ER-driven Contact Sites in Autophagosome Biogenesis Sequence

Eukaryote cells depend on membrane lipid trafficking from biogenic membranes, like the endoplasmic reticulum (ER), to other membranes in the cell. Two major routes for membrane lipid transport are recognized: vesicular trafficking and lipid transfer at zones of close contact between membranes. Specific ER regions involved in such membrane contact sites (MCSs) have been isolated, and lipid transfer at MCSs as well as protein-protein interactions between the partaking membranes have been demonstrated (reviewed by Holthuis, J. C. M., and Levine, T. P. (2005) Nat. Rev. 6, 209-220). Here we present the first demonstration of the physical association between membranes involved in MCSs: by using optical imaging and manipulation, strong attracting forces between ER and chloroplasts are revealed. We used Arabidopsis thaliana expressing green fluorescent protein in the ER lumen and observed leaf protoplasts by confocal microscopy. The ER network was evident, with ER branch end points apparently localized at chloroplast surfaces. After rupture of a protoplast using a laser scalpel, the cell content was released. ER fragments remained attached to the released chloroplasts and could be stretched out by optical tweezers. The applied force, 400 pN, could not drag a chloroplast free from its attached ER, which could reflect protein-protein interactions at the ER-chloroplast MCSs. As chloroplasts rely on import of ER-synthesized lipids, we propose that lipid transfer occurs at these MCSs. We suggest that lipid transfer at the MCSs also occurs in the opposite direction, for example to channel plastid-synthesized acyl groups to supply substrates for ER-localized synthesis of membrane and storage lipids.

Membrane contact sites (MCSs) are fundamental hubs of inter-organelle communication that mediate the non-vesicular exchange of lipids, ions, and metabolites, thereby sustaining cellular homeostasis. In plants, the "contactome"-the dynamic network of all membrane contact sites-has evolved distinctive features to accommodate the requirements of a sessile, photosynthetic lifestyle and the presence of plastids. Within this network, the endoplasmic reticulum (ER) functions as a central hub for lipid biosynthesis and distribution, forming functionally important contacts with multiple organelles. Recent advances in high-resolution imaging, lipidomics, and molecular genetics are beginning to uncover the complexity of these inter-organelle connections and their contribution to lipid homeostasis in plants. This review summarizes current knowledge of the plant contactome, with a focus on lipid transfer proteins and lipid-modifying enzymes that maintain lipid balance during organelle biogenesis, plant development, and stress adaptation. Plant lipid transfer at membrane contact sites can be broadly divided into two mechanistic modes: precision-regulated "shuttles," exemplified by the Ca2+-dependent SYT1-mediated diacylglycerol transfer at ER-plasma membrane interfaces, and high-capacity lipid transfer mechanisms, such those mediated by ATG2, that support rapid lipid flux during autophagosome biogenesis. Knowledge of lipid metabolism at plant membrane contact sites is still in its initial stages, and many of the underlying mechanisms remain unexplored. Major challenges include understanding how these sites integrate stress responses, metabolic fluxes, and organelle dynamics. Addressing these questions will be essential to unravel the unique aspects of plant lipid biology and may open opportunities for improving stress resilience and metabolic engineering in crops.

Lipid transport between membranes within cells involves vesicle and protein carriers, but as agents of nonvesicular lipid transfer, the role of membrane contact sites has received increasing attention. As zones for lipid metabolism and exchange, various membrane contact sites mediate direct associations between different organelles. In particular, membrane contact sites linking the plasma membrane (PM) and the endoplasmic reticulum (ER) represent important regulators of lipid and ion transfer. In yeast, cortical ER is stapled to the PM through membrane-tethering proteins, which establish a direct connection between the membranes. In this review, we consider passive and facilitated models for lipid transfer at PM–ER contact sites. Besides the tethering proteins, we examine the roles of an additional repertoire of lipid and protein regulators that prime and propagate PM–ER membrane association. We conclude that instead of being simple mediators of membrane association, regulatory components of membrane contact sites have complex and multilayered functions.

Autophagy is a tightly regulated process that eukaryotic cells use as a major survival mechanism to reallocate nutrients to essential processes in adverse conditions such as nutrient or energy deprivation. Autophagy is characterized by the formation of a phagophore, a double membrane organelle that matures into an autophagosome to capture damaged or surplus materials in the cytosol and deliver them to the lysosome for degradation and recycling. Yet, how the autophagosome is generated de novo remains a long-standing question in biology. Research in the last decades has suggested that autophagosome biogenesis requires the transfer of lipids from the endoplasmic reticulum (ER) to the nascent autophagosome (phagophore), which happens at the membrane contact site (MCS) between the two organelles. Several core autophagy initiation complexes and a lipid transfer machinery are recruited to the MCS to modulate autophagic membrane formation and elongation. However, profound questions remain: How does the MCS assemble at the right place and time? Is there a regulatory mechanism? How is lipid transfer modulated to support phagophore elongation? Answering these questions would provide fundamental insight into the mechanisms of autophagosome biogenesis. In this thesis, biochemical reconstitution is used as a reductionist approach to address these questions. One of the main challenges of this approach is the production of recombinant proteins. Here, we meet this challenge by purifying almost all full-length proteins of the core autophagy initiation complexes (ULK1 complex, PI3K complex 1, ATG9) and the lipid transfer unit (ATG2-WIPI4). Interestingly, we were able to reconstitute a seven-subunit autophagy initiation super-complex and found that a three-subunit complex of ATG9-ATG13-ATG101 serves as a core complex for the assembly of other four subunits, including ULK1, FIP200, ATG14L, and BECN1. Data from our lab also shows that ATG13 and ATG101 are metamorphic proteins, and their metamorphoses result in an incredibly slow self-assembly of the core complex. The slow assembly of the core complex thus acts as a rate-limiting step in the assembly of the super-complex and raises the possibility of a regulatory mechanism for on-demand assembly of the super-complex upon autophagy induction. Moreover, I found that the core complex also interacts with the lipid transfer unit, ATG2-WIPI4, to form a five-protein subcomplex. ATG2-WIPI4 was previously found to tether membranes and mediate lipid transfer at the MCS. Surprisingly, the lipid transfer efficiency of the lipid transfer unit can be significantly enhanced by both ATG9 and ATG13-ATG101 of the core complex. In summary, our findings pave the way for mechanistic models that explain how autophagosome biogenesis is regulated in space and time and how the co-incidence of the different functional complexes supports autophagosome expansion.

Lipid transfer proteins (LTPs) play a critical role in distributing lipids within eukaryotic cells. In yeast, Osh6 and Osh7, which belong to the oxysterol-binding protein-related protein family, transfer phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) in exchange for phosphatidylinositol 4-phosphate (PI(4)P). These proteins localize at ER–PM contact sites by associating with Ist2, an ER-resident TMEM16-like protein that bridges the ER and PM via a long intrinsically disordered region (IDR). Recent studies have shown that this association ensures accurate PS transfer by concentrating Osh6 and Osh7 at the ER–PM interface while preserving their ability to access both membranes. However, it remains unclear how these LTPs function when bound to the Ist2 IDR, whose length far exceeds the ER–PM distance at contact sites, and why they do not integrate both the tethering and the PS/PI(4)P exchange functions, like their human homologs. Additionally, it has been revealed that Ist2 can transfer lipids across the ER membrane via a scramblase activity. Yet, whether and why this activity is coupled to the PS/PI(4)P exchange activity of Osh6 and Osh7 remains unknown. The Ist2–Osh6/7 system emerges as a fascinating model that integrates tethering, scramblase, and lipid exchange functions. Future studies of this system are likely to provide important insights into how lipid transfer processes are coordinated at membrane contact sites.

Calcium ions play an important role as secondary messenger in cell biology therefore, regulation of its cellular concentration is key to maintain homeostasis and trigger signaling events. A multitude of pathways have been developed during evolution to tightly control the in and out of cellular calcium ions. Store-operated calcium entry (SOCE) is a major signaling pathway involved in proper development of muscle cells and immune system activation. During SOCE, an agonist stimulation generates inositol trisphosphate (IP3) from phosphatidylinositol 4,5-biphosphate (PIP2) hydrolysis. Following, IP3 induces depletion of endoplasmic reticulum (ER) calcium store through IP3 receptor (IP3R). Stromal interaction protein 1 (STIM1), an ER transmembrane protein, senses calcium depletion through its luminal domains which induce a conformational change exposing key cytosolic domains. STIM1 new intramolecular arrangement favors its oligomerization and in turn its translocation to cortical ER (cER), which is closely apposed to plasma membrane (PM). Within those ER-PM junctions, STIM1 binds and gate ORAI1 calcium channel on the PM and induce calcium entry. Refilling of the ER store and cytosolic calcium elevations are responsible for SOCE termination due to loss of STIM-ORAI complexes though calcium dependent inactivation processes. Physical interaction between STIM1 and ORAI1 occurs at ER-PM junctions where the gap distance is permissive and ranges from 10 nm to 25 nm. ER-PM junctions are part of the membrane contact sites (MCS) family which are membranes in close apposition between different organelles or subcellular structures, with an intermembrane distance usually below 30 nm. Interestingly, ER-PM contact sites play an important role in sustaining calcium signaling through replenishment of PIP2 in the PM. MCS are dynamic structures using tether proteins to adapt to cellular needs and are involved in the shaping of physiological responses. During SOCE, cER length increases in a STIM1 dependent manner and global coverage of ER-PM junctions increases by ~5 fold. Meanwhile, ER-PM gap distance is shortened from 21.8 nm to 14.8 nm by recruitment of ESyt1, an ER resident tether protein activated by calcium entry. MCS structure and function have been dependent one to another, however, a unifying model is missing. The first part of my thesis focuses on understanding of the relationship between SOCE and ER-PM contact site ultrastructure. To this end, we modified morphometric parameters of ER-PM contact sites by expressing known tether proteins (ESyt1 and ESyt2) or synthetic constructs from MAPPER family (MAPPER-Short and MAPPER-Long). Using electron microscopy (EM), we assessed ER-PM contact morphology upon expression of tether proteins. cER length and its associated ER-PM distance have been systematically measured and correlated at different time points of SOCE. Both parameters combined revealed that ER depletion is increasing cER length from 66 nm to 156 nm without significantly impacting gap distance. On the other hand, expression of tether proteins amplifies cER elongation upon calcium depletion. It is particularly dramatic with ESyt2 and MAPPER-Long which generate contacts longer than 1.5 µm. Additionally, these long contact sites were stabilized to an ER-PM distance specific to each tether family. As ESyt2 and MAPPER-L behave similarly at morphometric level, they were associated with a ~50% reduction of calcium entry in multiple cellular systems. These results support a role for ER-PM ultrastructure in regulation of SOCE through recruitment of tether proteins. Microscopy imaging is part of the core techniques used in the study of calcium signaling and MCS. Generation of high number of images represents new challenges in the way we deal with information and pipelines are now more and more essential for a smooth and reliable data processing. New approaches are developed every day to increase workflow from acquisition to extraction of key features from data. Due to the high amount of data generated in this thesis I included as a second part the development of new analytic tools to create a powerful and reliable pipeline for calcium, TIRF and ER-PM junction analysis. The objectivity and time saving opportunities offered by this pipeline are substantial and its reliability makes it used by most of my co-workers and even some collaborators. I additionally generated custom strategies to overcome limitation we were facing in data analysis of our microscopy timelapses. We could extract information that would have been out of reach without help from these analytic methods. Overall, using electron microscopy and calcium imaging with my pipeline I can conclude that cER expansion during SOCE is mediated by STIM1 and is enhanced by tether proteins recruited to contact sites. This might have potential application for regulation of SOCE during immune cell activation and open the way to a better understanding of how cells regulate their signaling through the MCS ultrastructure.

Endoplasmic reticulum-plasma membrane (ER-PM) contact sites play important roles in maintaining lipid homeostasis at the plasma membrane (PM), cellular calcium homeostasis, and cell signaling. Here, we show that MCTP1 and MCTP2 are at ER subdomains that form membrane contact sites (MCS) with multiple organelles using a proximity labeling assay. MCTPs are three C2 domain-containing transmembrane proteins. We show that upon overexpression, MCTPs promote ER-PM contact sites in a C2 domain-dependent manner. MCTP C2 domains bind to PI(4)P and PI(4,5)P2, phosphoinositides that are enriched in the PM. Furthermore, we show that deletion of MCTP1 or MCTP2 increases PI(4)P levels in the PM and promotes cell migration. Thus, our study identifies MCTPs as multiple ER-organelle contact site proteins and establishes its role at ER-PM contact sites in regulating lipid homeostasis and cell migration.

Membrane contact sites (MCS) are specialized structures where the endoplasmic reticulum (ER) and other organelle form come in close proximity (~10–30 nm). Functionally, these regions are thought to serve as hubs for cellular processes such as inter‐organellar exchange of lipids, calcium homeostasis, and the access of phosphatases to substrates. Consequently, the spatial‐temporal organization of these structures will impact numerous cellular functions. The formation of ER plasma membrane (PM) contact sites are controlled by a variety of membrane tether proteins as well as the morphology of the ER. We and others have found that the sub‐plasmalemmal cortical actin cytoskeleton, a mesh‐like network that occupies 100–200 nm below the PM, can also limit the formation of ER‐PM contact sites. Yet, how ER‐PM junctions are regulated in circumstances where cells have extensive cortical actin remodeling is not understood. An example of such cellular process involving dynamic cortical actin re‐arrangement is phagocytosis. Phagocytosis is an actin‐dependent processes used to internalize particulate material (≥0.5 μm) that serves both antimicrobial and homeostatic functions. The role of the ER in phagocytosis has been a matter of much controversy. Electron micrographs of macrophages undergoing phagocytosis revealed the presence of ER in extensive, close contact with the forming phagosome. It is speculated that the role for ER in this context is to form ER‐PM contact sites with the PM. My studies have revealed that during phagocytosis, the disassembly of F‐actin from the base of the phagocytic cup allows for the formation of new ER‐PM contact sites. ER‐PM contacts formed promptly and specifically where the polymerized actin has been cleared. As such I have found that the spatial occupancy of ER‐PM junction increased ~3 fold during phagocytosis. Finally, I identified PTP1B, a tyrosine phosphatase, as one of the ER‐PM contact proteins that may be functionally important during phagocytosis.Support or Funding InformationNSERC PGS‐DCIHR Project Grant

Interorganellar communication and membrane contact sites in protozoan parasites.

In this issue of EMBO reports, Loewen and colleagues reveal a role for plasma membrane–endoplasmic reticulum contact sites in regulating phosphatidylcholine synthesis in budding yeast.

Lipid transfer proteins (LTPs) that shuttle lipids at membrane contact sites (MCS) play an important role in maintaining cellular homeostasis. One such important LTP is the Retinal Degeneration B (RDGB) protein. RDGB is localized at the MCS formed between the endoplasmic reticulum (ER) and the apical plasma membrane (PM) in Drosophila photoreceptors where it transfers phosphatidylinositol (PI) during G-protein coupled phospholipase C signalling. Previously, the C-terminal domains of RDGB have been shown to be essential for its function and accurate localization. In this study, using in-silico integrative modelling we predict the structure of entire RDGB protein in complex with the ER membrane protein VAP. The structure of RDGB has then been used to decipher the structural features of the protein important for its orientation at the contact site. Using this structure, we identify two lysine residues in the C-terminal helix of the LNS2 domain important for interaction with the PM. Using molecular docking, we also identify an unstructured region USR1, immediately c-terminal to the PITP domain that is important for the interaction of RDGB with VAP. Overall the 10.06 nm length of the predicted RDGB-VAP complex spans the distance between the PM and ER and is consistent with the cytoplasmic gap between the ER and PM measured by transmission electron microscopy in photoreceptors. Overall our model explains the topology of the RDGB-VAP complex at this ER-PM contact site and paves the way for analysis of lipid transfer function in this setting. Communicated by Ramaswamy H. Sarma

Membrane contact sites (MCSs) are junctures that perform important roles including coordinating lipid metabolism. Previous studies have indicated that vacuolar fission/fusion processes are coupled with modifications in the membrane lipid composition. However, it has been still unclear whether MCS-mediated lipid metabolism controls the vacuolar morphology. Here we report that deletion of tricalbins (Tcb1, Tcb2, Tcb3), tethering proteins at endoplasmic reticulum (ER)-plasma membrane (PM) and ER-Golgi contact sites, alters fusion/fission dynamics and causes vacuolar fragmentation in the yeast Saccharomyces cerevisiae. In addition, we show that the sphingolipid precursor phytosphingosine accumulates in tricalbin-deleted cells, triggering the vacuolar division. Detachment of the nucleus vacuole junction (NVJ), an important contact site between the vacuole and the perinuclear ER, restored vacuolar morphology in both cells subjected to high exogenous phytosphingosine and Tcb3-deleted cells, supporting that phytosphingosine transport across the NVJ induces vacuole division. Thus, our results suggest that vacuolar morphology is maintained by MCSs through the metabolism of sphingolipids.

Membrane contact sites (MCSs) are junctures that perform important roles including coordinating lipid metabolism. Previous studies have indicated that vacuolar fission/fusion processes are coupled with modifications in the membrane lipid composition. However, it has been still unclear whether MCS-mediated lipid metabolism controls the vacuolar morphology. Here we report that deletion of tricalbins (Tcb1, Tcb2, Tcb3), tethering proteins at endoplasmic reticulum (ER)-plasma membrane (PM) and ER-Golgi contact sites, alters fusion/fission dynamics and causes vacuolar fragmentation in the yeast Saccharomyces cerevisiae. In addition, we show that the sphingolipid precursor phytosphingosine accumulates in tricalbin-deleted cells, triggering the vacuolar division. Detachment of the nucleus vacuole junction (NVJ), an important contact site between the vacuole and the perinuclear ER, restored vacuolar morphology in both cells subjected to high exogenous phytosphingosine and Tcb3-deleted cells, supporting that phytosphingosine transport across the NVJ induces vacuole division. Thus, our results suggest that vacuolar morphology is maintained by MCSs through the metabolism of sphingolipids.

Membrane contact sites (MCSs) are junctures that perform important roles including coordinating lipid metabolism. Previous studies have indicated that vacuolar fission/fusion processes are coupled with modifications in the membrane lipid composition. However, it has been still unclear whether MCS-mediated lipid metabolism controls the vacuolar morphology. Here, we report that deletion of tricalbins (Tcb1, Tcb2, and Tcb3), tethering proteins at endoplasmic reticulum (ER)-plasma membrane (PM) and ER-Golgi contact sites, alters fusion/fission dynamics and causes vacuolar fragmentation in the yeast Saccharomyces cerevisiae. In addition, we show that the sphingolipid precursor phytosphingosine (PHS) accumulates in tricalbin-deleted cells, triggering the vacuolar division. Detachment of the nucleus-vacuole junction (NVJ), an important contact site between the vacuole and the perinuclear ER, restored vacuolar morphology in both cells subjected to high exogenous PHS and Tcb3-deleted cells, supporting that PHS transport across the NVJ induces vacuole division. Thus, our results suggest that vacuolar morphology is maintained by MCSs through the metabolism of sphingolipids.

Membrane contact sites (MCSs) are junctures that perform important roles including coordinating lipid metabolism. Previous studies have indicated that vacuolar fission/fusion processes are coupled with modifications in the membrane lipid composition. However, it has been still unclear whether MCS-mediated lipid metabolism controls the vacuolar morphology. Here we report that deletion of tricalbins (Tcb1, Tcb2, Tcb3), tethering proteins at endoplasmic reticulum (ER)-plasma membrane (PM) and ER-Golgi contact sites, alters fusion/fission dynamics and causes vacuolar fragmentation in the yeast Saccharomyces cerevisiae. In addition, we show that the sphingolipid precursor phytosphingosine accumulates in tricalbin-deleted cells, triggering the vacuolar division. Detachment of the nucleus vacuole junction (NVJ), an important contact site between the vacuole and the perinuclear ER, restored vacuolar morphology in both cells subjected to high exogenous phytosphingosine and Tcb3-deleted cells, supporting that phytosphingosine transport across the NVJ induces vacuole division. Thus, our results suggest that vacuolar morphology is maintained by MCSs through the metabolism of sphingolipids.