Integrated regulation of the phosphatidylinositol cycle and phosphoinositide‐driven lipid transport at ER‐PM contact sites

Among the structural phospholipids that form the bulk of eukaryotic cell membranes, phosphatidylinositol (PtdIns) is unique in that it also serves as the common precursor for low‐abundance regulatory lipids, collectively referred to as polyphosphoinositides (PPIn). The metabolic turnover of PPIn species has received immense attention because of the essential functions of these lipids as universal regulators of membrane biology and their dysregulation in numerous human pathologies. The diverse functions of PPIn lipids occur, in part, by orchestrating the spatial organization and conformational dynamics of peripheral or integral membrane proteins within defined subcellular compartments. The emerging role of stable contact sites between adjacent membranes as specialized platforms for the coordinate control of ion exchange, cytoskeletal dynamics, and lipid transport has also revealed important new roles for PPIn species. In this review, we highlight the importance of membrane contact sites formed between the endoplasmic reticulum (ER) and plasma membrane (PM) for the integrated regulation of PPIn metabolism within the PM. Special emphasis will be placed on non‐vesicular lipid transport during control of the PtdIns biosynthetic cycle as well as toward balancing the turnover of the signaling PPIn species that define PM identity.     

PI4P/PS countertransport by ORP10 at ER–endosome membrane contact sites regulates endosome fission

Membrane contact sites (MCSs) serve as a zone for nonvesicular lipid transport by oxysterol-binding protein (OSBP)-related proteins (ORPs). ORPs mediate lipid countertransport, in which two distinct lipids are transported counterdirectionally. How such lipid countertransport controls specific biological functions, however, remains elusive. We report that lipid countertransport by ORP10 at ER–endosome MCSs regulates retrograde membrane trafficking. ORP10, together with ORP9 and VAP, formed ER–endosome MCSs in a phosphatidylinositol 4-phosphate (PI4P)-dependent manner. ORP10 exhibited a lipid exchange activity toward its ligands, PI4P and phosphatidylserine (PS), between liposomes in vitro, and between the ER and endosomes in situ. Cell biological analysis demonstrated that ORP10 supplies a pool of PS from the ER, in exchange for PI4P, to endosomes where the PS-binding protein EHD1 is recruited to facilitate endosome fission. Our study highlights a novel lipid exchange at ER–endosome MCSs as a nonenzymatic PI4P-to-PS conversion mechanism that organizes membrane remodeling during retrograde membrane trafficking.     

ER–endosome contact sites: molecular compositions and functions

Recent studies have revealed the existence of numerous contact sites between the endoplasmic reticulum (ER) and endosomes in mammalian cells. Such contacts increase during endosome maturation and play key roles in cholesterol transfer, endosome positioning, receptor dephosphorylation, and endosome fission. At least 7 distinct contact sites between the ER and endosomes have been identified to date, which have diverse molecular compositions. Common to these contact sites is that they impose a close apposition between the ER and endosome membranes, which excludes membrane fusion while allowing the flow of molecular signals between the two membranes, in the form of enzymatic modifications, or ion, lipid, or protein transfer. Thus, ER–endosome contact sites ensure coordination of molecular activities between the two compartments while keeping their general compositions intact. Here, we review the molecular architectures and cellular functions of known ER–endosome contact sites and discuss their implications for human health.     

Phosphoinositide transport and metabolism at membrane contact sites

Phosphoinositides are a family of signaling lipids that play a profound role in regulating protein function at the membrane-cytosol interface of all cellular membranes. Underscoring their importance, mutations or alterations in phosphoinositide metabolizing enzymes lead to host of developmental, neurodegenerative, and metabolic disorders that are devastating for human health. In addition to lipid enzymes, phosphoinositide metabolism is regulated and controlled at membrane contact sites (MCS). Regions of close opposition typically between the ER and other cellular membranes, MCS are non-vesicular lipid transport portals that engage in extensive communication to influence organelle homeostasis. This review focuses on lipid transport, specifically phosphoinositide lipid transport and metabolism at MCS.     

ER–plasma membrane contact sites contribute to autophagosome biogenesis by regulation of local PI3P synthesis

The double‐membrane‐bound autophagosome is formed by the closure of a structure called the phagophore, origin of which is still unclear. The endoplasmic reticulum (ER) is clearly implicated in autophagosome biogenesis due to the presence of the omegasome subdomain positive for DFCP1, a phosphatidyl‐inositol‐3‐phosphate (PI3P) binding protein. Contribution of other membrane sources, like the plasma membrane (PM), is still difficult to integrate in a global picture. Here we show that ER–plasma membrane contact sites are mobilized for autophagosome biogenesis, by direct implication of the tethering extended synaptotagmins (E‐Syts) proteins. Imaging data revealed that early autophagic markers are recruited to E‐Syt‐containing domains during autophagy and that inhibition of E‐Syts expression leads to a reduction in autophagosome biogenesis. Furthermore, we demonstrate that E‐Syts are essential for autophagy‐associated PI3P synthesis at the cortical ER membrane via the recruitment of VMP1, the stabilizing ER partner of the PI3KC3 complex. These results highlight the contribution of ER–plasma membrane tethers to autophagosome biogenesis regulation and support the importance of membrane contact sites in autophagy.     

The chlamydial organism Simkania negevensis forms ER vacuole contact sites and inhibits ER‐stress

Most intracellular bacterial pathogens reside within membrane‐surrounded host‐derived vacuoles. Few of these bacteria exploit membranes from the host's endoplasmic reticulum (ER) to form a replicative vacuole. Here, we describe the formation of ER–vacuole contact sites as part of the replicative niche of the chlamydial organism Simkania negevensis. Formation of ER–vacuole contact sites is evolutionary conserved in the distantly related protozoan host Acanthamoeba castellanii. Simkania growth is accompanied by mitochondria associating with the Simkania‐containing vacuole (SCV). Super‐resolution microscopy as well as 3D reconstruction from electron micrographs of serial ultra‐thin sections revealed a single vacuolar system forming extensive ER–SCV contact sites on the Simkania vacuolar surface. Simkania infection induced an ER‐stress response, which was later downregulated. Induction of ER‐stress with Thapsigargin or Tunicamycin was strongly inhibited in cells infected with Simkania. Inhibition of ER‐stress was required for inclusion formation and efficient growth, demonstrating a role of ER‐stress in the control of Simkania infection. Thus, Simkania forms extensive ER–SCV contact sites in host species evolutionary as diverse as human and amoeba. Moreover, Simkania is the first bacterial pathogen described to interfere with ER‐stress induced signalling to promote infection.     

Lipids at membrane contact sites: cell signaling and ion transport

Communication between organelles is essential to coordinate cellular functions and the cell's response to physiological and pathological stimuli. Organellar communication occurs at membrane contact sites (MCSs), where the endoplasmic reticulum (ER) membrane is tethered to cellular organelle membranes by specific tether proteins and where lipid transfer proteins and cell signaling proteins are located. MCSs have many cellular functions and are the sites of lipid and ion transfer between organelles and generation of second messengers. This review discusses several aspects of MCSs in the context of lipid transfer, formation of lipid domains, generation of Ca²⁺ and cAMP second messengers, and regulation of ion transporters by lipids.     

Reduced ER–mitochondria connectivity promotes neuroblastoma multidrug resistance

Most cancer deaths result from progression of therapy resistant disease, yet our understanding of this phenotype is limited. Cancer therapies generate stress signals that act upon mitochondria to initiate apoptosis. Mitochondria isolated from neuroblastoma cells were exposed to tBid or Bim, death effectors activated by therapeutic stress. Multidrug‐resistant tumor cells obtained from children at relapse had markedly attenuated Bak and Bax oligomerization and cytochrome c release (surrogates for apoptotic commitment) in comparison with patient‐matched tumor cells obtained at diagnosis. Electron microscopy identified reduced ER–mitochondria‐associated membranes (MAMs; ER–mitochondria contacts, ERMCs) in therapy‐resistant cells, and genetically or biochemically reducing MAMs in therapy‐sensitive tumors phenocopied resistance. MAMs serve as platforms to transfer Ca²⁺ and bioactive lipids to mitochondria. Reduced Ca²⁺ transfer was found in some but not all resistant cells, and inhibiting transfer did not attenuate apoptotic signaling. In contrast, reduced ceramide synthesis and transfer was common to resistant cells and its inhibition induced stress resistance. We identify ER–mitochondria‐associated membranes as physiologic regulators of apoptosis via ceramide transfer and uncover a previously unrecognized mechanism for cancer multidrug resistance.     

Synaptotagmins at the endoplasmic reticulum–plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress

Endoplasmic reticulum–plasma membrane contact sites (ER–PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER–PM protein tether synaptotagmin1 (SYT1) exhibit decreased PM integrity under multiple abiotic stresses, such as freezing, high salt, osmotic stress, and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER–PM tether that also functions in maintaining PM integrity. The ER–PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild-type while the levels of most glycerolipid species remain unchanged. In addition, the SYT1-green fluorescent protein fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

Arabidopsis synaptotagmins 1 and 3 are localized at endoplasmic reticulum–plasma membrane contact sites and during abiotic stress episodes control diacylglycerol homeostasis at the plasma membrane     

STARD3 mediates endoplasmic reticulum‐to‐endosome cholesterol transport at membrane contact sites

StAR‐related lipid transfer domain‐3 (STARD3) is a sterol‐binding protein that creates endoplasmic reticulum (ER)–endosome contact sites. How this protein, at the crossroad between sterol uptake and synthesis pathways, impacts the intracellular distribution of this lipid was ill‐defined. Here, by using in situ cholesterol labeling and quantification, we demonstrated that STARD3 induces cholesterol accumulation in endosomes at the expense of the plasma membrane. STARD3‐mediated cholesterol routing depends both on its lipid transfer activity and its ability to create ER–endosome contacts. Corroborating this, in vitro reconstitution assays indicated that STARD3 and its ER‐anchored partner, Vesicle‐associated membrane protein‐associated protein (VAP), assemble into a machine that allows a highly efficient transport of cholesterol within membrane contacts. Thus, STARD3 is a cholesterol transporter scaffolding ER–endosome contacts and modulating cellular cholesterol repartition by delivering cholesterol to endosomes.     

Vesicular and non-vesicular sterol transport in living cells. The endocytic recycling compartment is a major sterol storage organelle.

We examined the intracellular transport of sterol in living cells using a naturally fluorescent cholesterol analog, dehydroergosterol (DHE), which has been shown to mimic many of the properties of cholesterol. By using DHE loaded on methyl-beta-cyclodextrin, we followed this cholesterol analog in pulse-chase studies. At steady state, DHE co-localizes extensively with transferrin (Tf), a marker for the endocytic recycling compartment (ERC), and redistributes with Tf in cells with altered ERC morphology. Expression of a dominant-negative mutation of an ERC-associated protein, mRme-1 (G429R), results in the slowing of both DHE and Tf receptor return to the cell surface. [3H]Cholesterol is found in the same fraction as 125I-Tf on sucrose density gradients, and this fraction can be specifically shifted to a higher density based on the presence of horseradish peroxidase-conjugated Tf in the same organelle. Whereas vesicular transport of Tf and efflux of DHE from the ERC are entirely blocked in energy-depleted cells, delivery of DHE to the ERC from the plasma membrane is only slightly affected. Biochemical studies performed using [3H]cholesterol show that the energy dependence of cholesterol transport to and from the ERC is similar to DHE transport. We propose that a large portion of intracellular cholesterol is localized in the ERC, and this pool might be important in maintaining cellular cholesterol homeostasis.     

The p24 family and selective transport processes at the ER—Golgi interface

The secretory pathway is of vital importance for eukaryotic cells and has a pivotal role in the synthesis, sorting, processing and secretion of a large variety of bioactive molecules involved in intercellular communication. One of the key processes in the secretory pathway concerns the transport of cargo proteins from the ER (endoplasmic reticulum) to the Golgi. Type‐I transmembrane proteins of ～24 kDa are abundantly present in the membranes of the early secretory pathway, and bind the COPI and COPII coat complexes that cover vesicles travelling between the membranes. These p24 proteins are thought to play an important role in the selective transport processes at the ER—Golgi interface, although their exact functioning is still obscure. One model proposes that p24 proteins couple cargo selection in the lumen with vesicle coat recruitment in the cytosol. Alternatively, p24 proteins may furnish subcompartments of the secretory pathway with the correct subsets of machinery proteins. Here we review the current knowledge of the p24 proteins and the various roles proposed for the p24 family members.     

FUNDC1 regulates mitochondrial dynamics at the ER–mitochondrial contact site under hypoxic conditions

In hypoxic cells, dysfunctional mitochondria are selectively removed by a specialized autophagic process called mitophagy. The ER–mitochondrial contact site (MAM) is essential for fission of mitochondria prior to engulfment, and the outer mitochondrial membrane protein FUNDC1 interacts with LC3 to recruit autophagosomes, but the mechanisms integrating these processes are poorly understood. Here, we describe a new pathway mediating mitochondrial fission and subsequent mitophagy under hypoxic conditions. FUNDC1 accumulates at the MAM by associating with the ER membrane protein calnexin. As mitophagy proceeds, FUNDC1/calnexin association attenuates and the exposed cytosolic loop of FUNDC1 interacts with DRP1 instead. DRP1 is thereby recruited to the MAM, and mitochondrial fission then occurs. Knockdown of FUNDC1, DRP1, or calnexin prevents fission and mitophagy under hypoxic conditions. Thus, FUNDC1 integrates mitochondrial fission and mitophagy at the interface of the MAM by working in concert with DRP1 and calnexin under hypoxic conditions in mammalian cells.     

Dynamin is primed at endocytic sites for ultrafast endocytosis

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Syndapin oligomers interconnect the machineries for endocytic vesicle formation and actin polymerization

Syndapins were proposed to interconnect the machineries for vesicle formation and actin polymerization, as they interact with dynamin and the Arp2/3 complex activator N-WASP (neural Wiskott-Aldrich syndrome protein). Syndapins, however, have only one Src homology 3 domain mediating both interactions. Here we show that syndapins self-associate via direct syndapin/syndapin interactions, providing a molecular mechanism for the coordinating role of syndapin. Cross-link studies with overexpressed and endogenous syndapins suggest that predominantly dimers form in vivo. Our analyses show that the N-terminal Fes/Cip4 homology domain but not the central coiled-coil domain is sufficient for oligomerization. Additionally, a second interface located further C-terminally mediated interactions with the N terminus. The Src homology 3 domain and the NPF region are not involved and thus available for further interactions interconnecting different syndapin binding partners. Our analyses showed that self-association is crucial for syndapin function. Both syndapin-mediated cytoskeletal rearrangements and endocytosis were disrupted by a self-association-deficient mutant. Consistent with a role of syndapins in linking actin polymerization bursts with endocytic vesicle formation, syndapin-containing complexes had a size of 300-500 kDa in gel filtration analysis and contained both dynamin and N-WASP. The existence of an interconnection of the GTPase dynamin with N-WASP via syndapin oligomers was demonstrated both by coimmunoprecipitations and by reconstitution at membranes in intact cells. The interconnection was disrupted by coexpression of syndapin mutants incapable of self-association. Syndapin oligomers may thus act as multivalent organizers spatially and temporally coordinating vesicle fission with local actin polymerization.     

Nir1 constitutively localizes at ER–PM junctions and promotes Nir2 recruitment for PIP2 homeostasis

Homeostatic regulation of plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate (PIP₂) in receptor-stimulated cells is mediated by the lipid transfer protein Nir2. Nir2 is dynamically recruited to endoplasmic reticulum–plasma membrane (ER–PM) junctions to facilitate replenishment of PM PIP₂ hydrolyzed during receptor-mediated signaling. However, our knowledge regarding the activation and sustainment of Nir2-mediated replenishment of PM PIP₂ is limited. Here, we describe the functions of Nir1 as a positive regulator of Nir2 and PIP₂ homeostasis. In contrast to the family proteins Nir2 and Nir3, Nir1 constitutively localizes at ER–PM junctions. Nir1 potentiates Nir2 targeting to ER–PM junctions during receptor-mediated signaling and is required for efficient PM PIP₂ replenishment. Live-cell imaging and biochemical analysis reveal that Nir1 interacts with Nir2 via a region between the FFAT motif and the DDHD domain. Combined, results from this study identify Nir1 as an ER–PM junction localized protein that promotes Nir2 recruitment for PIP₂ homeostasis.