A new perspective on liver diseases: Focusing on the mitochondria-associated endoplasmic reticulum membranes.

The pathogenesis of liver diseases is multifaceted and intricate, posing a persistent global public health challenge with limited therapeutic options. Therefore, further research into liver diseases is imperative for better comprehension and advancement in treatment strategies. Numerous studies have confirmed the endoplasmic reticulum (ER) and mitochondria as key organelles driving liver diseases. Notably, the mitochondrial-associated ER membranes (MAMs) establish a physical and functional connection between the ER and mitochondria, highlighting the importance of inter-organelle communication in maintaining their functional homeostasis. This review delves into the intricate architecture and regulative mechanism of the integrated MAM that facilitate the physiological transfer of signals and substances between organelles. Additionally, we also provide a detailed overview regarding the varied pathogenic roles of malfunctioning MAM in liver diseases, focusing on its involvement in the progression of ER stress and mitochondrial dysfunction, the regulation of mitochondrial dynamics and Ca2+ transfer, as well as the disruption of lipid and glucose homeostasis. Furthermore, the current challenges and prospects associated with MAM in liver disease research are thoroughly discussed. In conclusion, elucidating the specific structure and function of MAM in different liver diseases may pave the way for novel therapeutic strategies.

Protocol for detecting lysosome quantity and membrane permeability in acute myeloid leukemia cell lines.

Lysosomal function and activity are essential to support cellular adaptation to multiple stresses. For example, certain drugs can induce increased lysosomal membrane permeability to exert their anti-cancer effects. Here, we present a protocol to evaluate the lysosome alterations induced by drug treatment. We first describe the steps for inducing lysosomal alterations in cultured cells. We then show how to quantify the number of lysosomes, assess the integrity of lysosomal membranes, and determine lysosomal membrane permeabilization by using galectin puncta assay. For complete details on the use and execution of this protocol, please refer to Jiang et al.1.

Mild Heat Stress Alters the Physical State and Structure of Membranes in Triacylglycerol-Deficient Fission Yeast, Schizosaccharomyces pombe.

We investigated whether the elimination of two major enzymes responsible for triacylglycerol synthesis altered the structure and physical state of organelle membranes under mild heat shock conditions in the fission yeast, Schizosaccharomyces pombe. Our study revealed that key intracellular membrane structures, lipid droplets, vacuoles, the mitochondrial network, and the cortical endoplasmic reticulum were all affected in mutant fission yeast cells under mild heat shock but not under normal growth conditions. We also obtained direct evidence that triacylglycerol-deficient cells were less capable than wild-type cells of adjusting their membrane physical properties during thermal stress. The production of thermoprotective molecules, such as HSP16 and trehalose, was reduced in the mutant strain. These findings suggest that an intact system of triacylglycerol metabolism significantly contributes to membrane protection during heat stress.

Molecular architecture of coronavirus double-membrane vesicle pore complex.

Coronaviruses remodel the intracellular host membranes during replication, forming double-membrane vesicles (DMVs) to accommodate viral RNA synthesis and modifications1,2. SARS-CoV-2 non-structural protein 3 (nsp3) and nsp4 are the minimal viral components required to induce DMV formation and to form a double-membrane-spanning pore, essential for the transport of newly synthesized viral RNAs3-5. The mechanism of DMV pore complex formation remains unknown. Here we describe the molecular architecture of the SARS-CoV-2 nsp3-nsp4 pore complex, as resolved by cryogenic electron tomography and subtomogram averaging in isolated DMVs. The structures uncover an unexpected stoichiometry and topology of the nsp3-nsp4 pore complex comprising 12 copies each of nsp3 and nsp4, organized in 4 concentric stacking hexamer rings, mimicking a miniature nuclear pore complex. The transmembrane domains are interdigitated to create a high local curvature at the double-membrane junction, coupling double-membrane reorganization with pore formation. The ectodomains form extensive contacts in a pseudo-12-fold symmetry, belting the pore complex from the intermembrane space. A central positively charged ring of arginine residues coordinates the putative RNA translocation, essential for virus replication. Our work establishes a framework for understanding DMV pore formation and RNA translocation, providing a structural basis for the development of new antiviral strategies to combat coronavirus infection.

Spatially confined photoacoustic effects of responsive nanoassembly boosts lysosomal membrane permeabilization and immunotherapy of triple-negative breast cancer.

Although immunogenic cell death (ICD) induced by lysosomal membrane permeabilization (LMP) evidently enhance the effectiveness of antitumor immunity for triple-negative breast cancer (TNBC) with poor immunogenicity, their potential is increasingly restricted by the development of other death pathways and the repair of lysosomes by endoplasmic reticulum (ER) during LMP induction. Herein, a polydopamine nanocomposite with i-motif DNA modified and BNN6 loaded is prepared toward boosting LMP and immunotherapy of TNBC by synergy of spatially confined photoacoustic (PA) effects and nitric oxide. Combining the high-frequency pulsed laser (4000 kHz) with the intra-lysosomal assembly of nanocomposites produced spatially confined and significantly boosted PA effects (4.8-fold higher than the individually dispersed particles extracellular), suppressing damage to other cellular components and selectively reducing lysosomal integrity to 19.2 %. Simultaneously, the releasing of nitric oxide inhibited the repair of lysosomes by ER stress, causing exacerbated LMP. Consequently, efficient immune activation was achieved, including the abundant releasing of CRT/HMGB1 (5.93-6.8-fold), the increasing maturation of dendritic cells (3.41-fold), and the fostered recruitment of CD4+/CD8+T cells (3.99-3.78-fold) in vivo. The study paves a new avenue for the rational design and synergy of confined energy conversion and responsive nanostructures to achieve the treatment of low immunogenicity tumors. STATEMENT OF SIGNIFICANCE: A strategy of boosting lysosomal membrane permeabilization (LMP) and concomitantly preventing the repair was developed to address the immunotherapy challenge of triple-negative breast cancer. Spatially confined and significantly enhanced photoacoustic (PA) effects were achieved through DNA-guided pH-responsive assembly of polydopamine nanocomposites in lysosomes and application of a high-frequency pulsed laser. Efficient immunogenic cell death was guaranteed by selective and powerful damage of lysosomal membranes through the significant contrast of PA intensities for dispersed/assembled particles and nitric oxide release induced endoplasmic reticulum stress. The study paves a new avenue for the rational design and synergy of confined energy conversion and responsive nanostructures to achieve the treatment of low immunogenicity tumors.

Endocytosed dsRNAs induce lysosomal membrane permeabilization that allows cytosolic dsRNA translocation for Drosophila RNAi responses.

RNA interference (RNAi) is a gene-silencing mechanism triggered by the cytosolic entry of double-stranded RNAs (dsRNAs). Many animal cells internalize extracellular dsRNAs via endocytosis for RNAi induction. However, it is not clear how the endocytosed dsRNAs are translocated into the cytosol across the endo/lysosomal membrane. Herein, we show that in Drosophila S2 cells, endocytosed dsRNAs induce lysosomal membrane permeabilization (LMP) that allows cytosolic dsRNA translocation. LMP mediated by dsRNAs requires the lysosomal Cl-/H+ antiporter ClC-b/DmOstm1. In clc-b or dmostm1 knockout S2 cells, extracellular dsRNAs are endocytosed and reach the lysosomes normally but fail to enter the cytosol. Pharmacological induction of LMP restores extracellular dsRNA-directed RNAi in clc-b or dmostm1-knockout cells. Furthermore, clc-b or dmostm1 mutant flies are defective in extracellular dsRNA-directed RNAi and its associated antiviral immunity. Therefore, endocytosed dsRNAs have an intrinsic ability to induce ClC-b/DmOstm1-dependent LMP that allows cytosolic dsRNA translocation for RNAi responses in Drosophila cells.

Pharmaceutical Analysis in the Plant Endomembrane System.

Vesicle trafficking is an essential cellular process conserved in eukaryotes to precisely transport proteins to their destinations. The plant endomembrane system plays a pivotal role in orchestrating this vesicle-mediated protein transport process, making its study essential for a comprehensive understanding of plant growth and development. Pharmaceutical analysis proves highly useful in investigating the plant endomembrane system. To facilitate further studies in this area, we present a summary of several commonly used chemical inhibitors in this chapter, providing a practical resource for researchers interested in the plant endomembrane system.

Lysosomal membrane contact sites: Integrative hubs for cellular communication and homeostasis.

Lysosomes are more than just cellular recycling bins; they play a crucial role in regulating key cellular functions. Proper lysosomal function is essential for growth pathway regulation, cell proliferation, and metabolic homeostasis. Impaired lysosomal function is associated with lipid storage disorders and neurodegenerative diseases. Lysosomes form extensive and dynamic close contacts with the membranes of other organelles, including the endoplasmic reticulum, mitochondria, peroxisomes, and lipid droplets. These membrane contacts sites (MCSs) are vital for many lysosomal functions. In this chapter, we will explore lysosomal MCSs focusing on the machinery that mediates these contacts, how they are regulated, and their functional implications on physiology and pathology.

The astrovirus N-terminal nonstructural protein anchors replication complexes to the perinuclear ER membranes.

An essential aspect of positive-sense RNA virus replication is anchoring the replication complex (RC) to cellular membranes. Positive-sense RNA viruses employ diverse strategies, including co-translational membrane targeting through signal peptides and co-opting cellular membrane trafficking components. Often, N-terminal nonstructural proteins play a crucial role in linking the RC to membranes, facilitating the early association of the replication machinery. Astroviruses utilize a polyprotein strategy to synthesize nonstructural proteins, relying on subsequent processing to form replication-competent complexes. This study provides evidence for the perinuclear ER membrane association of RCs in five distinct human astrovirus strains. Using tagged recombinant classical human astrovirus 1 and neurotropic MLB2 strains, we establish that the N-terminal domain guides the ER membrane association. We identified di-arginine motifs responsible for the perinuclear ER retention and formation of functional RCs through mutational analysis of the N-terminal domain in replicon and reverse genetics systems. In addition, we demonstrate the association of key components of the astrovirus replication complex: double-stranded RNA, RNA-dependent RNA polymerase, protease, and N-terminal protein. Our findings highlight the intricate virus-ER interaction mechanism employed by astroviruses, potentially leading to the development of novel antiviral intervention strategies.

Inhibition of PLA2G4A attenuated valproic acid- induced lysosomal membrane permeabilization and restored impaired autophagic flux: Implications for hepatotoxicity.

Valproic acid (VPA) has broad efficacy against several seizures but causes liver injury limiting its prolonged clinical use. Some studies have demonstrated that VPA-induced hepatotoxicity is characterized by microvesicular hepatic steatosis. However, novel detailed mechanisms to explain VPA-induced hepatic steatosis and experimentally rigorously validated protective agents are still lacking. In this study, 8-week-old C57BL/6J mice were gavaged with VPA (500 mg/kg/d) for 4 weeks to establish an in vivo model of VPA-induced chronic liver injury. Quantitative proteomic and non-targeted lipidomic analyses were performed to explore the underlying mechanisms of VPA-induced hepatotoxicity. As a result, VPA-induced hepatotoxicity is associated with impaired autophagic flux, which is attributed to lysosomal dysfunction. Further studies revealed that VPA-induced lysosomal membrane permeabilization (LMP), allows soluble lysosomal enzymes to leak into the cytosol, which subsequently led to impaired lysosomal acidification. A lower abundance of glycerophospholipids and an increased abundance of lysophospholipids in liver tissues of mice in the VPA group strongly indicated that VPA-induced LMP may be mediated by the activation of phospholipase PLA2G4A. Metformin (Met) acted as a potential protective agent attenuating VPA-induced liver dysfunction and excessive lipid accumulation. Molecular docking and cellular thermal shift assays demonstrated that Met inhibited the activity of PLA2G4A by directly binding to it, thereby ameliorating VPA-induced LMP and autophagic flux impairment. In conclusion, this study highlights the therapeutic potential of targeting PLA2G4A-mediated lysosomal dysfunction in VPA-induced hepatotoxicity.

SigmaR1 shapes rough endoplasmic reticulum membrane sheets.

Rough endoplasmic reticulum (ER) sheets are a fundamental domain of the ER and the gateway into the secretory pathway. Although reticulon proteins stabilize high-curvature ER tubules, it is unclear whether other proteins scaffold the flat membranes of rough ER sheets. Through a proteomics screen using ER sheet-localized RNA-binding proteins as bait, we identify the sigma-1 receptor (SigmaR1) as an ER sheet-shaping factor. High-resolution live cell imaging and electron tomography assign SigmaR1 as an ER sheet-localized factor whose levels determine the amount of rough ER sheets in cells. Structure-guided mutagenesis and in vitro reconstitution on giant unilamellar vesicles further support a mechanism whereby SigmaR1 oligomers use their extended arrays of amphipathic helices to bind and flatten the lumenal leaflet of ER membranes to oppose membrane curvature and stabilize rough ER sheets.

Golgi pH homeostasis stabilizes the lysosomal membrane through N-glycosylation of membrane proteins.

Protein glycosylation plays a vital role in various cellular functions, many of which occur within the Golgi apparatus. The Golgi pH regulator (GPHR) is essential for the proper functioning of the Golgi apparatus. The lysosomal membrane contains highly glycosylated membrane proteins in abundance. This study investigated the role of the Golgi luminal pH in N-glycosylation of lysosomal membrane proteins and the effect of this protein modification on membrane stability using Gphr-deficient MEFs. We showed that Gphr deficiency causes an imbalance in the Golgi luminal pH, resulting in abnormal protein N-glycosylation, indicated by a reduction in sialylated glycans and markedly reduced molecular weight of glycoproteins. Further experiments using FRAP and PLA revealed that Gphr deficiency prevented the trafficking dynamics and proximity condition of glycosyltransferases in the Golgi apparatus. In addition, incomplete N-glycosylation of lysosomal membrane proteins affected lysosomal membrane stability, as demonstrated by the increased susceptibility to lysosomal damage. Thus, this study highlights the critical role of Golgi pH regulation in controlling protein glycosylation and the impact of Golgi dysfunction on lysosomal membrane stability.

Does deacclimation reverse the changes in structural/physicochemical properties of the chloroplast membranes that are induced by cold acclimation in oilseed rape?

Winter crops acquire frost tolerance during the process of cold acclimation when plants are exposed to low but non-freezing temperatures that is connected to specific metabolic adjustments. Warm breaks during/after cold acclimation disturb the natural process of acclimation, thereby decreasing frost tolerance and can even result in a resumption of growth. This phenomenon is called deacclimation. In the last few years, studies that are devoted to deacclimation have become more important (due to climate changes) and necessary to be able to understand the mechanisms that occur during this phenomenon. In the acclimation of plants to low temperatures, the importance of plant membranes is indisputable; that is why the main aim of our studies was to answer the question of whether (and to what extent) deacclimation alters the physicochemical properties of the plant membranes. The studies were focused on chloroplast membranes from non-acclimated, cold-acclimated and deacclimated cultivars of winter oilseed rape. The analysis of the membranes (formed from chloroplast lipid fractions) using the Langmuir technique revealed that cold acclimation increased membrane fluidity (expressed as the Alim values), while deacclimation generally decreased the values that were induced by cold. Moreover, because the chloroplast membranes were penetrated by lipophilic molecules such as carotenoids or tocopherols, the relationships between the structure of the lipids and the content of these antioxidants in the chloroplast membranes during the process of the cold acclimation and deacclimation of oilseed rape are discussed.

RNA scaffolds the Golgi ribbon by forming condensates with GM130.

The mammalian Golgi is composed of stacks that are laterally connected into a continuous ribbon-like structure. The integrity and function of the ribbon is disrupted under stress conditions, but the molecular mechanisms remain unclear. Here we show that the ribbon is maintained by biomolecular condensates of RNA and the Golgi matrix protein GM130 (GOLGA2). We identify GM130 as a membrane-bound RNA-binding protein, which directly recruits RNA and associated RNA-binding proteins to the Golgi membrane. Acute degradation of RNA or GM130 in cells disrupts the ribbon. Under stress conditions, RNA dissociates from GM130 and the ribbon is disjointed, but after the cells recover from stress the ribbon is restored. When overexpressed in cells, GM130 forms RNA-dependent liquid-like condensates. GM130 contains an intrinsically disordered domain at its amino terminus, which binds RNA to induce liquid-liquid phase separation. These co-condensates are sufficient to link purified Golgi membranes, reconstructing lateral linking of stacks into a ribbon-like structure. Together, these studies show that RNA acts as a structural biopolymer that together with GM130 maintains the integrity of the Golgi ribbon.

Organelle-Targeted Laurdans Measure Heterogeneity in Subcellular Membranes and Their Responses to Saturated Lipid Stress.

Organelles feature characteristic lipid compositions that lead to differences in membrane properties. In cells, membrane ordering and fluidity are commonly measured using the solvatochromic dye Laurdan, whose fluorescence is sensitive to lipid packing. As a general lipophilic dye, Laurdan stains all hydrophobic environments in cells; therefore, it is challenging to characterize membrane properties in specific organelles or assess their responses to pharmacological treatments in intact cells. Here, we describe the synthesis and application of Laurdan-derived probes that read out the membrane packing of individual cellular organelles. The set of organelle-targeted Laurdans (OTL) localizes to the ER, mitochondria, lysosomes, and Golgi compartments with high specificity while retaining the spectral resolution needed to detect biological changes in membrane ordering. We show that ratiometric imaging with OTLs can resolve membrane heterogeneity within organelles as well as changes in lipid packing resulting from inhibition of trafficking or bioenergetic processes. We apply these probes to characterize organelle-specific responses to saturated lipid stress. While the ER and lysosomal membrane fluidity is sensitive to exogenous saturated fatty acids, that of mitochondrial membranes is protected. We then use differences in ER membrane fluidity to sort populations of cells based on their fatty acid diet, highlighting the ability of organelle-localized solvatochromic probes to distinguish between cells based on their metabolic state. These results expand the repertoire of targeted membrane probes and demonstrate their application in interrogating lipid dysregulation.

Rft1 catalyzes lipid-linked oligosaccharide translocation across the ER membrane.

The eukaryotic asparagine (N)-linked glycan is pre-assembled as a fourteen-sugar oligosaccharide on a lipid carrier in the endoplasmic reticulum (ER). Seven sugars are first added to dolichol pyrophosphate (PP-Dol) on the cytoplasmic face of the ER, generating Man5GlcNAc2-PP-Dol (M5GN2-PP-Dol). M5GN2-PP-Dol is then flipped across the bilayer into the lumen by an ER translocator. Genetic studies identified Rft1 as the M5GN2-PP-Dol flippase in vivo but are at odds with biochemical data suggesting Rft1 is dispensable for flipping in vitro. Thus, the question of whether Rft1 plays a direct or an indirect role during M5GN2-PP-Dol translocation has been controversial for over two decades. We describe a completely reconstituted in vitro assay for M5GN2-PP-Dol translocation and demonstrate that purified Rft1 catalyzes the translocation of M5GN2-PP-Dol across the lipid bilayer. These data, combined with in vitro results demonstrating substrate selectivity and rft1∆ phenotypes, confirm the molecular identity of Rft1 as the M5GN2-PP-Dol ER flippase.

Structural and mechanistic insights into a lysosomal membrane enzyme HGSNAT involved in Sanfilippo syndrome.

Heparan sulfate (HS) is degraded in lysosome by a series of glycosidases. Before the glycosidases can act, the terminal glucosamine of HS must be acetylated by the integral lysosomal membrane enzyme heparan-α-glucosaminide N-acetyltransferase (HGSNAT). Mutations of HGSNAT cause HS accumulation and consequently mucopolysaccharidosis IIIC, a devastating lysosomal storage disease characterized by progressive neurological deterioration and early death where no treatment is available. HGSNAT catalyzes a unique transmembrane acetylation reaction where the acetyl group of cytosolic acetyl-CoA is transported across the lysosomal membrane and attached to HS in one reaction. However, the reaction mechanism remains elusive. Here we report six cryo-EM structures of HGSNAT along the reaction pathway. These structures reveal a dimer arrangement and a unique structural fold, which enables the elucidation of the reaction mechanism. We find that a central pore within each monomer traverses the membrane and controls access of cytosolic acetyl-CoA to the active site at its luminal mouth where glucosamine binds. A histidine-aspartic acid catalytic dyad catalyzes the transfer reaction via a ternary complex mechanism. Furthermore, the structures allow the mapping of disease-causing variants and reveal their potential impact on the function, thus creating a framework to guide structure-based drug discovery efforts.

Giant organelle vesicles to uncover intracellular membrane mechanics and plasticity.

Tools for accessing and studying organelles remain underdeveloped. Here, we present a method by which giant organelle vesicles (GOVs) are generated by submitting cells to a hypotonic medium followed by plasma membrane breakage. By this means, GOVs ranging from 3 to over 10 µm become available for micromanipulation. GOVs are made from organelles such as the endoplasmic reticulum, endosomes, lysosomes and mitochondria, or in contact with one another such as giant mitochondria-associated ER membrane vesicles. We measure the mechanical properties of each organelle-derived GOV and find that they have distinct properties. In GOVs procured from Cos7 cells, for example, bending rigidities tend to increase from the endoplasmic reticulum to the plasma membrane. We also found that the mechanical properties of giant endoplasmic reticulum vesicles (GERVs) vary depending on their interactions with other organelles or the metabolic state of the cell. Lastly, we demonstrate GERVs' biochemical activity through their capacity to synthesize triglycerides and assemble lipid droplets. These findings underscore the potential of GOVs as valuable tools for studying the biophysics and biology of organelles.

Mfn2 regulates mitochondria and mitochondria-associated endoplasmic reticulum membrane function in neurodegeneration induced by repeated sevoflurane exposure.

Repeated sevoflurane exposure in neonatal mice can leads to neuronal apoptosis and mitochondrial dysfunction. The mitochondria are responsible for energy production to maintain homeostasis in the central nervous system. The mitochondria-associated endoplasmic reticulum membrane (MAM) is located between the mitochondria and endoplasmic reticulum (ER), and it is critical for mitochondrial function and cell survival. MAM malfunction contributes to neurodegeneration, however, whether it is involved in sevoflurane-induced neurotoxicity remains unknown. Our study demonstrated that repeated sevoflurane exposure induced mitochondrial dysfunction and dampened the MAM structure. The upregulated ER-mitochondria tethering enhanced Ca2+ transition from the cytosol to the mitochondria. Overload of mitochondrial Ca2+ contributed to opening of the mitochondrial permeability transition pore (mPTP), which caused neuronal apoptosis. Mitofusin 2(Mfn2), a key regulator of ER-mitochondria contacts, was found to be suppressed after repeated sevoflurane exposure, while restoration of Mfn2 expression alleviated cognitive dysfunction due to repeated sevoflurane exposure in the adult mice. These evidences suggest that sevoflurane-induced MAM malfunction is vulnerable to Mfn2 suppression, and the enhanced ER-mitochondria contacts promotes mitochondrial Ca2+ overload, contributing to mPTP opening and neuronal apoptosis. This paper sheds light on a novel mechanism of sevoflurane-induced neurotoxicity. Furthermore, targeting Mfn2-mediated regulation of the MAM structure and mitochondrial function may provide a therapeutic advantage in sevoflurane-induced neurodegeneration.

Structure and mechanism of the human CTDNEP1-NEP1R1 membrane protein phosphatase complex necessary to maintain ER membrane morphology.

C-terminal Domain Nuclear Envelope Phosphatase 1 (CTDNEP1) is a noncanonical protein serine/threonine phosphatase that has a conserved role in regulating ER membrane biogenesis. Inactivating mutations in CTDNEP1 correlate with the development of medulloblastoma, an aggressive childhood cancer. The transmembrane protein Nuclear Envelope Phosphatase 1 Regulatory Subunit 1 (NEP1R1) binds CTDNEP1, but the molecular details by which NEP1R1 regulates CTDNEP1 function are unclear. Here, we find that knockdown of NEP1R1 generates identical phenotypes to reported loss of CTDNEP1 in mammalian cells, establishing CTDNEP1-NEP1R1 as an evolutionarily conserved membrane protein phosphatase complex that restricts ER expansion. Mechanistically, NEP1R1 acts as an activating regulatory subunit that directly binds and increases the phosphatase activity of CTDNEP1. By defining a minimal NEP1R1 domain sufficient to activate CTDNEP1, we determine high-resolution crystal structures of the CTDNEP1-NEP1R1 complex bound to a peptide sequence acting as a pseudosubstrate. Structurally, NEP1R1 engages CTDNEP1 at a site distant from the active site to stabilize and allosterically activate CTDNEP1. Substrate recognition is facilitated by a conserved Arg residue in CTDNEP1 that binds and orients the substrate peptide in the active site. Together, this reveals mechanisms for how NEP1R1 regulates CTDNEP1 and explains how cancer-associated mutations inactivate CTDNEP1.