Mapping autophagy-related membrane contact site proteins and complexes with AutoMCS Navigator.

In eukaryotic cells, membrane contact sites (MCSs) mediate interactions and communication between organelles by bringing their membranes into close proximity without fusion. These sites play crucial roles in intracellular transport, signal transduction, and the regulation of organelle functions. In a recent study, we compiled data on MCS proteins and complexes from publications to create the MCSdb database. During data compilation, we discovered that many MCSs, their associated proteins, and complexes are highly relevant to macroautophagy/autophagy. To elucidate the role of MCSs in autophagy, we reorganized the autophagy-related MCS proteins and complexes from MCSdb, creating a data map called AutoMCS Navigator. The current version of this map includes 30 complexes and 84 proteins, covering 13 different MCSs and 7 species. Meanwhile, we embedded a dedicated webpage for AutoMCS Navigator on the MCSdb website. This webpage features an orchestrated visual guide that hierarchically displays MCS proteins and complexes involved in autophagy. In summary, our research has developed a user-friendly visual map for querying, browsing, and visualizing detailed information on autophagy-related MCS proteins and complexes. This tool offers researchers easy access to understand autophagy-related MCS structure, assembly, functions, and therapeutic strategies for related diseases. AutoMCS Navigator is freely available at https://cellknowledge.com.cn/mcsdb/autophagy.html.

Getting to Grips with the Oxysterol-Binding Protein Family - a Forty Year Perspective.

This review discusses how research around the oxysterol-binding protein family has evolved. We briefly summarize how this protein family, designated OSBP-related (ORP) or OSBP-like (OSBPL) proteins, was discovered, how protein domains highly conserved among family members between taxa paved the way for understanding their mechanisms of action, and how insights into protein structural and functional features help to understand their versatility as lipid transporters. We also discuss questions and future avenues of research opened by these findings. The investigations on oxysterol-binding protein family serve as a real-life example of the notion that science often advances as a collective effort of multiple lines of enquiry, including serendipitous routes. While original articles invariably explain the motivation of the research undertaken in rational terms, the actual paths to findings may be less intentional. Fortunately, this does not reduce the impact of the discoveries made. Besides hopefully providing a useful account of ORP family proteins, we aim to convey this message.

Protection of Membrane Contact Protein by the Methionine Sulfoxide Reductases.

In this News and Views, I discuss our recent publication that established how steroidogenic acute regulatory-related lipid transfer domain-3 (STARD3), a membrane contact protein situated at lysosomal membranes, plays a role in the detoxification of cholesterol hydroperoxide. STARD3's methionine residues can be oxidized to methionine sulfoxide by cholesterol hydroperoxide, after which methionine sulfoxide reductases reduce the methionine sulfoxide residues back to methionine. The reaction also results in the reduction of the cholesterol hydroperoxide to an alcohol. The cyclic oxidation and reduction of methionine residues in STARD3 at membrane contact sites creates a catalytically efficient mechanism for detoxification of cholesterol hydroperoxide during cholesterol transport, thus protecting membrane contact sites and the entire cell against the toxicity of cholesterol hydroperoxide.

Proposed dual membrane contact with full-length Osh4.

Membrane contacts sites (MCSs) play important roles in lipid trafficking across cellular compartments and maintain the widespread structural diversity of organelles. We have utilized microsecond long all-atom (AA) molecular dynamics (MD) simulations and enhanced sampling techniques to unravel the MCS structure targeting by yeast oxysterol binding protein (Osh4) in an environment that mimics the interface of membranes with an increased proportion of anionic lipids using CHARMM36m forcefield with additional CUFIX parameters for lipid-protein electrostatic interactions. In a dual-membrane environment, unbiased MD simulations show that Osh4 briefly interacts with both membranes, before aligning itself with a single membrane, adopting a ß-crease-bound conformation similar to observations in a single-membrane scenario. Targeted molecular dynamics simulations followed by microsecond-long AA MD simulations have revealed a distinctive dual-membrane bound state of Osh4 at MCS, wherein the protein interacts with the lower membrane via the ß-crease surface, featuring its PHE-239 residue positioned below the phosphate plane of membrane, while concurrently establishing contact with the opposite membrane through the extended α6-α7 region. Osh4 maintains these dual membrane contacts simultaneously over the course of microsecond-long MD simulations. Moreover, binding energy calculations highlighted the essential roles played by the phenylalanine loop and the α6 helix in dynamically stabilizing dual-membrane bound state of Osh4 at MCS. Our computational findings were corroborated through frequency of contact analysis, showcasing excellent agreement with past experimental cross-linking data. Our computational study reveals a dual-membrane bound conformation of Osh4, providing insights into protein-membrane interactions at membrane contact sites and their relevance to lipid transfer processes.

The endoplasmic reticulum connects to the nucleus by constricted junctions that mature after mitosis.

Junctions between the endoplasmic reticulum (ER) and the outer membrane of the nuclear envelope (NE) physically connect both organelles. These ER-NE junctions are essential for supplying the NE with lipids and proteins synthesized in the ER. However, little is known about the structure of these ER-NE junctions. Here, we systematically study the ultrastructure of ER-NE junctions in cryo-fixed mammalian cells staged in anaphase, telophase, and interphase by correlating live cell imaging with three-dimensional electron microscopy. Our results show that ER-NE junctions in interphase cells have a pronounced hourglass shape with a constricted neck of 7-20 nm width. This morphology is significantly distinct from that of junctions within the ER network, and their morphology emerges as early as telophase. The highly constricted ER-NE junctions are seen in several mammalian cell types, but not in budding yeast. We speculate that the unique and highly constricted ER-NE junctions are regulated via novel mechanisms that contribute to ER-to-NE lipid and protein traffic in higher eukaryotes.

KRAP regulates mitochondrial Ca2+ uptake by licensing IP3 receptor activity and stabilizing ER-mitochondrial junctions.

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are high-conductance channels that allow the regulated redistribution of Ca2+ from the endoplasmic reticulum (ER) to the cytosol and, at specialized membrane contact sites (MCSs), to other organelles. Only a subset of IP3Rs release Ca2+ to the cytosol in response to IP3. These 'licensed' IP3Rs are associated with Kras-induced actin-interacting protein (KRAP, also known as ITPRID2) beneath the plasma membrane. It is unclear whether KRAP regulates IP3Rs at MCSs. We show, using simultaneous measurements of Ca2+ concentration in the cytosol and mitochondrial matrix, that KRAP also licenses IP3Rs to release Ca2+ to mitochondria. Loss of KRAP abolishes cytosolic and mitochondrial Ca2+ signals evoked by stimulation of IP3Rs via endogenous receptors. KRAP is located at ER-mitochondrial membrane contact sites (ERMCSs) populated by IP3R clusters. Using a proximity ligation assay between IP3R and voltage-dependent anion channel 1 (VDAC1), we show that loss of KRAP reduces the number of ERMCSs. We conclude that KRAP regulates Ca2+ transfer from IP3Rs to mitochondria by both licensing IP3R activity and stabilizing ERMCSs.

Nucleus-Mitochondria Contact Sites Are Associated With Asynthetic Fission in Zebrafish Skin.

Rapid increase in body surface area of growing zebrafish larvae (Danio rario) is partially accomplished by asynthetic fission of superficial epithelial cells (SECs) of the skin. There are two cycles of this atypical form of cell division which is unaccompanied by DNA replication; resulting in cells with a variable DNA content. Here, electron microscopy of basal epithelium cells that give rise to these SECs in zebrafish larvae shows aggregation of mitochondria around the nucleus and the formation of nucleus-mitochondria membrane contact sites. Membrane aggregates appear in the lumen of the nuclear envelope at these sites of membrane contact in some cells, suggesting lipid turnover in this vicinity. As the epithelial cells mature and stratify, the mitochondria are engulfed by extensions arising from the nuclear envelope. The mitochondrial outer membrane fragments and mitochondria fuse with the nuclear envelope and parts of the endoplasmic reticulum. Other organelles, including the Golgi apparatus, progressively localize to a central region of the cell and lose their integrity. Thus, asynthetic fission is accompanied by an atypical pattern of organelle destruction and a prelude to this is the formation of nucleus-mitochondria membrane contact sites.

The Role of ATG9 Vesicles in Autophagosome Biogenesis.

Autophagy mediates the degradation and recycling of cellular material in the lysosomal system. Dysfunctional autophagy is associated with a plethora of diseases including uncontrolled infections, cancer and neurodegeneration. In macroautophagy (hereafter autophagy) this material is encapsulated in double membrane vesicles, the autophagosomes, which form upon induction of autophagy. The precursors to autophagosomes, referred to as phagophores, first appear as small flattened membrane cisternae, which gradually enclose the cargo material as they grow. The assembly of phagophores during autophagy initiation has been a major subject of investigation over the past decades. A special focus has been ATG9, the only conserved transmembrane protein among the core machinery. The majority of ATG9 localizes to small Golgi-derived vesicles. Here we review the recent advances and breakthroughs regarding our understanding of how ATG9 and the vesicles it resides in serve to assemble the autophagy machinery and to establish membrane contact sites for autophagosome biogenesis. We also highlight open questions in the field that need to be addressed in the years to come.

Stay in touch with the endoplasmic reticulum.

The endoplasmic reticulum (ER), which is composed of a continuous network of tubules and sheets, forms the most widely distributed membrane system in eukaryotic cells. As a result, it engages a variety of organelles by establishing membrane contact sites (MCSs). These contacts regulate organelle positioning and remodeling, including fusion and fission, facilitate precise lipid exchange, and couple vital signaling events. Here, we systematically review recent advances and converging themes on ER-involved organellar contact. The molecular basis, cellular influence, and potential physiological functions for ER/nuclear envelope contacts with mitochondria, Golgi, endosomes, lysosomes, lipid droplets, autophagosomes, and plasma membrane are summarized.

The role of the HORMA domain proteins ATG13 and ATG101 in initiating autophagosome biogenesis.

Autophagy is a process of regulated degradation. It eliminates damaged and unnecessary cellular components by engulfing them with a de novo-generated organelle: the double-membrane autophagosome. The past three decades have provided us with a detailed parts list of the autophagy initiation machinery, have developed important insights into how these processes function and have identified regulatory proteins. It is now clear that autophagosome biogenesis requires the timely assembly of a complex machinery. However, it is unclear how a putative stable machine is assembled and disassembled and how the different parts cooperate to perform its overall function. Although they have long been somewhat enigmatic in their precise role, HORMA domain proteins (first identified in Hop1p, Rev7p and MAD2 proteins) autophagy-related protein 13 (ATG13) and ATG101 of the ULK-kinase complex have emerged as important coordinators of the autophagy-initiating subcomplexes. Here, we will particularly focus on ATG13 and ATG101 and the role of their unusual metamorphosis in initiating autophagosome biogenesis. We will also explore how this metamorphosis could potentially be purposefully rate-limiting and speculate on how it could regulate the spontaneous self-assembly of the autophagy-initiating machinery.

Peroxisome population control by phosphoinositide signaling at the endoplasmic reticulum-plasma membrane interface.

Phosphoinositides are lipid signaling molecules acting at the interface of membranes and the cytosol to regulate membrane trafficking, lipid transport and responses to extracellular stimuli. Peroxisomes are multicopy organelles that are highly responsive to changes in metabolic and environmental conditions. In yeast, peroxisomes are tethered to the cell cortex at defined focal structures containing the peroxisome inheritance protein, Inp1p. We investigated the potential impact of changes in cortical phosphoinositide levels on the peroxisome compartment of the yeast cell. Here we show that the phosphoinositide, phosphatidylinositol-4-phosphate (PI4P), found at the junction of the cortical endoplasmic reticulum and plasma membrane (cER-PM) acts to regulate the cell's peroxisome population. In cells lacking a cER-PM tether or the enzymatic activity of the lipid phosphatase Sac1p, cortical PI4P is elevated, peroxisome numbers and motility are increased, and peroxisomes are no longer firmly tethered to Inp1p-containing foci. Reattachment of the cER to the PM through an artificial ER-PM "staple" in cells lacking the cER-PM tether does not restore peroxisome populations to the wild-type condition, demonstrating that integrity of PI4P signaling at the cell cortex is required for peroxisome homeostasis.

Pharmacological interventions for lipid transport disorders.

The recent discovery that defects in inter-organelle lipid transport are at the heart of several neurological and neurodegenerative disorders raises the challenge of identifying therapeutic strategies to correct lipid transport defects. This perspective highlights two potential strategies suggested by the study of lipid transport in budding yeast. In the first approach, small molecules are proposed that enhance the lipid transfer activity of VPS13 proteins and thereby compensate for reduced transport. In the second approach, molecules that act as inter-organelle tethers could be used to create artificial contact sites and bypass the loss of endogenous contacts.

ER-dependent membrane repair of mycobacteria-induced vacuole damage.

IMPORTANCE: Tuberculosis still remains a global burden and is one of the top infectious diseases from a single pathogen. Mycobacterium tuberculosis, the causative agent, has perfected many ways to replicate and persist within its host. While mycobacteria induce vacuole damage to evade the toxic environment and eventually escape into the cytosol, the host recruits repair machineries to restore the MCV membrane. However, how lipids are delivered for membrane repair is poorly understood. Using advanced fluorescence imaging and volumetric correlative approaches, we demonstrate that this involves the recruitment of the endoplasmic reticulum (ER)-Golgi lipid transfer protein OSBP8 in the Dictyostelium discoideum/Mycobacterium marinum system. Strikingly, depletion of OSBP8 affects lysosomal function accelerating mycobacterial growth. This indicates that an ER-dependent repair pathway constitutes a host defense mechanism against intracellular pathogens such as M. tuberculosis.

Ultrastructural analysis of prostate cancer tissue provides insights into androgen-dependent adaptations to membrane contact site establishment.

Membrane trafficking and organelle contact sites are important for regulating cell metabolism and survival; processes often deregulated in cancer. Prostate cancer is the second leading cause of cancer-related death in men in the developed world. While early-stage disease is curable by surgery or radiotherapy there is an unmet need to identify prognostic biomarkers, markers to treatment response and new therapeutic targets in intermediate-late stage disease. This study explored the morphology of organelles and membrane contact sites in tumor tissue from normal, low and intermediate histological grade groups. The morphology of organelles in secretory prostate epithelial cells; including Golgi apparatus, ER, lysosomes; was similar in prostate tissue samples across a range of Gleason scores. Mitochondrial morphology was not dramatically altered, but the number of membrane contacts with the ER notably increased with disease progression. A three-fold increase of tight mitochondria-ER membrane contact sites was observed in the intermediate Gleason score group compared to normal tissue. To investigate whether these changes were concurrent with an increased androgen signaling in the tissue, we investigated whether an anti-androgen used in the clinic to treat advanced prostate cancer (enzalutamide) could reverse the phenotype. Patient-derived explant tissues with an intermediate Gleason score were cultured ex vivo in the presence or absence of enzalutamide and the number of ER-mitochondria contacts were quantified for each matched pair of tissues. Enzalutamide treated tissue showed a significant reduction in the number and length of mitochondria-ER contact sites, suggesting a novel androgen-dependent regulation of these membrane contact sites. This study provides evidence for the first time that prostate epithelial cells undergo adaptations in membrane contact sites between mitochondria and the ER during prostate cancer progression. These adaptations are androgen-dependent and provide evidence for a novel hormone-regulated mechanism that support establishment and extension of MAMs. Future studies will determine whether these changes are required to maintain pro-proliferative signaling and metabolic changes that support prostate cancer cell viability.

Metamorphosis by ATG13 and ATG101 in human autophagy initiation.

ABBREVIATIONS: ATG, Autophagy-related, HORMA, protein domain named after HOP1-MAD2-REV7; RB1CC1, RB1 inducible coiled-coil 1; ULK, Unc-51-like kinase.

Yeast Models of Amyotrophic Lateral Sclerosis Type 8 Mimic Phenotypes Seen in Mammalian Cells Expressing Mutant VAPBP56S.

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disease that results in the loss of motor neurons and can occur sporadically or due to genetic mutations. Among the 30 genes linked to familial ALS, a P56S mutation in VAPB, an ER-resident protein that functions at membrane contact sites, causes ALS type 8. Mammalian cells expressing VAPBP56S have distinctive phenotypes, including ER collapse, protein and/or membrane-containing inclusions, and sensitivity to ER stress. VAPB is conserved through evolution and has two homologs in budding yeast, SCS2 and SCS22. Previously, a humanized version of SCS2 bearing disease-linked mutations was described, and it caused Scs2-containing inclusions when overexpressed in yeast. Here, we describe a yeast model for ALS8 in which the two SCS genes are deleted and replaced with a single chromosomal copy of either wild-type or mutant yeast SCS2 or human VAPB expressed from the SCS2 promoter. These cells display ER collapse, the formation of inclusion-like structures, and sensitivity to tunicamycin, an ER stress-inducing drug. Based on the phenotypic similarity to mammalian cells expressing VAPBP56S, we propose that these models can be used to study the molecular basis of cell death or dysfunction in ALS8. Moreover, other conserved ALS-linked genes may create opportunities for the generation of yeast models of disease.

Bridge-Like Lipid Transfer Proteins (BLTPs) in C. elegans: From Genetics to Structures and Functions.

In eukaryotic cells, lipid transfer can occur at membrane contact sites (MCS) to facilitate the exchange of various lipids between two adjacent cellular organelle membranes. Lipid transfer proteins (LTPs), including shuttle LTP or bridge-like LTP (BLTP), transport lipids at MCS and are critical for diverse cellular processes, including lipid metabolism, membrane trafficking, and cell signaling. BLTPs (BLTP1-5, including the ATG2 and VPS13 family proteins) contain lipid-accommodating hydrophobic repeating ß-groove (RBG) domains that allow the bulk transfer of lipids through MCS. Compared with vesicular lipid transfer and shuttle LTP, BLTPs have been only recently identified. Their functions and regulatory mechanisms are currently being unraveled in various model organisms and by diverse approaches. In this review, we summarize the genetics, structural features, and biological functions of BLTP in the genetically tractable model organism C. elegans. We discuss our recent studies and findings on C. elegans LPD-3, a prototypical megaprotein ortholog of BLTP1, with identified lipid transfer functions that are evolutionarily conserved in multicellular organisms and in human cells. We also highlight areas for future research of BLTP using C. elegans and complementary model systems and approaches. Given the emerging links of BLTP to several human diseases, including Parkinson's disease and Alkuraya-Kucinskas syndrome, discovering evolutionarily conserved roles of BLTPs and their mechanisms of regulation and action should contribute to new advances in basic cell biology and potential therapeutic development for related human disorders.

Gluing yeast peroxisomes - composition and function of membrane contact sites.

Membrane contact sites are defined as regions of close proximity between two membranes; this association is mediated by protein-protein and/or protein-lipid interactions. Contact sites are often involved in lipid transport, but also can perform other functions. Peroxisomal membrane contact sites have obtained little attention compared to those of other cell organelles. However, recent studies resulted in a big leap in our knowledge of the occurrence, composition and function of peroxisomal contact sites. Studies in yeast strongly contributed to this progress. In this Review, we present an overview of our current knowledge on peroxisomal membrane contact sites in various yeast species, including Hansenula polymorpha, Saccharomyces cerevisiae, Pichia pastoris and Yarrowia lipolytica. Yeast peroxisomes form contacts with almost all other cellular organelles and with the plasma membrane. The absence of a component of a yeast peroxisomal contact site complex results in a range of peroxisomal phenotypes, including metabolic and biogenesis defects and alterations in organelle number, size or position.

Pathogen vacuole membrane contact sites - close encounters of the fifth kind.

Vesicular trafficking and membrane fusion are well-characterized, versatile, and sophisticated means of 'long range' intracellular protein and lipid delivery. Membrane contact sites (MCS) have been studied in far less detail, but are crucial for 'short range' (10-30 nm) communication between organelles, as well as between pathogen vacuoles and organelles. MCS are specialized in the non-vesicular trafficking of small molecules such as calcium and lipids. Pivotal MCS components important for lipid transfer are the VAP receptor/tether protein, oxysterol binding proteins (OSBPs), the ceramide transport protein CERT, the phosphoinositide phosphatase Sac1, and the lipid phosphatidylinositol 4-phosphate (PtdIns(4)P). In this review, we discuss how these MCS components are subverted by bacterial pathogens and their secreted effector proteins to promote intracellular survival and replication.

The ultrastructural function of MLN64 in the late endosome-mitochondria membrane contact sites in placental cells.

The close apposition between two different organelles is critical in essential processes such as ion homeostasis, signaling, and lipid transition. However, information related to the structural features of membrane contact sites (MCSs) is limited. This study used immuno-electron microscopy and immuno-electron tomography (I-ET) to analyze the two- and three-dimensional structures of the late endosome-mitochondria contact sites in placental cells. Filamentous structures or tethers were identified that connected the late endosomes and mitochondria. Lamp1 antibody-labeled I-ET revealed enrichment of tethers in the MCSs. The cholesterol-binding endosomal protein metastatic lymph node 64 (MLN64) encoded by STARD3 was required for the formation of this apposition. The distance of the late endosome-mitochondria contact sites was <20 nm, shorter than that in STARD3-knockdown cells (<150 nm). The perturbation of cholesterol egress from the endosomes induced by U18666A treatment produced a longer distance in the contact sites than that in knockdown cells. The late endosome-mitochondria tethers failed to form correctly in STARD3-knockdown cells. Our results unravel the role of MLN64 involved in MCSs between late endosomes and mitochondria in placental cells.