Golgi Apparatus Research Articles

Robust reference gene selection in Norway spruce: essential for real-time quantitative PCR across different tissue, stress and developmental conditions

Accurate gene expression analysis in Norway spruce (Picea abies) under diverse stress conditions requires the identification of stable reference genes for normalization. Notably, the literature lacks reports on suitable reference genes in Norway spruce. Here, we aimed to address this gap by identifying suitable reference genes for quantitative real-time PCR in Norway spruce across various stress conditions (drought, heat, pathogen infection) in seedlings, tissues (needle, phloem, root), and developmental stages (seedlings, mature trees). We evaluated the stability of 15 candidate reference genes and assessed their expression stability using five statistical algorithms (ΔCt, geNorm, NormFinder, BestKeeper, and RefFinder). Our results highlight ubiquitin-protein ligase (SP1), conserved oligomeric Golgi complex (COG7), and tubby-like F-box protein (TULP6) as the most stable reference genes, while succinate dehydrogenase (SDH5) and heat shock protein 90 (HSP90) were the least stable under various experimental conditions. COG7 and TULP6 are novel candidate reference genes reported for the first time. The expression stability of the identified reference genes was further validated using dehydrin-like protein 5 (PaDhn5) under drought conditions in Norway spruce. Pairwise variation analysis suggests that two reference genes were sufficient to normalize gene expression across all sample sets. This study provides a comprehensive analysis of reference gene stability under different experimental conditions and a catalog of genes for each condition, facilitating future functional genomic research in Norway spruce and related conifers.

Stairway to the Golgi: Two paths VPS13B can go by

VPS13 proteins mediate lipid transfer across membranes. Among them, VPS13B is associated with Golgi membranes, and VPS13B mutations cause Cohen syndrome. In this issue, Ugur et al. (https://doi.org/10.1083/jcb.202311189) and Du et al. (https://doi.org/10.1083/jcb.202402083) reveal new VPS13B interactors and their functions in Golgi organization and trafficking.

Abstract 4138754: Analysis of Polarization Changes in Ascending Aortic Endothelial Cells Induced by Aortic Valve Regurgitation

Background: Fragility of the ascending aortic wall has been reported in patients with aortic regurgitation (AR), thought to be affected by turbulence in the ascending aorta. However, the mechanism behind this effect is unclear. We hypothesized that turbulence in the ascending aorta of AR patients causes changes in the polarization of endothelial cells. This study aimed to clarify that the polarization of endothelial cells in the ascending aortic wall is altered in AR patients. Methods: Twenty patients who underwent ascending aortic replacement at our institution from October 2022 to July 2023 were included: ten with thoracic aortic aneurysms (TAA) and ten with TAA and AR. The polarization of endothelial cells in the greater curvature of the ascending aortic wall collected during surgery was evaluated by measuring the ratio of the long axis to the short axis, the angle between the long axis and the blood flow axis, and the alignment of the Golgi apparatus and nucleus parallel to the blood flow. Results: There were no significant differences in patient background between the AR and non-AR groups. Additionally, there was no significant difference in the diameter of the ascending aorta between the two groups (AR vs. non-AR, 47.7±3.8 mm vs. 44.5±2.2 mm, p=0.479). The long axis to short axis ratio (aspect ratio) of endothelial cells in the ascending aorta was significantly smaller in the AR group than in the non-AR group. The angle (θ) between the long axis of the endothelial cells and the blood flow axis was significantly smaller in the AR group than in the non-AR group. The ratio of Golgi apparatus and nucleus alignment parallel to the blood flow was significantly lower in the AR group than in the non-AR group. Degeneration of the aortic tunica media was also observed in the AR group compared to the non-AR group. Conclusion: The results of this study suggest that the presence of AR alters the polarization of endothelial cells in the ascending aortic wall.

An automated high-content screening and assay platform for the analysis of spheroids at subcellular resolution

The endomembrane system is essential for healthy cell function, with the various compartments carrying out a large number of specific biochemical reactions. To date, almost all of our understanding of the endomembrane system has come from the study of cultured cells growing as monolayers. However, monolayer-grown cells only poorly represent the environment encountered by cells in the human body. As a first step to address this disparity, we have developed a platform that allows us to investigate and quantify changes to the endomembrane system in three-dimensional (3D) cell models, in an automated and highly systematic manner. HeLa Kyoto cells were grown on custom-designed micropatterned 96-well plates to facilitate spheroid assembly in the form of highly uniform arrays. Fully automated high-content confocal imaging and analysis were then carried out, allowing us to measure various spheroid-, cellular- and subcellular-level parameters relating to size and morphology. Using two drugs known to perturb endomembrane function, we demonstrate that cell-based assays can be carried out in these spheroids, and that changes to the Golgi apparatus and endosomes can be quantified from individual cells within the spheroids. We also show that image texture measurements are useful tools to discriminate cellular phenotypes. The automated platform that we show here has the potential to be scaled up, thereby allowing large-scale robust screening to be carried out in 3D cell models.

Visualization of endogenous G proteins on endosomes and other organelles

Classical G-protein-coupled receptor (GPCR) signaling takes place in response to extracellular stimuli and involves receptors and heterotrimeric G proteins located at the plasma membrane. It has recently been established that GPCR signaling can also take place from intracellular membrane compartments, including endosomes that contain internalized receptors and ligands. While the mechanisms of GPCR endocytosis are well understood, it is not clear how well internalized receptors are supplied with G proteins. To address this gap, we use gene editing, confocal microscopy, and bioluminescence resonance energy transfer to study the distribution and trafficking of endogenous G proteins. We show here that constitutive endocytosis is sufficient to supply newly internalized endocytic vesicles with 20–30% of the G protein density found at the plasma membrane. We find that G proteins are present on early, late, and recycling endosomes, are abundant on lysosomes, but are virtually undetectable on the endoplasmic reticulum, mitochondria, and the medial-trans Golgi apparatus. Receptor activation does not change heterotrimer abundance on endosomes. Our findings provide a subcellular map of endogenous G protein distribution, suggest that G proteins may be partially excluded from nascent endocytic vesicles, and are likely to have implications for GPCR signaling from endosomes and other intracellular compartments.

Visualization of endogenous G proteins on endosomes and other organelles.

Classical G-protein-coupled receptor (GPCR) signaling takes place in response to extracellular stimuli and involves receptors and heterotrimeric G proteins located at the plasma membrane. It has recently been established that GPCR signaling can also take place from intracellular membrane compartments, including endosomes that contain internalized receptors and ligands. While the mechanisms of GPCR endocytosis are well understood, it is not clear how well internalized receptors are supplied with G proteins. To address this gap, we use gene editing, confocal microscopy, and bioluminescence resonance energy transfer to study the distribution and trafficking of endogenous G proteins. We show here that constitutive endocytosis is sufficient to supply newly internalized endocytic vesicles with 20-30% of the G protein density found at the plasma membrane. We find that G proteins are present on early, late, and recycling endosomes, are abundant on lysosomes, but are virtually undetectable on the endoplasmic reticulum, mitochondria, and the medial-trans Golgi apparatus. Receptor activation does not change heterotrimer abundance on endosomes. Our findings provide a subcellular map of endogenous G protein distribution, suggest that G proteins may be partially excluded from nascent endocytic vesicles, and are likely to have implications for GPCR signaling from endosomes and other intracellular compartments.

Rab2A-mediated Golgi-lipid droplet interactions support very-low-density lipoprotein secretion in hepatocytes.

Lipid droplets (LDs) serve as crucial hubs for lipid trafficking and metabolic regulation through their numerous interactions with various organelles. While the interplay between LDs and the Golgi apparatus has been recognized, their roles and underlying mechanisms remain poorly understood. Here, we reveal the role of Ras-related protein Rab-2A (Rab2A) in mediating LD-Golgi interactions, thereby contributing to very-low-density lipoprotein (VLDL) lipidation and secretion in hepatocytes. Mechanistically, our findings identify a selective interaction between Golgi-localized Rab2A and 17-beta-hydroxysteroid dehydrogenase 13 (HSD17B13) protein residing on LDs. This complex facilitates dynamic organelle communication between the Golgi apparatus and LDs, thus contributing to lipid transfer from LDs to the Golgi apparatus for VLDL2 lipidation and secretion. Attenuation of Rab2A activity via AMP-activated protein kinase (AMPK) suppresses the Rab2A-HSD17B13 complex formation, impairing LD-Golgi interactions and subsequent VLDL secretion. Furthermore, genetic inhibition of Rab2A and HSD17B13 in the liver reduces the serum triglyceride and cholesterol levels. Collectively, this study provides a new perspective on the interactions between the Golgi apparatus and LDs.

Treatment of Conjunctival Melanoma Cell Lines With a Light-Activated Virus-Like Drug Conjugate Induces Immunogenic Cell Death.

Conjunctival melanoma (CJM) is a rare malignant ocular surface tumor, which often leads to local recurrences and metastases. In murine models of subcutaneous tumors, treatment with a novel virus-like drug conjugate (VDC; Bel-sar) showed a dual mechanism of action with direct tumor cell killing as well as stimulation of an antitumoral immune response. Bel-sar is currently being evaluated for the treatment of primary uveal melanoma and indeterminate nevi in a phase III clinical trial. We determined whether Bel-sar also has direct antitumor efficiency and a potential immunostimulatory capacity in CJM cells. Three human tumor-derived CJM lines were used. Bel-sar's subcellular and intracellular locations were determined with tracers. Following light activation of Bel-sar, cytotoxicity and exposure of damage-associated molecular patterns (DAMPs) were assessed. Treated tumor cells were co-cultured with THP-1 derived macrophages to assess tumor-cell phagocytosis. Bel-sar was bound and internalized by CJM cells and subsequently found in the cell membrane, lysosome, Golgi apparatus, and mitochondria. Bel-sar activation induced near complete cell death with half-maximal inhibitory concentration (IC50) values between 30 pM and 60 pM. Finally, light-activated Bel-sar enhanced exposure of DAMPs, including calreticulin, heat shock protein 90, and stimulated phagocytosis by macrophages. Treatment with a novel VDC (Bel-sar) induced pro-immunogenic cell death in all three CJM cell lines. The in vitro cytotoxicity was accompanied by exposure of DAMPs, suggesting Bel-sar is a potential treatment for CJM by a dual mechanism of action. This dual mechanism may provide a targeted and direct killing of tumor cells and induce an immune response which might decrease local recurrences and metastasis.

Betula platyphylla glucosyltransferase BpGT14;6 is essential for cell wall development and stress response

Glycosyltransferases (GTs) constitute a diverse family of synthetic polysaccharides with important roles in plant growth and development. This study characterized the GT14 family gene BpGT14;6 of birch (Betula platyphylla Suk.). BpGT14;6 was highly expressed in the xylem and stem of birch plants. Subcellular localization analysis suggested that BpGT14;6 was located in the Golgi apparatus. RNA interference (RNAi) silencing of BpGT14;6 revealed lower lignin, hemicellulose, and pectin contents compared to wild type (WT) plants. Following treatment with abscisic acid (ABA), compared to WT plants, RNAi-BpGT14;6 plants were more sensitive to ABA, suffered more membrane lipid damage, and accumulated more reactive oxygen species. The inhibition of BpGT14;6 expression narrowed the birch xylem and thinned the cell wall, and increased the expression of multiple ABA pathway-related genes in birch under ABA treatment. Compared to WT plants, RNAi-BpGT14;6 plants showed increased tolerance to drought stress. Promoter analysis revealed that BpGT14;6 is involved in hormone regulation and adaptation to adversity. Using the 1 156 bp BpGT14;6 promoter as bait, two potential transcription factors, BpWRKY1 and BpARF2, were identified through Y1H screening that may regulate its expression. EMSA confirmed that BpWRKY1 and BpARF2 can directly bind to the W-BOX and AuxRE cis-acting elements on the BpGT14;6 promoter, respectively. The collective results suggest that BpGT14;6 affects birch xylem and cell wall development by affecting lignin, hemicellulose, and pectin synthesis, and participates in birch adversity adaptation.

Linking secretion and cytoskeleton in immunity- a case for Arabidopsis TGNap1.

In plants, robust defense depends on the efficient and resilient trafficking supply chains to the site of pathogen attack. Though the importance of intracellular trafficking in plant immunity has been well established, a lack of clarity remains regarding the contribution of the various trafficking pathways in transporting immune-related proteins. We have recently identified a trans-Golgi network protein, TGN-ASSOCIATED PROTEIN 1 (TGNap1), which functionally links post-Golgi vesicles with the cytoskeleton to transport immunity-related proteins in the model plant species Arabidopsis thaliana. We propose new hypotheses on the various functional implications of TGNap1 and then elaborate on the surprising heterogeneity of TGN vesicles during immunity revealed by the discovery of TGNap1 and other TGN-associated proteins in recent years.

Organelle-Level Toxicity of Nanometals Relevant to Titanium Implants. Original Research and Comprehensive Literature Overview

ObjectiveThis study analyzed organelle toxicities of nanometals applied as free formulations or titanium rod-coating materials in rats. MethodsAll materials were injected intraperitoneally, including the physiological saline applied to the control group. The first experimental group was implanted with nanosilver-coated titanium rods, and the second, third, and fourth groups received free nanosilver at rising levels. The fifth group was implanted with nanosilver, nanocopper, and nanozinc-coated titanium rods, and the sixth group received the same nanometals as free formulations. Light and electron microscopy and ICP-Mass Spectrometry were utilized to determine the neural, hepatic, and renal toxicities and tissue metal levels. ResultsIn brains, neuropil, myelin, and cellular damages occurred, especially in groups receiving high-dose nanosilver or nanometal combinations. Histiocyte accumulation and dark mitochondria within hepatocytes were discernible in the liver. Kidneys were the organs that were most severely affected by nanometal toxicity. The nephrotoxicity was apparent with the perturbations of the membrane infoldings and mitochondrial damage in the proximal and distal convoluted epithelia. Large angular peroxisomes developed inside the mesangial cells, and Golgi bodies increased in epithelial cells. Systemic metal levels increased on the thirtieth and prominently dropped on the sixtieth day. ConclusionThese results provide insights into the extent of injury and organelle targets of nanometals and will guide optimizing the nanomaterials and implants used in the surgical practice.

Genome-wide analysis of the Amorphophallus konjac AkCSLA gene family and its functional characterization in drought tolerance of transgenic arabidopsis

BackgroundAmorphophallus konjac (A. konjac), a perennial tuberous plant, is widely cultivated for its high konjac glucomannan (KGM) content, a heteropolysaccharide with diverse applications. The cellulose synthase-like (CSL) gene family is known to be a group of processive glycan synthases involved in the synthesis of cell-wall polysaccharides and plays an important role in the biological process of KGM. However, in A. konjac the classification, structure, and function of the AkCSLA superfamily have been studied very little.ResultsBioinformatics methods were used to identify the 11 AkCSLA genes from the whole genome of Amorphophallus konjac and to systematically analyze their characteristics, phylogenetic evolution, promoter cis-elements, expression patterns, and subcellular locations. Phylogenetic analysis revealed that the AkCSLA gene family can be divided into three subfamilies (Groups I- III), which have close relationships with Arabidopsis. The promoters of most AkCSLA family members contain MBS elements and ABA response elements. Analysis of expression patterns in different tissues showed that most AkCSLAs are highly expressed in the corms. Notably, PEG6000 induced down-regulation of the expression of most AkCSLAs, including AkCSLA11. Subcellular localization results showed that AkCSLA11 was localized to the plasma membrane, Golgi apparatus and endoplasmic reticulum. Transgenic Arabidopsis experiments demonstrated that overexpression of AkCSLA11 reduced the plant’s drought tolerance. This overexpression also inhibited the expression of drought response genes and altered the sugar components of the cell wall. These findings provide new insights into the response mechanisms of A. konjac to drought stress and may offer potential genetic resources for improving crop drought resistance.ConclusionIn conclusion, the study reveals that the AkCSLA11 gene from A. konjac negatively impacts drought tolerance when overexpressed in Arabidopsis. This discovery provides valuable insights into the mechanisms of plant response to drought stress and may guide future research on crop improvement for enhanced resilience.

Susceptibility of Leishmania amazonensis Axenic Amastigotes to the Calpain Inhibitor MDL28170

Leishmaniasis encompasses a group of neglected diseases caused by flagellated protozoa belonging to the Leishmania genus, associated with high morbidity and mortality. The search for compounds with anti-Leishmania activity that exhibit lower toxicity and can overcome the emergence of resistant strains remains a significant goal. In this context, the calpain inhibitor MDL28170 has previously demonstrated deleterious effects against promastigote forms of Leishmania amazonensis, which led us to investigate its role on axenic amastigote forms. The calpain inhibitor MDL28170 was able to decrease the viability of amastigotes in a typically dose-dependent manner. The treatment with the IC50 dose (13.5 μM) for 72 h led to significant amastigote lysis and increased cell-to-cell aggregation. Ultrastructural analysis revealed several cellular alterations, including disruption of the trans-Golgi network and the formation of autophagosomes when treated with MDL28170 at ½ × IC50 dose. Additionally, mitochondrial swelling and the formation of concentric membranous structures inside the mitochondrion were observed after incubation with the IC50 dose. These results reinforce the potential application of the calpain inhibitor MDL28170 against L. amazonensis, highlighting its effectiveness and possible mechanism of action against the parasite.

Arabidopsis KNS3 and its two homologs mediate endoplasmic reticulum-to-plasma membrane traffic of boric acid channels.

Membrane proteins targeted to the plasma membrane are first transported from the endoplasmic reticulum (ER) to the Golgi apparatus. This study explored the mechanisms controlling plasma membrane trafficking of the boric acid channel AtNIP5;1 from the ER. Imaging-based screening using transgenic Arabidopsis identified six mutants in which GFP-NIP5;1 was localized in the ER in addition to the plasma membrane. Genetic mapping and whole-genome resequencing identified the responsible gene in four among the six mutants as KAONASHI3 (KNS3)/SPOTTY1/IMPERFECTIVE EXINE FORMATION. Among the plasma membrane-localized proteins tested, NIP5;1 and its homolog NIP6;1 were retained in the ER of the kns3 mutants. Our genetic analysis further discovered that two homologs of KNS3, KNSTH1 and KNSTH2, were also involved in the ER exit of NIP5;1. In Arabidopsis protoplasts and tobacco leaves, mCherry-fused KNS3 localized to the ER and Golgi, whereas KNSTH2 localized to the ER. The cytosolic C-terminal tail of KNS3 contains amino acids important for Golgi-to-ER trafficking. Furthermore, the ER-to-Golgi trafficking of KNS3 depended on KNSTH1 and KNSTH2, and the accumulation of these three proteins in Arabidopsis roots depended on each other. We propose that KNS3, KNSTH1, and KNSTH2 function as a cargo-receptor complex mediating the ER exit of NIP5;1.

Comprehensive Proteomic Characterization of Intra-Golgi Trafficking Intermediates.

The Conserved Oligomeric Golgi (COG) complex is critical for efficient intra-Golgi trafficking and glycosylation. Prior research has demonstrated that COG dysfunction or RNAi-induced depletion leads to the accumulation of non-tethered COG complex-dependent (CCD) vesicles. However, the precise connection between COG deficiency, degradation of Golgi enzymes, and its impact on vesicular trafficking has not been fully elucidated. In this study, we conducted a comprehensive proteomic analysis of Golgi-derived vesicles from both wild-type and COG-depleted cells. We specifically analyzed three distinct populations of vesicles immunoisolated with antibodies targeting transmembrane proteins from the cis, medial, and trans-Golgi sub-compartments. Our findings reveal that, while the vesicle content encompasses the entire Golgi proteome, the molecular signatures of vesicles derived from wild- type cells were markedly distinct, underscoring a robust recycling mechanism for Golgi- dependent proteins. Notably, these vesicles retained various vesicular coats, and COG depletion significantly accelerated their uncoating. Furthermore, the increased overlap in molecular signatures upon COG depletion indicates that persistent defects in vesicle tethering severely compromise intra-Golgi sorting mechanisms. Crucially, our analysis highlights that the entire Golgi glycosylation machinery recycles within CCD vesicles in a COG-dependent manner, while secretory proteins and components involved in ER-Golgi and Golgi-endosome trafficking were not enriched. These results strongly support a model of multi-step intra-Golgi vesicular recycling of the glycosylation machinery, orchestrated by the COG complex in concert with a cisternae-specific array of vesicular coats, coiled-coil tethers, Rabs, and SNARE proteins.

N-glycosylation of the envelope glycoprotein I is essential for the proliferation and virulence of the duck plague virus

Duck plague virus (DPV) causes the highly pathogenic duck plague, and the envelope glycoprotein I (gI), as one of the key virulence genes, has not yet had its critical virulence sites identified through screening. This study used reverse genetics technology to target the gI, specifically within the DPV genome. Four DPV mutants with gI N-glycosylation site mutations were designed and constructed, and these mutant strains were successfully rescued. Our results confirmed that three asparagine residues of gI (N69, N78, and N265) are N-glycosylation sites, and western blot analysis substantiated that glycosylation at each predicted N-glycosylation site was compromised. The deglycosylation of gI leads to the protein misfolding and subsequent retention in the endoplasmic reticulum (ER). The subsequent deglycosylated gI is carried into the Golgi apparatus (GM130) in the interaction of gE. Compared to the parental virus, the mutated virus shows a 66.3% reduction in intercellular transmission capability. In ducks, the deglycosylation of gI significantly reduces DPV replication in vivo, thereby weakening the virulence of DPV. This study represents the first successful creation of a weak DPV virus strain by specific mutation at the N-glycosylation site. The findings provide a foundational understanding of DPV pathogenesis and form the basis for developing live attenuated vaccines against the disease.

The Dsc ubiquitin ligase complex identifies transmembrane degrons to degrade orphaned proteins at the Golgi

The Golgi apparatus is essential for protein sorting, yet its quality control mechanisms are poorly understood. Here we show that the Dsc ubiquitin ligase complex uses its rhomboid pseudo-protease subunit, Dsc2, to assess the hydrophobic length of α-helical transmembrane domains (TMDs) at the Golgi. Thereby the Dsc complex likely interacts with orphaned ER and Golgi proteins that have shorter TMDs and ubiquitinates them for targeted degradation. Some Dsc substrates will be extracted by Cdc48 for endosome and Golgi associated proteasomal degradation (EGAD), while others will undergo ESCRT dependent vacuolar degradation. Some substrates are degraded by both, EGAD- or ESCRT pathways. The accumulation of Dsc substrates entails a specific increase in glycerophospholipids with shorter and asymmetric fatty acyl chains. Hence, the Dsc complex mediates the selective degradation of orphaned proteins at the sorting center of cells, which prevents their spreading across other organelles and thereby preserves cellular membrane protein and lipid composition.

Comparative Transcriptome Analysis of Non-Organogenic and Organogenic Tissues of Gaillardia pulchella Revealing Genes Regulating De Novo Shoot Organogenesis

Gaillardia pulchella is an important plant species with pharmacological and ornamental applications. It contains a wide array of phytocompounds which play roles against diseases. In vitro propagation requires callogenesis and differentiation of plant organs, which offers a sustainable, alternative synthesis of compounds. The morphogenetic processes and the underlying mechanisms are, however, known to be under genetic regulation and are little understood. The present study investigated these events by generating transcriptome data, with de novo assembly of sequences to describe shoot morphogenesis molecularly in G. pulchella. The RNA was extracted from the callus of pre- and post-shoot organogenesis time. The callus induction was optimal using leaf segments cultured onto MS medium containing α-naphthalene acetic acid (NAA; 2.0 mg/L) and 6-benzylaminopurine (BAP; 0.5 mg/L) and further exhibited a high shoot regeneration/caulogenesis ability. A total of 68,366 coding sequences were obtained using Illumina150bpPE sequencing and transcriptome assembly. Differences in gene expression patterns were noted in the studied samples, showing opposite morphogenetic responses. Out of 10,108 genes, 5374 (53%) were downregulated, and there were 4734 upregulated genes, representing 47% of the total genes. Through the heatmap, the top 100 up- and downregulating genes’ names were identified and presented. The up- and downregulated genes were identified using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Important pathways, operative during G. pulchella shoot organogenesis, were signal transduction (13.55%), carbohydrate metabolism (8.68%), amino acid metabolism (5.11%), lipid metabolism (3.75%), and energy metabolism (3.39%). The synthesized proteins displayed phosphorylation, defense response, translation, regulation of DNA-templated transcription, carbohydrate metabolic processes, and methylation activities. The genes’ product also exhibited ATP binding, DNA binding, metal ion binding, protein serine/threonine kinase -, ATP hydrolysis activity, RNA binding, protein kinase, heme and GTP binding, and DNA binding transcription factor activity. The most abundant proteins were located in the membrane, nucleus, cytoplasm, ribosome, ribonucleoprotein complex, chloroplast, endoplasmic reticulum membrane, mitochondrion, nucleosome, Golgi membrane, and other organellar membranes. These findings provide information for the concept of molecular triggers, regulating programming, differentiation and reprogramming of cells, and their uses.

Live-Cell Identification of Inhibitors of the Lipid Transfer Protein CERT Using Nanoluciferase Bioluminescence Resonance Energy Transfer (NanoBRET).

A BRET system is described, in which Nanoluciferase was fused to the lipid transfer protein CERT for efficient energy transfer to a Nile red-labeled ceramide, which is either directly bound to CERT or transported to the adjacent Golgi membrane. Bulk formation of sphingomyelin, a major plasma membrane component in mammals, is dependent on CERT-mediated transfer of its predecessor ceramide. CERT is considered a promising drug target but no direct cell-based methods exist to efficiently identify inhibitors. The utility off the method was demonstrated by a library of 140 derivatives of the CERT inhibitor HPA-12. These were obtained in a combinatorial synthesis using solid-phase transacylation. Screening of the library led to six compounds that were picked and confirmed to be superior to HPA-12 in a subsequent dose-response study and also in an orthogonal lipidomics analysis.

Live‐Cell Identification of Inhibitors of the Lipid Transfer Protein CERT Using Nanoluciferase Bioluminescence Resonance Energy Transfer (NanoBRET)

A BRET system is described, in which Nanoluciferase was fused to the lipid transfer protein CERT for efficient energy transfer to a Nile red‐labeled ceramide, which is either directly bound to CERT or transported to the adjacent Golgi membrane. Bulk formation of sphingomyelin, a major plasma membrane component in mammals, is dependent on CERT‐mediated transfer of its predecessor ceramide. CERT is considered a promising drug target but no direct cell‐based methods exist to efficiently identify inhibitors. The utility off the method was demonstrated by a library of 140 derivatives of the CERT inhibitor HPA‐12. These were obtained in a combinatorial synthesis using solid‐phase transacylation. Screening of the library led to six compounds that were picked and confirmed to be superior to HPA‐12 in a subsequent dose‐response study and also in an orthogonal lipidomics analysis.