually the endoplasmic reticulum (ER) plus a partner organelle, come into close apposition. These sites of intimate contact are places where lipids and small molecules such as calcium are exchanged. However, MCS are very poorly understood because so few of their components have been identified so far. Moreover, our understanding of the function of lipid trafficking at MCS, and how it is integrated into other cellular communication systems, is in its infancy.
Contacts between the ER and PM are particularly intriguing, as they affect PM signaling, endocytosis, phagocytosis, cell migration and other processes important for cellular functions during development and in specialized cell types such as neurons, adipocytes and immune cells. My studies have identified new components of ER-PM MCS, the ER-localized extended-synaptotagmin proteins (E-Syt1, E-Syt2 and E-Syt3) whose overexpression induces formation of cortical ER while their loss of function reduces the number of ER-PM contacts. One of the important conclusions of my studies on the E-Syts is that they form a novel class of ER-PM contacts. However their functions remains to be elucidated. E-Syts have multiple lipid binding domains, including an SMP domain and multiple C2 domains. The last C2 domain of E-Syt2 and E-Syt3 bind specifically and with high affinity to PI(4,5)P2, thus bringing the ER to the PM. The SMP domain is predicted to adopt a hydrophobic lipid-binding tunnel structure, and possibly function to transfer lipids between membranes. The central objective of this proposal is to understand the functions of E-Syts at ER-PM contact sites in mammalian cells. Our working hypothesis is that regulation of PM lipid metabolism is a key function of E-Syt-mediated ER-PM contact.
My preliminary results have revealed interactions between E-Syts and proteins involved in lipid metabolism, including an ER-localized phosphatidylserine synthase and two members of the oxysterol-binding protein (OSBP)-related protein (ORP) family. One of these ORPs has recently been shown to bind to phosphatidylserine rather than sterols, and likely transports this phospholipid using a PI4P exchange mechanism similar to that demonstrated for Osh4 and OSBP. These exciting preliminary results suggest a function for the E-Syts in coordinating multiple lipid synthesis and transfer activities at ER-PM MCS, opening the door to novel lipid-based regulatory mechanisms for PM membrane remodeling processes. I will explore whether the presence of these contact sites might regulate endocytosis and phagocytosis, for example by locally altering PM lipid composition. PI(4,5)P2 is required at multiple stages during clathrin-mediated endocytosis, binding to numerous endocytic adaptors and regulators. Indeed, my preliminary studies have shown that EGF receptor endocytosis is controlled by the E-Syts. I will pursue these initial studies by completing screens for interacting partners, and validating partners identified. I will also determine whether E-Syt-mediated ER-PM contacts regulate the lipid composition of the PM, and elucidate the mechanisms involved. Using my expertise in electron microscopy, live cell imaging and fluorescence microscopy, optogenetics, as well as biochemical approaches, I will study the roles of E-Syts and their lipid regulatory functions in PM remodelling processes including endocytosis and phagocytosis in macrophages.
Institut Jacques Monod (Laboratoire public)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
Institut Jacques Monod
Help of the ANR 261,604 euros
Beginning and duration of the scientific project: December 2014 - 48 Months