Endoplasmic Reticulum Membrane Research Articles

Co-opted cytosolic proteins form condensate substructures within membranous replication organelles of a positive-strand RNA virus.

Positive-strand RNA viruses co-opt organellar membranes for biogenesis of viral replication organelles (VROs). Tombusviruses also co-opt pro-viral cytosolic proteins to VROs. It is currently not known what type of molecular organization keeps co-opted proteins sequestered within membranous VROs. In this study, we employed tomato bushy stunt virus (TBSV) and carnation Italian ringspot virus (CIRV) - Nicotiana benthamiana pathosystems to identify biomolecular condensate formation in VROs. We show that TBSV p33 and the CIRV p36 replication proteins sequester glycolytic and fermentation enzymes in unique condensate substructures associated with membranous VROs. We find that p33 and p36 form droplets in vitro driven by intrinsically disordered region. The replication protein organizes partitioning of co-opted host proteins into droplets. VRO-associated condensates are critical for local adenosine triphosphate production to support energy for virus replication. We find that co-opted endoplasmic reticulum membranes and actin filaments form meshworks within and around VRO condensates, contributing to unique composition and structure. We propose that p33/p36 organize liquid-liquid phase separation of co-opted concentrated host proteins in condensate substructures within membranous VROs. Overall, we demonstrate that subverted membranes and condensate substructures co-exist and are critical for VRO functions. The replication proteins induce and connect the two substructures within VROs.

STIM2 variants regulate Orai1/TRPC1/TRPC4-mediated store-operated Ca2+ entry and mitochondrial Ca2+ homeostasis in cardiomyocytes

The stromal interaction molecules (STIMs) are the sarcoplasmic reticulum (SR) Ca2+ sensors that trigger store-operated Ca2+ entry (SOCE) in a variety of cell types. While STIM1 isoform has been the focus of the research in cardiac pathophysiology, the function of the homolog STIM2 remains unknown. Using Ca2+ imaging and patch-clamp techniques, we showed that knockdown (KD) of STIM2 by siRNAs increased SOCE and the ISOC current in neonatal rat ventricular cardiomyocytes (NRVMs). Within this cardiomyocyte model, we identified the transcript expression of Stim2.1 and Stim2.2 splice variants, with predominance for Stim2.2. Using conventional and super-resolution confocal microscopy (STED), we found that exogenous STIM2.1 and STIM2.2 formed pre-clusters with a reticular organization at rest. Following SR Ca2+ store depletion, some STIM2.1 and STIM2.2 clusters were translocated to SR-plasma membrane (PM) junctions and co-localized with Orai1. The overexpression strategy revealed that STIM2.1 suppressed Orai1-mediated SOCE and the ISOC current while STIM2.2 enhanced SOCE. STIM2.2-enhanced SOCE was also dependent on TRPC1 and TRPC4. Even if STIM2 KD or splice variants overexpression did not affect cytosolic Ca2+ cycling, we observed, using Rhod-2/AM Ca2+ imaging, that Orai1 inhibition or STIM2.1 overexpression abolished the mitochondrial Ca2+ (mCa2+) uptake, as opposed to STIM2 KD. We also found that STIM2 was present in the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) by interacting with the inositol trisphosphate receptors (IP3Rs), voltage-dependent anion channel (VDAC), mitochondrial Ca2+ uniporter (MCU), and mitofusin-2 (MNF2). Our results suggested that, in NRVMs, STIM2.1 constitutes the predominant functional variant that negatively regulates Orai1-generated SOCE. It participates in the control of mCa2+ uptake capacity possibly via the STIM2-IP3Rs-VDAC-MCU and MNF2 complex.

Ethanol-Induced Hepatic Ferroptosis Is Mediated by PERK-Dependent MAMs Formation: Preventive Role of Quercetin.

Iron deposition is frequently observed in alcoholic liver disease (ALD), which indicates a potential role of ferroptosis in its development. This study aims to explore the effects of quercetin on ferroptosis in ALD and elucidates the underlying mechanism involving the formation of mitochondria-associated endoplasmic reticulum membranes (MAMs) mediated by protein kinase RNA-like endoplasmic reticulum kinase (PERK). C57BL/6J mice are fed either a regular or an ethanol-containing liquid diet (with 28% energy form ethanol) with or without quercetin supplementation (100mgkg-1 BW) for 12 weeks. Ethanol feeding or treatment induced ferroptosis in mice and AML12 cells, which is associated with increased MAMs formation and PERK expression within MAMs. Quercetin attenuates these changes and protects against ethanol-induced liver injury. The antiferroptotic effect of quercetin is abolished by ferroptosis inducers, but mimicked by ferroptosis inhibitors and PERK knockdown. The study demonstrates that PERK structure, rather than its kinase activity (transfected with the K618A site mutation that inhibits kinase activity-ΔK plasmid or protein C terminal knockout-ΔC plasmid of PERK), mediates the enhanced MAMs formation and ferroptosis during the ethanol exposure. Quercetin ameliorates ethanol-induced liver injury by inhibiting ferroptosis via modulating PERK-dependent MAMs formation.

ER membrane complex (EMC): Structure, functions, and roles in diseases.

The endoplasmic reticulum (ER) is the largest membrane system in eukaryotic cells and is the primary site for the biosynthesis of lipids and carbohydrates, as well as for the folding, assembly, modification, and transport of secreted and integrated membrane proteins. The ER membrane complex (EMC) on the ER membrane is an ER multiprotein complex that affects the quality control of membrane proteins, which is abundant and widely preserved. Its disruption has been found to affect a wide range of processes, including protein and lipid synthesis, organelle communication, endoplasmic reticulum stress, and viral maturation, and may lead to neurodevelopmental disorders and cancer. Therefore, EMC has attracted the attention of many scholars and become a hot field. In this paper, we summarized the main contributions of the research of EMC in the past nearly 15 years, and reviewed the structure and function of EMC as well as its related diseases. We hope this review will promote further progress of research on EMC.

Steroidogenesis Upregulation through Mitochondria-Associated Endoplasmic Reticulum Membranes and Mitochondrial Dynamics in Rat Testes: The Role of D-Aspartate.

Mitochondria-Associated Endoplasmic Reticulum Membranes (MAMs) mediate the communication between the Endoplasmic Reticulum (ER) and the mitochondria, playing a fundamental role in steroidogenesis. This study aimed to understand how D-aspartate (D-Asp), a well-known stimulator of testosterone biosynthesis and spermatogenesis, affects the mechanism of steroidogenesis in rat testes. Our results suggested that D-Asp exerts this function through MAMs, affecting lipid trafficking, calcium signaling, ER stress, and mitochondrial dynamics. After 15 days of oral administration of D-Asp to rats, there was an increase in both antioxidant enzymes (SOD and Catalase) and in the protein expression levels of ATAD3A, FACL4, and SOAT1, which are markers of lipid transfer, as well as VDAC and GRP75, which are markers of calcium signaling. Additionally, there was a decrease in protein expression levels of GRP78, a marker of aging that counteracts ER stress. The effects of D-Asp on mitochondrial dynamics strongly suggested its active role as well. It induced the expression levels of proteins involved in fusion (MFN1, MFN2, and OPA1) and in biogenesis (NRF1 and TFAM), as well as in mitochondrial mass (TOMM20), and decreased the expression level of DRP1, a crucial mitochondrial fission marker. These findings suggested D-Asp involvement in the functional improvement of mitochondria during steroidogenesis. Immunofluorescent signals of ATAD3A, MFN1/2, TFAM, and TOMM20 confirmed their localization in Leydig cells showing an intensity upgrade in D-Asp-treated rat testes. Taken together, our results demonstrate the involvement of D-Asp in the steroidogenesis of rat testes, acting at multiple stages of both MAMs and mitochondrial dynamics, opening new opportunities for future investigation in other steroidogenic tissues.

MemPrep, a new technology for isolating organellar membranes provides fingerprints of lipid bilayer stress

Biological membranes have a stunning ability to adapt their composition in response to physiological stress and metabolic challenges. Little is known how such perturbations affect individual organelles in eukaryotic cells. Pioneering work has provided insights into the subcellular distribution of lipids in the yeast Saccharomyces cerevisiae, but the composition of the endoplasmic reticulum (ER) membrane, which also crucially regulates lipid metabolism and the unfolded protein response, remains insufficiently characterized. Here, we describe a method for purifying organelle membranes from yeast, MemPrep. We demonstrate the purity of our ER membrane preparations by proteomics, and document the general utility of MemPrep by isolating vacuolar membranes. Quantitative lipidomics establishes the lipid composition of the ER and the vacuolar membrane. Our findings provide a baseline for studying membrane protein biogenesis and have important implications for understanding the role of lipids in regulating the unfolded protein response (UPR). The combined preparative and analytical MemPrep approach uncovers dynamic remodeling of ER membranes in stressed cells and establishes distinct molecular fingerprints of lipid bilayer stress.

Structural insights into human EMC and its interaction with VDAC.

The endoplasmic reticulum (ER) membrane protein complex (EMC) is a conserved, multi-subunit complex acting as an insertase at the ER membrane. Growing evidence shows that the EMC is also involved in stabilizing and trafficking membrane proteins. However, the structural basis and regulation of its multifunctionality remain elusive. Here, we report cryo-electron microscopy structures of human EMC in apo- and voltage-dependent anion channel (VDAC)-bound states at resolutions of 3.47 Å and 3.32 Å, respectively. We discovered a specific interaction between VDAC proteins and the EMC at mitochondria-ER contact sites, which is conserved from yeast to humans. Moreover, we identified a gating plug located inside the EMC hydrophilic vestibule, the substrate-binding pocket for client insertion. Conformation changes of this gating plug during the apo-to-VDAC-bound transition reveal that the EMC unlikely acts as an insertase in the VDAC1-bound state. Based on the data analysis, the gating plug may regulate EMC functions by modifying the hydrophilic vestibule in different states. Our discovery offers valuable insights into the structural basis of EMC's multifunctionality.

Exocytic plasma membrane flows remodel endoplasmic reticulum-plasma membrane tethering for septin collar assembly.

Endoplasmic reticulum (ER)-plasma membrane (PM) tethering is crucial for the non-vesicular lipid transport between the ER membrane and the PM. However, the PM-associated ER can impede the PM binding of cytoskeletons and other organelles. It is poorly understood how the competition between the ER and cytoskeletons/organelles on the PM is resolved. Here, we show that, upon septin collar assembly, ER-PM tethering proteins are excluded from the yeast bud sites, and the PM-associated ER is locally detached from the PM. Our results suggest that PM flows by polarized exocytosis extrude PM proteins, including ER-PM tethering proteins, from the bud sites. When the reorganization of the ER-PM tethering was inhibited by exocytosis repression, septin localization was restricted to the PM sites poor in ER-PM tethering proteins. This study proposes machinery reconciling ER-septin competition on the PM, providing mechanistic insights into the spatial organization of PM-associated organelles and cytoskeletons.

Neuronal Nitric Oxide Synthase Regulates Cerebellar Parallel Fiber Slow EPSC in Purkinje Neurons by Modulating STIM1-Gated TRPC3-Containing Channels.

Responding to burst stimulation of parallel fibers (PFs), cerebellar Purkinje neurons (PNs) generate a convolved synaptic response displaying a fast excitatory postsynaptic current (EPSCFast) followed by a slow EPSC (EPSCSlow). The latter is companied with a rise of intracellular Ca2+ and critical for motor coordination. The genesis of EPSCSlow in PNs results from activation of metabotropic type 1 glutamate receptor (mGluR1), oligomerization of stromal interaction molecule 1 (STIM1) on the membrane of endoplasmic reticulum (ER) and opening of transient receptor potential canonical 3 (TRPC3) channels on the plasma membrane. Neuronal nitric oxide synthase (nNOS) is abundantly expressed in PFs and granule neurons (GNs), catalyzing the production of nitric oxide (NO) hence regulating PF-PN synaptic function. We recently found that nNOS/NO regulates the morphological development of PNs through mGluR1-regulated Ca2+-dependent mechanism. This study investigated the role of nNOS/NO in regulating EPSCSlow. Electrophysiological analyses showed that EPSCSlow in cerebellar slices of nNOS knockout (nNOS-/-) mice was significantly larger than that in wildtype (WT) mice. Activation of mGluR1 in cultured PNs from nNOS-/- mice evoked larger TRPC3-channel mediated currents and intracellular Ca2+ rise than that in PNs from WT mice. In addition, nNOS inhibitor and NO-donor increased and decreased, respectively, the TRPC3-current and Ca2+ rise in PNs. Moreover, the NO-donor effectively decreased TRPC3 currents in HEK293 cells expressing WT STIM1, but not cells expressing a STIM1 with cysteine mutants. These novel findings indicate that nNOS/NO inhibits TRPC3-containig channel mediated cation influx during EPSCSlow, at least in part, by S-nitrosylation of STIM1.

Polystyrene microplastics impair the functions of cultured mouse Leydig (TM3) and Sertoli (TM4) cells by inducing mitochondrial-endoplasmic reticulum damage

Many laboratory studies demonstrated that the exposure to microplastics causes testosterone deficiency and spermatogenic impairment in mammals; however, the mechanism underlying this process remains still unclear. In this study, we investigated the effects of polystyrene microplastics (PS-MP) on the proliferation and functionality of cultured Leydig (TM3) and Sertoli (TM4) cells, focusing on the mitochondrial compartment and its association with the endoplasmic reticulum (ER). The in vitro exposure to PS-MP caused a substantial reduction in cellular viability in TM3 and TM4 cells. In TM3 cells PS-MP inhibited the protein levels of StAR and of steroidogenic enzymes 3β-HSD and 17β-HSD, and in TM4 cells PS-MP inhibited the protein levels of the androgen receptors other than the activity of lactate dehydrogenase (LDH). PS-MP inhibited the functions of TM3 and TM4, as evidenced by the decrease of the phosphorylation of ERK1/2 and Akt in both cell lines. The oxidative stress caused by PS-MP decreased antioxidant defense in TM3 and TM4 cells, promoting autophagic and apoptotic processes. Furthermore, we found mitochondrial dysfunction and activation of ER stress. It is known that mitochondria are closely associated with ER to form the Mitochondrial-Associated Endoplasmic Reticulum Membranes (MAM), the site of calcium ions transfer as well as of lipid biosynthesis-involved enzymes and cholesterol transport from ER to the mitochondria. For the first time, we studied this aspect in PS-MP-treated TM3 and TM4 cells and MAMs dysregulation was observed. This study is the first to elucidate the intracellular mechanism underlying the effects of PS-MPs in somatic testicular cells, corroborating that PS-MP might be one of the causes of an increase in male infertility through the impairment of steroidogenesis in Leydig cells and of the nurse function of Sertoli cells. Thus, our findings contributed with new information to the mechanism underlying the effects of PS-MP on the male reproductive system.

4-Phenylbutyric Acid Attenuates Soybean Glycinin/β-Conglycinin-Induced IPEC-J2 Cells Apoptosis by Regulating the Mitochondria-Associated Endoplasmic Reticulum Membrane and NLRP-3.

Glycinin (11S) and β-conglycinin (7S) from soybean (glycine max) cause diarrhea and intestinal barrier damage in young animals. Understanding the mechanisms underlying the damage caused by 7S and 11S, it is vital to develop strategies to eliminate allergenicity. Consequently, we investigated 7S/11S-mediated apoptosis in porcine intestinal epithelial (IPEC-J2) cells. IPEC-J2 cells suffered endoplasmic reticulum stress (ERS) in response to 7S and 11S, activating protein kinase RNA-like ER kinase, activating transcription factor 6, C/EBP homologous protein, and inositol-requiring enzyme 1 alpha. 4-Phenylbutyric acid (4-PBA) treatment alleviated ERS; reduced the NLR family pyrin domain containing 3, interleukin-1β, and interleukin-18 levels; inhibited apoptosis; increased mitofusin 2 expression; and mitigated Ca2+ overload and mitochondria-associated ER membrane (MAM) dysfunction, thereby ameliorating IPEC-J2 injury. We demonstrated the pivotal role of ERS in MAM dysfunction and 7S- and 11S-mediated apoptosis, providing insights into 7S- and 11S-mediated intestinal barrier injury prevention and treatment.

Genome-wide identification of SWEET genes reveals their roles during seed development in peanuts

Sugar Will Eventually be Exported Transporter (SWEET) proteins are highly conserved in various organisms and play crucial roles in sugar transport processes. However, SWEET proteins in peanuts, an essential leguminous crop worldwide, remain lacking in systematic characterization. Here, we identified 94 SWEET genes encoding the conservative MtN3/saliva domains in three peanut species, including 47 in Arachis hypogea, 23 in Arachis duranensis, and 24 in Arachis ipaensis. We observed significant variations in the exon-intron structure of these genes, while the motifs and domain structures remained highly conserved. Phylogenetic analysis enabled us to categorize the predicted 286 SWEET proteins from eleven species into seven distinct groups. Whole genome duplication/segment duplication and tandem duplication were the primary mechanisms contributing to the expansion of the total number of SWEET genes. In addition, an investigation of cis-elements in the potential promoter regions and expression profiles across 22 samples uncovered the diverse expression patterns of AhSWEET genes in peanuts. AhSWEET24, with the highest expression level in seeds from A. hypogaea Tifrunner, was observed to be localized on both the plasma membrane and endoplasmic reticulum membrane. Moreover, qRT-PCR results suggested that twelve seed-expressed AhSWEET genes were important in the regulation of seed development across four different peanut varieties. Together, our results provide a foundational basis for future investigations into the functions of SWEET genes in peanuts, especially in the process of seed development.

Emerging Evidence of Golgi Stress Signaling for Neuropathies.

The Golgi apparatus is an intracellular organelle that modifies cargo, which is transported extracellularly through the nucleus, endoplasmic reticulum, and plasma membrane in order. First, the general function of the Golgi is reviewed and, then, Golgi stress signaling is discussed. In addition to the six main Golgi signaling pathways, two pathways that have been increasingly reported in recent years are described in this review. The focus then shifts to neurological disorders, examining Golgi stress reported in major neurological disorders, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. The review also encompasses findings related to other diseases, including hypomyelinating leukodystrophy, frontotemporal spectrum disorder/amyotrophic lateral sclerosis, microcephaly, Wilson's disease, and prion disease. Most of these neurological disorders cause Golgi fragmentation and Golgi stress. As a result, strong signals may act to induce apoptosis.

Dietary α-Ketoglutarate Alleviates Escherichia coli LPS-Induced Intestinal Barrier Injury by Modulating the Endoplasmic Reticulum-Mitochondrial System Pathway in Piglets

Backgroundα-Ketoglutarate (AKG) plays a pivotal role in mitigating inflammation and enhancing intestinal health. ObjectivesThis study aimed to investigate whether AKG could protect against lipopolysaccharide (LPS)-induced intestinal injury by alleviating disorders in mitochondria-associated endoplasmic reticulum (MAM) membranes, dysfunctional mitochondrial dynamics, and endoplasmic reticulum (ER) stress in a piglet model. MethodsTwenty-four piglets were subjected to a 2 × 2 factorial design with dietary factors (basal diet or 1% AKG diet) and LPS treatment (LPS or saline). After 21 d of consuming either the basal diet or AKG diet, piglets received injections of LPS or saline. The experiment was divided into 4 treatment groups [control (CON) group: basal diet + saline; LPS group: basal diet +LPS; AKG group: AKG diet + saline; and AKG_LPS group: AKG + LPS], each consisting of 6 piglets. ResultsThe results demonstrated that compared with the CON group, AKG enhanced jejunal morphology, antioxidant capacity, and the messenger RNA and protein expression of tight junction proteins. Moreover, it has shown a reduction in serum diamine oxidase activity and D-lactic acid content in piglets. In addition, fewer disorders in the ER-mitochondrial system were reflected by AKG, as evidenced by AKG regulating the expression of key molecules of mitochondrial dynamics (mitochondrial calcium uniporter, optic atrophy 1, fission 1, and dynamin-related protein 1), ER stress [activating transcription factor (ATF) 4, ATF 6, CCAAT/enhancer binding protein homologous protein, eukaryotic initiation factor 2α, glucose-regulated protein (GRP) 78, and protein kinase R-like ER kinase], and MAM membranes [mitofusin (Mfn)-1, Mfn-2, GRP 75, and voltage-dependent anion channel-1]. ConclusionsDietary AKG can prevent mitochondrial dynamic dysfunction, ER stress, and MAM membrane disorder, ultimately alleviating LPS-induced intestinal damage in piglets.

Mitochondria-associated endoplasmic reticulum membrane as a mediator of vanadium-induced endoplasmic reticulum quality control in duck brains.

Vanadium (V) plays a crucial role in normal cells, but excess V causes multi-organ toxicity, including neurotoxicity. Mitochondria-associated endoplasmic reticulum membrane (MAM) is a dynamic structure between endoplasmic reticulum (ER) and mitochondria that mediates ER quality control (ERQC). To explore the effects of excess V on MAM and ERQC in the brain, 72 ducks were randomly divided into two groups: the control group (basal diet) and the V group (30mgV/kg basal diet). On days 22 and 44, brain tissues were collected for histomorphological observation and determination of trace element contents. In addition, the mRNA and protein levels of MAM and ERQC-related factors in the brain were analyzed. Results show that excessive V causes the imbalance of trace elements, the integrity disruption of MAM, rupture of ER and autophagosomes formation. Moreover, it inhibits IP3R and VDAC1 co-localization, down-regulates the expression levels of MAM-related factors, but up-regulates the expression levels of ERQC and autophagy related factors. Together, results indicate that V exposure causes disruption of MAM and activates ERQC, which is further causing autophagy.

The derlin Dfm1 couples retrotranslocation of a folded protein domain to its proteasomal degradation.

Endoplasmic reticulum (ER) proteins are degraded by proteasomes in the cytosol through ER-associated degradation (ERAD). This process involves the retrotranslocation of substrates across the ER membrane, their ubiquitination, and membrane extraction by the Cdc48/Npl4/Ufd1 ATPase complex prior to delivery to proteasomes for degradation. How the presence of a folded luminal domain affects substrate retrotranslocation and this event is coordinated with subsequent ERAD steps remains unknown. Here, using a model substrate with a folded luminal domain, we showed that Cdc48 ATPase activity is sufficient to drive substrate retrotranslocation independently of ERAD membrane components. However, the complete degradation of the folded luminal domain required substrate-tight coupling of retrotranslocation and proteasomal degradation, which was ensured by the derlin Dfm1. Mutations in Dfm1 intramembrane rhomboid-like or cytosolic Cdc48-binding regions resulted in partial degradation of the substrate with accumulation of its folded domain. Our study revealed Dfm1 as a critical regulator of Cdc48-driven retrotranslocation and highlights the importance of coordinating substrate retrotranslocation and degradation during ERAD.

Generation of homozygous and heterozygous REEP1 knockout induced pluripotent stem cell lines by CRISPR/Cas9 gene editing

REEP1 is a transmembrane protein in the endoplasmic reticulum (ER) membrane that is involved in shaping and remodeling of the ER. Mutations in REEP1 cause SPG31, an autosomal dominant form of hereditary spastic paraplegia (HSP). Here we show the generation of a homozygous and a heterozygous REEP1 knockout induced pluripotent stem cell line suitable for in vitro disease modelling using the CRISPR/Cas9 editing system.

Combinatorial selective ER-phagy remodels the ER during neurogenesis

The endoplasmic reticulum (ER) employs a diverse proteome landscape to orchestrate many cellular functions, ranging from protein and lipid synthesis to calcium ion flux and inter-organelle communication. A case in point concerns the process of neurogenesis, where a refined tubular ER network is assembled via ER shaping proteins into the newly formed neuronal projections to create highly polarized dendrites and axons. Previous studies have suggested a role for autophagy in ER remodelling, as autophagy-deficient neurons in vivo display axonal ER accumulation within synaptic boutons, and the membrane-embedded ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy. However, our understanding of the mechanisms underlying selective removal of the ER and the role of individual ER-phagy receptors is limited. Here we combine a genetically tractable induced neuron (iNeuron) system for monitoring ER remodelling during in vitro differentiation with proteomic and computational tools to create a quantitative landscape of ER proteome remodelling via selective autophagy. Through analysis of single and combinatorial ER-phagy receptor mutants, we delineate the extent to which each receptor contributes to both the magnitude and selectivity of ER protein clearance. We define specific subsets of ER membrane or lumenal proteins as preferred clients for distinct receptors. Using spatial sensors and flux reporters, we demonstrate receptor-specific autophagic capture of ER in axons, and directly visualize tubular ER membranes within autophagosomes in neuronal projections by cryo-electron tomography. This molecular inventory of ER proteome remodelling and versatile genetic toolkit provide a quantitative framework for understanding the contributions of individual ER-phagy receptors for reshaping ER during cell state transitions.

Involvement of nucleus accumbens SERCA2b in methamphetamine-induced conditioned place preference.

Methamphetamine (METH) is a highly addictive psycho-stimulant that induces addictive behaviour by stimulating increased dopamine release in the nucleus accumbens (NAc). The sarco/endoplasmic reticulum calcium ion transport ATPases (SERCA or ATP2A) is a calcium ion (Ca2+) pump in the endoplasmic reticulum (ER) membrane. SERCA2b is a SERCA subtype mainly distributed in the central nervous system. This study used conditioned place preference (CPP), a translational drug reward model, to observe the effects of SERCA and SERCA2b on METH-CPP in mice. Result suggested that the activity of SERCA was significantly decreased in NAc after METH-CPP. Intraperitoneal SERCA agonist CDN1163 injection or bilateral CDN1163 microinjection in the NAc inhibited METH-CPP formation. SERCA2b overexpression by the Adeno-associated virus can reduce the DA release of NAc and inhibit METH-CPP formation. Although microinjection of SERCA inhibitor thapsigargin in the bilateral NAc did not significantly aggravate METH-CPP, interference with SERCA2b expression in NAc by adeno-associated virus increased DA release and promoted METH-CPP formation. METH reduced the SERCA ability to transport Ca2+ into the ER in SHSY5Y cells in vitro, which was reversed by CDN1163. This study revealed that METH dysregulates intracellular calcium balance by downregulating SERCA2b function, increasing DA release in NAc and inducing METH-CPP formation. Drugs that target SERCA2b may have the potential to treat METH addiction.

OrthoID: profiling dynamic proteomes through time and space using mutually orthogonal chemical tools

Identifying proteins at organelle contact sites, such as mitochondria-associated endoplasmic reticulum membranes (MAM), is essential for understanding vital cellular processes, yet challenging due to their dynamic nature. Here we report “OrthoID”, a proteomic method utilizing engineered enzymes, TurboID and APEX2, for the biotinylation (Bt) and adamantylation (Ad) of proteins close to the mitochondria and endoplasmic reticulum (ER), respectively, in conjunction with high-affinity binding pairs, streptavidin-biotin (SA-Bt) and cucurbit[7]uril-adamantane (CB[7]-Ad), for selective orthogonal enrichment of Bt- and Ad-labeled proteins. This approach effectively identifies protein candidates associated with the ER-mitochondria contact, including LRC59, whose roles at the contact site were—to the best of our knowledge—previously unknown, and tracks multiple protein sets undergoing structural and locational changes at MAM during mitophagy. These findings demonstrate that OrthoID could be a powerful proteomics tool for the identification and analysis of spatiotemporal proteins at organelle contact sites and revealing their dynamic behaviors in vital cellular processes.