The bridge and the shuttle : structure and dynamics of the membrane contact site formed by the sterol/PI(4)P exchanger OSBP – SwingBridge

we will explore OSBP-mediated MCS by two approaches: 1. Cryo-EM to get structural information (Partner 1). The goal is to determine the first 3D model of MCS. 2. Real time measurements to get information on dynamics of assembly/disassembly of MCS and of lipid transfer (Partner 2). In both cases, we will design new in vitro membrane systems. We rely on our complementary expertise in cell biology, membrane biochemistry and structural biology as well as on solid preliminary data.

experiments in progress

Contacts between cellular compartments membranes and especially between the ER where lipid are synthetized and other organelles are identified and their major role in lipid homeostasis is accepted. Disorders of this great cell function are increasingly cited in serious human diseases and some involved proteins are already identified as target of pharmacophores. The clinical translation is not yet accomplished and the field continuously feeds of fundamental advances. There is a network of results concerning various proteins involved in the MCS and structural and functional homologies are being understood. This project addresses two issues that hinder the understanding of the dual function of MCSs between ER and trans-Golgi. First visualizing, at the molecular level, these MCSs must fill the incomplete 3D models built on atomic data of sub-domains and establish the structure-function relationships necessary to understand the roles of each protein component in the tethering and in the transport of lipids. Second, this 3D model only makes sense if it is correlated with the dynamics, at the molecular level, of assembly and disassembly of MCSs which is a key element of their function during cell life. These two issues are common to other MCSs and given the strong homology between lipid transfer proteins, it is very likely that our results will be universal.

in preparation

Cellular compartmentalization is a hallmark of eukaryotic cells. It allows specialized functions to be confined in a specific environment. However, it also implies exchange of material between compartments. Intracellular trafficking mediated by vesicles contributes to these exchanges and the associated mechanisms have been heavily explored (Nobel Prize in Physiology and Medicine 2013). Another important mechanism supported by recent studies involves the formation of membrane junctions by which organelles communicate without membrane fusion. These narrow (<30 nm) cytosolic gaps, refereed as to membrane contact sites (MCS), are of key importance for intracellular lipid transport. They also play significant roles in intracellular signaling, organelle inheritance, and lipid metabolism and have received considerable attention in recent years due to their implication in some metabolic diseases.
MCS are conserved in all eukaryotic cells and generally join the endoplasmic reticulum (ER) with other organelles. As a result, a newly biosynthesized lipid molecule in the ER can rapidly reach another organelle by trafficking through a MCS. Lipid transfer proteins present in MCS have recently been identified as the intracellular targets of several anticancer and antiviral compounds, emphasizing their important role in cellular homeostasis. Not surprisingly, some intracellular bacterial pathogens hijack these proteins to exploit their lipid transport activities.
Cell biology and electron microscopy studies have delineated the key characteristics of MCS: 1. MCS are associated with specific functions including lipid transfer, calcium or apoptosis signaling. 2. MCS consist in assemblies of integral or membrane bound proteins present on each side of membranes and joined by cytoplasmic domains. 3. MCS are highly dynamic and regulated.
Current challenges in MCS research are thus clearly identified. At the structural level, the structure of some individual soluble proteins or domains has been solved but a structural description of complete architecture of MCS is lacking. Cryo-electron microscopy (cryo-EM), the expertise of the Partner 1 (Institut Curie, Paris), is the most suitable structural approach to visualize the full protein complex between two facing membranes. Moreover, MCS are now accessible to high resolution cryo-EM thanks to the current resolution in resolution. At the functional level, the dynamics and regulation of these junctions remain to be explored. Resolving these two issues requires the design of new cellular and in vitro assays adapted to the confined environment of MCS.
The Partner 2 (IPMC, Valbonne) has recently reconstituted the first functional MCS (Mesmin, Cell 2013). This MCS bridges the endoplasmic reticulum to the trans side of the Golgi apparatus and consists of a complex between oxysterol binding protein (OSBP), the ER protein VAP-A and the Golgi protein Arf1-GTP.
Based on this finding, we will explore OSBP-mediated MCS by two approaches: 1. Cryo-EM to get structural information (Partner 1). The goal is to determine the first 3D model of MCS. 2. Real time measurements to get information on dynamics of assembly/disassembly of MCS and of lipid transfer (Partner 2). In both cases, we will design new in vitro membrane systems. We rely on our complementary expertise in cell biology, membrane biochemistry and structural biology as well as on solid preliminary data. Deciphering the functional and structural principles underlying the action of OSBP will have a global impact, given specific domains among lipid transfer proteins are conserved. The tools and concepts developed here will also enrich the current vision of the dynamics of intracellular compartments and should trigger significant findings in pharmacological therapies of associated diseases. Thus, this proposal is in line with the goals of “Défi 4, Axe 1” for decrypting the spatial and temporal multi-scales mechanisms of the cell.