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Biochemical and functional characterization of ER-plasma membrane contact sites mediated by a novel class of tethering proteins, the Extended-Synaptotagmins

Periodic Report Summary 2 - ERPMCONTACTS (Biochemical and functional characterization of ER-plasma membrane contact sites mediated by a novel class of tethering proteins, the Extended-Synaptotagmins)

Close proximities between organelles have been described for over 50 years. However, only in the last decade a specific field dealing with organelle communication at membrane contact sites, or zones where the membranes of two different organelles come in a very close apposition without fusing, has gained wide acceptance. The endoplasmic reticulum (ER), the major site of lipid synthesis in eukaryotic cells, forms a membrane network that spreads throughout the cell, and makes contact with virtually every organelle including the plasma membrane (PM). Membrane contact sites have been shown to be important places where small molecules such as calcium and/or lipids can be exchanged. As molecular determinant have started to be uncovered, membrane contact-sites are more widely becoming implicated in a variety of cellular and pathophysiological processes and are becaming center-stage in cell biology research. However, membrane contact sites are still not well understood because most of their components still remain to be identified. Moreover, our understanding of the function of lipid trafficking at membrane contact sites, and how it is integrated into other cellular communication systems, is in its infancy.
I have previously identified the ER-anchored extended-synaptotagmin proteins (E-Syts) as components of a novel class of ER-PM contacts, functionally distinct from the ones responsible for capacitive calcium-entry. Recent studies have started to uncover a role of E-Syts in lipid transport, however E-Syts functions at ER-PM contacts and how they are regulated remain to be elucidated. The original objective of my project, carried out in collaboration with T. Galli’s group, was to identify interacting partners of E-Syts and study how they work all together to regulate lipid transport and related cellular processes at ER-PM contact sites in mammalian cells. Our biochemical data uncovered a novel interaction between E-Syts and a newly-identified non-fusogenic Sec22b/Stx SNARE complex, in both neuronal and non-neuronal cells. Our new data reveal a role of E-Syts in membrane expansion and neurite growth, dependent on their lipid transfer function. Moreover, in a collaborative study with E. Morel and coworkers we have uncovered an additional function of the E-Syt-mediated ER-PM contact sites in the regulation of autophagy in mammalian cells.
In the course of this project I have also found a novel interaction between E-Syts and the Oxysterol-binding proteins ORP5/8 recently shown to transport phosphatydilserine (PS) from the ER to the PM. Interestingly, I have discovered that ORP5/8 additionally localize at ER-mitochondria contacts where they are required to maintain mitochondria morphology and respiratory function. The new finding that ORP5/ORP8 localize also at ER-mitochondria contact sites represented the starting point of a parallel study that I leaded and carried out since I was at the Institut Jacques Monod and that I pursued after moving to the I2BC, where I established my new indipendent team through an ATIP-Avenir program. PS, synthesized in the ER, is transferred to mitochondria membranes for the synthesis of mitochondrial PE, a phospholipid that plays an essential role in maintaining mitochondria membrane integrity and respiratory function. We hypothesized that ORP5/ORP8 are the lipid transfer proteins involved in transport of PS at ER-mitochondria contact-sites, which identity was still unknown.
By using a combination of in vitro biochemical and in situ cell biology and imaging approaches including electron microscopy, we have revealed that ORP5 and ORP8 are the lipid transfer proteins mediating PS transfer from the ER to the mitochondria at ER-mitochondria contact sites. Moreover, we have found that they cooperate with the intra-mitochondria membrane contact sites MICOS complex, for efficient transport of PS to the inner mitochondria membrane, where it is converted in PE. Our study represents the first evidence that ER-Mitochondria contact sites and intra-mitochondrial contact sites are physically and functionally connected, and that they cooperate for efficient non-vesicular lipid transport.
Overall, these exciting results suggest a function for E-Syt, ORPs and for their binding partners in coordinating many lipid transfer activities at multiple inter-organelle membrane contact sites, opening the door to novel lipid-based regulatory mechanisms for membrane remodelling processes in a new inter-organelle network of cooperative activities. Given the important role of mitochondria and PM in many physiological processes, it is not surprising that dysfunction of these organelles/membranes and associated membrane contact sites are linked to numerous pathological conditions including obesity, diabetes, lipodistrophy, neurodegenerative disorders and cancer. ORPs are also the target of the “ORPphilins”, natural products that have been shown to strongly inhibit growth of cancer cell lines in culture, and ORP5 is highly expressed in some tumors. Hence, we believe that the research associated to this project will have a major impact not only in the lipid trafficking and membrane contact sites field, but also on our understanding of the pathophysiological mechanisms of metabolic diseases.