Piecing together the structural organisation of lipid exchange at membrane contact sites

Membrane contact sites (MCSs) are areas of close proximity between organelles, implicated in transport of small molecules and in organelle biogenesis. Lipid transfer proteins at MCSs facilitate the distribution of lipid species between organelle membranes. Such exchange processes rely on the apposition of two different membranes delimiting distinct compartments and a cytosolic intermembrane space. Maintaining organelle identity while transferring molecules therefore implies control over MCS architecture both on the ultrastructural and molecular levels. Factors including intermembrane distance, density of resident proteins, and contact surface area fine-tune MCS function. Furthermore, the structural arrangement of lipid transfer proteins and associated proteins underpins the molecular mechanisms of lipid fluxes at MCSs. Thus, the architecture of MCSs emerges as an essential aspect of their function.     

A close-up view of membrane contact sites between the endoplasmic reticulum and the endolysosomal system: From yeast to man

Abstract

Maintenance of organelle identity is crucial for the functionality of eukaryotic cells. Hence, transfer reactions between different compartments must be highly efficient and tightly regulated at the same time. Membrane contact sites (MCSs) represent an important route for inter-organelle transport and communication independent of vesicular trafficking. Due to extensive research, the mechanistic understanding of these sites increases constantly. However, how the formation and the versatile functions of MCSs are regulated is mainly unclear. Within this review, we focus on one well-known MCS, the nucleus–vacuole junction in yeast and discuss its analogy to endoplasmic reticulum-late endosome contacts in metazoan. Formation of the junction in yeast requires Vac8, a protein that is involved in various cellular processes at the yeast vacuole and a target of multiple posttranslational modifications. We discuss the possibility that dual functionality of proteins involved in contact formation is a common principle to coordinate inter-organelle transfer with organellar biogenesis.     

Regulation and physiology of membrane contact sites

Membrane contact sites (MCSs) in addition to impacting the functions of membrane-limited organelles also have a role in the spatial and functional organization of cells, tissues and whole organisms. MCSs have been identified between all organelles and the identification of their molecular composition has progressed significantly in recent years. Equally important is how MCSs respond dynamically to physiological stimuli, how this is regulated, and the physiological roles of MCSs in tissues and at the organismal level, an area that still remains relatively unexplored. In the present review, we focus on the regulation of MCSs, considerations of their function at the organismal level, and how mutations of MCS components linked to genetic diseases might inform us about their physiological relevance.     

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.     

Pex30-like proteins function as adaptors at distinct ER membrane contact sites

Membrane lipids and proteins synthesized in the ER are used for de novo assembly of organelles, such as lipid droplets and peroxisomes. After assembly, the growth of these organelles is supported by ER-derived lipids transferred at membrane contact sites (MCSs). How ER sites for organelle biogenesis and lipid transfer are established and regulated is unclear. Here, we investigate how the ER membrane protein Pex30 and its family members Pex28, Pex29, Pex31, and Pex32 target and function at multiple MCSs. We show that different Pex30 complexes function at distinct ER domains and MCSs. Pex30 targets ER–peroxisome MCSs when bound to Pex28 and Pex32, organizes the nuclear–vacuolar junction when bound to Pex29, and promotes the biogenesis of lipid droplets independently of other family members. Importantly, the reticulon homology domain (RHD) mediates the assembly of the various Pex30 complexes. Given the role of RHD in membrane shaping, our findings offer a mechanistic link between MCS and regulation of membrane curvature.     

Short-range intracellular trafficking of small molecules across endoplasmic reticulum junctions

Intracellular trafficking is not mediated exclusively by vesicles. Additional, non-vesicular mechanisms transport material, in particular small molecules such as lipids and Ca(2+) ions, from one organelle to another. This transport occurs at narrow cytoplasmic gaps called membrane contact sites (MCSs), at which two organelles come into close apposition. Despite the conservation of these structures throughout evolution, little is known about this transport, largely because of a lack of knowledge of almost all molecular components of MCSs. Recently, this situation has started to change because the structural proteins that bridge an MCS are now known in a single case, and proteins implicated in lipid trafficking have been localized to MCSs. In the light of these advances, I hypothesize that the endoplasmic reticulum has a central role in the trafficking of lipids and ions by forming a network of MCSs with most other intracellular organelles.     

Organelle contacts: Sub‐organelle zones to facilitate rapid and accurate inter‐organelle trafficking of lipids

When one person wants to communicate securely with another, he/she should contact the other person directly. This rule applies not only to human society, but also to the intracellular micro‐society. In the past two decades, it has become increasingly clear that the sub‐organelle regions called membrane contact sites (MCSs) are pivotal for inter‐organelle transport of lipids in cells, as highlighted in the thematic review series “Interorganelle trafficking of lipids” held in Traffic in 2014–2015. In this commentary, we will describe how the currently prevailing model for lipid trafficking at MCSs was generated, and comment on three important issues that have not been explored: (a1) the principles guiding the generation of an asymmetrical inter‐organelle flow of lipids in cells, (b2) the advantages in lipid trafficking at organelle contacts, and (c3) the dynamic network of inter‐organelle lipid trafficking.     

Maintaining social contacts: The physiological relevance of organelle interactions

Membrane-bound organelles in eukaryotic cells form an interactive network to coordinate and facilitate cellular functions. The formation of close contacts, termed “membrane contact sites” (MCSs), represents an intriguing strategy for organelle interaction and coordinated interplay. Emerging research is rapidly revealing new details of MCSs. They represent ubiquitous and diverse structures, which are important for many aspects of cell physiology and homeostasis. Here, we provide a comprehensive overview of the physiological relevance of organelle contacts. We focus on mitochondria, peroxisomes, the Golgi complex and the plasma membrane, and discuss the most recent findings on their interactions with other subcellular organelles and their multiple functions, including membrane contacts with the ER, lipid droplets and the endosomal/lysosomal compartment.     

Re-evaluation of physical interaction between plant peroxisomes and other organelles using live-cell imaging techniques

The dynamic behavior of organelles is essential for plant survival under various environmental conditions. Plant organelles, with various functions,migrate along actin filaments and contact other types of organelles, leading to physical interactions at a specific site called the membrane contact site. Recent studies have revealed the importance of physical interactions in maintaining efficient metabolite flow between organelles.In this review, we first summarize peroxisome function under different environmental conditions and growth stages to understand organelle interactions. We then discuss current knowledge regarding the interactions between peroxisome and other organelles, i.e., the oil bodies, chloroplast, and mitochondria from the perspective of metabolic and physiological regulation, with reference to various organelle interactions and techniques for estimating organelle interactions occurring in plant cells.     

Interorganellar communication and membrane contact sites in protozoan parasites

A key characteristic of eukaryotic cells is the presence of organelles with discrete boundaries and functions. Such subcellular compartmentalization into organelles necessitates platforms for communication and material exchange between each other which often involves vesicular trafficking and associated processes. Another way is via the close apposition between organellar membranes, called membrane contact sites (MCSs). Apart from lipid transfer, MCSs have been implicated to mediate in various cellular processes including ion transport, apoptosis, and organelle dynamics. In mammalian and yeast cells, contact sites have been reported between the membranes of the following: the endoplasmic reticulum (ER) and the plasma membrane (PM), ER and the Golgi apparatus, ER and endosomes (i.e., vacuoles, lysosomes), ER and lipid droplets (LD), the mitochondria and vacuoles, the nucleus and vacuoles, and the mitochondria and lipid droplets, whereas knowledge of MCSs in non-model organisms such as protozoan parasites is extremely limited. Growing evidence suggests that MCSs play more general and conserved roles in cell physiology. In this mini review, we summarize and discuss representative MCSs in divergent parasitic protozoa, and highlight the universality, diversity, and the contribution of MCSs to parasitism.     

Cnm1: A bridge between mitochondria and nuclear ER

Few membrane contact sites have been defined at the molecular level. By using a high-throughput, microscopy-based screen, Eisenberg-Bord, Zung et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202104100) identify Cnm1 as a novel tethering protein that mediates contact between mitochondria and the nuclear ER in response to phospholipid levels.

Organelles communicate through the exchange of biological materials by vesicular trafficking or at sites of close membrane apposition known as membrane contact sites (MCSs). While the molecular machinery mediating vesicular trafficking has been well characterized, our knowledge of the molecules involved in forming and regulating MCSs is limited. MCSs physically tether two or more organelles via protein–protein or protein–lipid interactions, contain defined proteomes, and perform specific biological functions (1). While MCSs have been appreciated microscopically since the 1950s, only recently have advances in technology permitted the discovery of the molecular composition of some MCSs (2). A major breakthrough occurred when a synthetic biology screen identified the ER–mitochondria encounter structure (ERMES), which forms an MCS between the ER and mitochondria (3). ERMES has since been shown to be involved in phospholipid transport between mitochondria and the ER (4). While ERMES is one of the best characterized MCSs, there are still many questions as to the precise molecules being transported at ER–mitochondria contacts and how directionality of transport is achieved. Subsequent studies using split fluorescent proteins revealed that nearly all organelles appear to form MCSs of some kind (5). Thus, despite progress in defining the components and functions of a few MCSs, there are still many MCSs whose molecular identities are completely unknown.Recently, a study in mammalian cells identified an MCS between the nucleus and mitochondria that plays a role in adapting cells to stress via the mitochondrial retrograde signaling response (6). The proteins that form this MCS are not conserved in yeast, however, suggesting that alternative mechanisms for nucleus–mitochondria contacts exist in other organisms. In this issue, Eisenberg-Bord, Zung et al., set out to identify proteins involved in forming an MCS between mitochondria and the nuclear ER that is distinct from ERMES-mediated ER–mitochondria contacts (7). First, high-resolution cryo-electron tomographs revealed that mitochondria form contacts with the nucleus that have an average separation of ∼20 nm, which is within the expected range for a bona fide MCS (1). To identify the molecular composition of this contact site, the authors generated a synthetic reporter that is specific to nucleus-mitochondria contacts by fusing one part of a split fluorescent protein to an outer mitochondrial membrane protein and the other to a peripheral nuclear protein. A high-throughput, microscopy-based genetic screen was then used to compare the localization of the synthetic reporter to fluorescently tagged versions of all yeast proteins. Candidates were refined by determining which proteins caused an expansion of the nucleus–mitochondria contact site upon overexpression, a phenotype that has been observed with other MCS proteins (8). Based on these results, the best candidate for a molecular tether between mitochondria and the nucleus was Ybr063c.Ybr063c is a 46-kD nonessential protein of uncharacterized function that contains predicted transmembrane domains. The authors first demonstrated that Ybr063c is an integral membrane protein residing on the nuclear membrane. In support of Ybr063c forming a nucleus–mitochondria contact site that is distinct from ERMES, Ybr063c did not colocalize with ERMES subunits nor did overexpression of Ybr063c alter the size of ERMES patches. Remarkably, overexpression of Ybr063c resulted in the mitochondrial network becoming tightly associated with the nuclear membrane. Based on these results, the authors concluded that Ybr063c functions as a molecular tether between mitochondria and the nucleus and the protein was renamed Cnm1 for contact nucleus mitochondria 1.Through further genetic screens, Eisenberg-Bord, Zung et al., identified several genes that are required to cluster mitochondria around the nucleus when Cnm1 is overexpressed. Interestingly, several of these genes are known to function in phosphatidylcholine (PC) metabolism. Deletion of these components resulted in a decrease in Cnm1 expression, which alters the extent of nucleus-mitochondria contacts. Overexpression of Cnm1 in genetic conditions that reduce PC levels resulted in exaggerated growth defects. These results raise the possibility that Cnm1-mediated nuclear–mitochondria contacts may be involved in the transport of PC from the ER to mitochondria. Thus, while the functional importance is unknown, Cnm1-mediated nuclear–mitochondria contacts respond to PC levels.The genetic screens also identified a single resident mitochondrial protein, Tom70, as affecting the ability of overexpressed Cnm1 to cluster mitochondria around the nucleus. Subsequent experiments demonstrated that localization of Cnm1 to the nuclear membrane and Tom70 to the mitochondrial membrane is required to tether mitochondria to the nucleus upon overexpression of Cnm1. Thus, Cnm1 and Tom70 mediate an MCS between mitochondria and the nucleus.The identification of Cnm1-mediated nucleus–mitochondria contacts opens many questions about the function and composition of the contact site and how it operates in the broader context of mitochondrial–nuclear communication. While identifying the functions of MCSs has proven challenging, the genetic screens conducted in this study provide an excellent starting point by elucidating a link between Cnm1 and PC metabolism. The authors propose that Cnm1-mediated contacts could function in the direct transport of PC from the ER to mitochondria (Fig. 1). In this model, ERMES, which likely functions in earlier steps of PC synthesis by transporting phosphatidylethanolamine (PE) or phosphatidylserine (PS), would have a distinct but related function in organizing and maintaining a pipeline for the transport of lipids between the ER and mitochondria (Fig. 1). This model is speculative, however, and future experiments will be necessary to define the role of Cnm1 in PC metabolism.Open in a separate windowFigure 1.The ER and vacuole form multiple MCSs with mitochondria in budding yeast. The ER is depicted in green, and the mitochondrial network is depicted in gray. ERMES mediates an MCS between tubular ER and mitochondria. In addition to functions that are distinct from lipid trafficking, ERMES-mediated MCSs likely function to transport PS or PE between the organelles. Cnm1 mediates an MCS specifically between the nuclear ER and mitochondria and potentially functions in PC transport. The Vps13-Mcp1 vCLAMP mediates an MCS between mitochondria and the vacuole that likely functions in lipid transport and may have redundant functions with ERMES. The Vps39-Tom40 vCLAMP is a separate MCS between mitochondria and the vacuole that responds to different stress conditions, though its function is unknown.There is a growing body of evidence that two organelles can form multiple MCSs that are spatially and functionally distinct. In addition to ERMES and Cnm1-mediated mitochondria–ER contacts, in yeast, two distinct MCSs have been described between mitochondria and the vacuole that are referred to as vacuolar and mitochondrial patches, or vCLAMPs. One, mediated by Vam6 and Tom40, has been implicated in responding to cellular stress while the other, mediated by Mcp1 and Vps13, may have overlapping functions with the ERMES complex (8, 9; Fig. 1). Interestingly, many of the proteins present at MCSs have been shown to be multifunctional (2). For example, the vCLAMP component Vam6 is also a subunit of the homotypic fusion and protein-sorting (HOPS) complex while its binding partner Tom40 is the central subunit of the translocase of outer membrane (TOM) complex (8). Thus, while these complexes have distinct biological functions in vacuolar protein sorting and mitochondrial protein import respectively, individual subunits have moonlighting functions in the formation, and perhaps function, of MCSs. Eisenberg-Bord, Zung et al., now reveal that Tom70, another component of the TOM complex, also plays a role in the formation of nucleus–mitochondria contacts. This raises the exciting possibility that cells use these multifunctional proteins to coordinate functions such as mitochondrial protein import with lipid trafficking. A crucial next step will be to determine how the multiple functions of these proteins are coordinated to maintain organelle homeostasis.Nuclear–mitochondrial communication is a critical aspect of eukaryotic cellular life that allows cells to adapt to different environmental conditions and energy needs. A breakdown in communication between mitochondria and the nucleus has been implicated in several diseases, including cancers (10). The formation of a nucleus–mitochondria MCS likely facilitates the exchange of lipids or small molecules that stimulate signaling pathways to help cells respond to environmental changes or mitochondrial damage (6, 7). Identifying the molecules that regulate these contacts and clarifying the physiological contexts under which these contacts function is crucial to our understanding of human disease. Thus, the identification of a nucleus-mitochondria MCS represents a significant breakthrough in our understanding of nucleus–mitochondria communication.     

Ultrastructural relationship of the phagophore with surrounding organelles

Phagophore nucleates from a subdomain of the endoplasmic reticulum (ER) termed the omegasome and also makes contact with other organelles such as mitochondria, Golgi complex, plasma membrane and recycling endosomes during its formation. We have used serial block face scanning electron microscopy (SB-EM) and electron tomography (ET) to image phagophore biogenesis in 3 dimensions and to determine the relationship between the phagophore and surrounding organelles at high resolution. ET was performed to confirm whether membrane contact sites (MCSs) are evident between the phagophore and those surrounding organelles. In addition to the known contacts with the ER, we identified MCSs between the phagophore and membranes from putative ER exit sites, late endosomes or lysosomes, the Golgi complex and mitochondria. We also show that one phagophore can have simultaneous MCSs with more than one organelle. Future membrane flux experiments are needed to determine whether membrane contacts also signify lipid translocation.     

Fixation of conserved sequences shapes human intron size and influences transposon-insertion dynamics

The basis for intron expansion in humans is largely unexplored. In this article, we demonstrate that intron expansion has primarily been determined by fixation of multispecies conserved sequences (MCSs) over time. The presence of MCSs has shaped intron features: the insertion of transposable elements (TEs) has been constrained as more MCSs were fixed. Analysis of TE and MCS distribution suggested an unprecedented estimate of information requirements for proper splicing of long introns with indication of sequence constraints extending up to >3 kb downstream 5' splice sites.     

New insights into the OSBP‒VAP cycle

VAP-A is a major endoplasmic reticulum (ER) receptor that allows this organelle to engage numerous membrane contact sites with other organelles. One highly studied example is the formation of contact sites through VAP-A interaction with Oxysterol-binding protein (OSBP). This lipid transfer protein transports cholesterol from the ER to the trans-Golgi network owing to the counter-exchange of the phosphoinositide PI(4)P. In this review, we highlight recent studies that advance our understanding of the OSBP cycle and extend the model of lipid exchange to other cellular contexts and other physiological and pathological conditions.     

Generalized concept of minimal cut sets in biochemical networks

Recently, the concept of minimal cut sets has been introduced for studying structural fragility and identifying knock-out strategies in biochemical reaction networks. A minimal cut set (MCS) has been defined as a minimal set of reactions whose removal blocks the operation of a chosen objective reaction. In this report the theoretical framework of MCSs is refined and extended increasing the practical applicability significantly. An MCS is now defined as a minimal (irreducible) set of structural interventions (removal of network elements) repressing a certain functionality specified by a deletion task. A deletion task describes unambiguously the flux patterns (or the functionality) to be repressed. It is shown that the MCSs can be computed from the set of target modes, which comprises all elementary modes that exhibit the functionality to be attacked. Since a deletion task can be specified by several Boolean rules, MCSs can now be determined for a large variety of complex deletion problems and may be utilized for inhibiting very special flux patterns. It is additionally shown that the other way around is also possible: the elementary modes belonging to a certain functionality can be computed from the respective set of MCSs. Therefore, elementary modes and MCSs can be seen as dual representations of network functions and both can be converted into each other. Moreover, there exist a strong relationship to minimal hitting sets known from set theory: the MCSs are the minimal hitting sets of the collection of target modes and vice versa. Another generalization introduced herein is that MCSs need not to be restricted to the removal of reactions they may also contain network nodes. In the light of the extended framework of MCSs, applications for assessing, manipulating, and designing metabolic networks in silico are discussed.     

Perspective on architecture and assembly of membrane contact sites

Membrane contact sites (MCS) are platforms of physical contact between different organelles. They are formed through interactions involving lipids and proteins, and function in processes such as calcium and lipid exchange, metabolism and organelle biogenesis. In this article, we discuss emerging questions regarding the architecture, organisation and assembly of MCS, such as: What is the contribution of different components to the interaction between organelles? How is the specific composition of different types of membrane contacts sites established and maintained? How are proteins and lipids spatially organised at MCS and how does that influence their function? How dynamic are MCS on the molecular and ultrastructural level? We highlight current state of research and point out experimental approaches that promise to contribute to a spatiomechanistic understanding of MCS functions.     

In vitro three-dimensional modelling of human ovarian surface epithelial cells

Objectives:  Ninety percent of malignant ovarian cancers are epithelial and thought to arise from the ovarian surface epithelium (OSE). We hypothesized that biological characteristics of primary OSE cells would more closely resemble OSE in vivo if established as three-dimensional (3D) cultures.
Materials and methods:  OSE cells were cultured as multicellular spheroids (MCS) (i) in a rotary cell culture system (RCCS) and (ii) on polyHEMA-coated plastics. The MCSs were examined by electron microscopy and compared to OSE from primary tissues and cells grown in 2D. Annexin V FACS analysis was used to evaluate apoptosis and expression of extracellular matrix (ECM) proteins was analysed by immunohistochemical staining.
Results:  On polyHEMA-coated plates, OSE spheroids had defined internal architecture. RCCS MCSs had disorganized structure and higher proportion of apoptotic cells than polyHEMA MCSs and the same cells grown in 2D culture. In 2D, widespread expression of AE1/AE3, laminin and vimentin were undetectable by immunohistochemistry, whereas strong expression of these proteins was observed in the same cells grown in 3D culture and in OSE on primary tissues.
Conclusions:  Physiological and biological features of OSE cells grown in 3D culture more closely resemble characteristics of OSE cells in vivo than when grown by classical 2D approaches. It is likely that establishing in vitro 3D OSE models will lead to greater understanding of the mechanisms of neoplastic transformation in epithelial ovarian cancers.     

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.     

Chemo- and opto-genetic tools for dissecting the role of membrane contact sites in living cells: Recent advances and limitations

Membrane contact sites (MCSs) are morphologically defined intracellular structures where cellular membranes are closely apposed. Recent progress has significantly advanced our understanding of MCSs with the use of new tools and techniques. Visualization of MCSs in living cells by split fluorescence proteins or FRET-based techniques tells us the dynamic property of MCSs. Manipulation of MCSs by chemically-induced dimerization (CID) or light-induced dimerization (LID) greatly contributes to our understanding of their functional aspects including inter-organelle lipid transport mediated by lipid transfer proteins (LTPs). Here we highlight recent advances in these tools and techniques as applied to MCSs, and we discuss their advantages and limitations.