Endoplasmic Reticulum Marker Research Articles

Molecular characterization and functional analysis of apoptosis-inducing factor (AIF) in palmitic acid-induced apoptosis in Ctenopharyngodon idellus kidney (CIK) cells.

Palmitic acid (PA), the most common saturated free fatty acid, may cause apoptosis when overloaded in non-fat cells. Apoptosis-inducing factor (AIF) is known to translocate from the mitochondria into the nucleus to induce apoptosis. However, it remains to be investigated whether AIF involved in palmitic acid-induced lipoapoptosis in fish. In the present study, we cloned a coding sequence of grass carp (Ctenopharyngodon idella) AIF (CiAIF) gene, and determined its function in Ctenopharyngodon idellus kidney (CIK) cells. The open reading frame (ORF) of CiAIF gene is 1863 bp, encoding a precursor protein of 620 amino acids (aa). Sequence analysis indicated that CiAIF contains a mitochondrial localization sequence, a conserved Pyr_redox and a C-terminal domain. Phylogenetic analyses showed that the CiAIF gene tended to cluster with sequences from Danio rerio. CiAIF gene was ubiquitously expressed in all tested tissues, including heart, liver, spleen, muscle, brain, eye, kidney, intestine, and fat. Moreover, we demonstrated that PA treatment induced the expression level of CiAIF and increases in markers of endoplasmic reticulum (ER) stress and apoptosis. Meanwhile, ER stress-inducing agent thapsigargin (TG) induced CiAIF translocated into the nucleus in CIK cells, whereas the suppression of ER stress inhibited PA-induced CiAIF expression and apoptosis. In addition, overexpression of CiAIF caused apoptosis by upregulating capase9, capase8, and capase3b, and affects protein translation via directly interacting with CieIF3g. Taken together, our data indicate that in Ctenopharyngodon idellus, PA is key elements that affect not only ER stress and mitochondrial apoptosis but also different physiological functions, such as protein translation, and CiAIF might play a key role in this progress.

Functional Genomic Characterization of Endoplasmic Reticulum-Associated Dependencies in Multiple Myeloma - Biologic and Therapeutic Implications

Background: Endoplasmic reticulum (ER)-associated degradation (ERAD) for unfolded proteins represents an important biological vulnerability for multiple myeloma (MM) cells, given the high proteostatic stress in these cells due to immunoglobulin secretion. This helps explain the efficacy of proteasome inhibitors in MM compared to most other neoplasms. Aim: To systematically investigate ER-associated genes that mediate the essential role of ERAD in MM. Methods: We integrated results from (i) genome-scale studies for CRISPR-gene knockout in 19 MM cell lines (Avana library) and CRISPR-activation (Calabrese library) in 5 MM lines, to define ER-associated dependencies and drivers of increased MM cell growth; (ii) publicly-available CRISPR-based studies for genes regulating ERAD of substrates located in the luminal (ERAD-L), membrane (ERAD-M) or cytosolic (ERAD-C) side of the ER in non-MM models; (iii) functional characterization of candidate genes in MM models in vitro and in vivo. Results: While ER function in general is globally required for all normal and malignant cells, a surprisingly large proportion of ER-related genes are not "pan-essential" genes (e.g. CERES scores <-0.4 are for <80% of human cell lines): in fact CRISPR-mediated disruption of many ER genes elicits no growth disadvantage for most non-MM tumor cell lines. Notably, though, several of these latter non-"pan- essential" genes are recurrently essential for MM cell lines, or even preferential dependencies for MM compared to other cancers. By overlaying the patterns of CRISPR essentiality in MM lines vs. the functional modules of ER function, we observe that recurrent or preferential ER-associated dependencies for MM cells include genes involved in (a) ER membrane protein complexes mediating dislocation of misfolded ER proteins to the cytosol (e.g. HERPUD1, SEL1L, AUP1 for ERAD-L substrates; and AMFR and RNF139 for ERAD-M and -C substrates,) and associated ER-specific E2/E3 enzymes (SYVN1, UBE2J1, UBE2J2, and UBE2G2); (b) upstream of the ER-to-cytosol dislocation complexes, several enzymes (e.g. DPM1, DPM3, ALG3, ALG9, ALG12, ALG6, ALG8) required for N-glycan-dependent surveillance of quality control for luminal ER glycoproteins; (c) chaperones (e.g. DNAJB11, DNAJBC3) for BiP complexes with misfolded proteins; (d) the known ER stress-sensor IRE1a (ERN1) and its downstream transcription factor XBP1. CRISPR-activation of nearly all these recurrent/MM-preferential ER-associated genes led to no significant growth advantage for MM cell lines. Several of the MM-preferential ER-associated genes (HERPUD1, SEL1L, SYVN1, UBE2J1) are expressed at significantly higher levels in MM vs. non-MM cell lines (CCLE) or patient derived samples (CoMMpass study vs. TCGA) and are proximal to binding sites of key transcription factors for plasma cell identity such as IRF4 and PRDM1. By contrast, CRISPR-mediated LOF of such genes in MM cells decreases their growth in vitro (e.g. competition assays vs. CRISPR LOF of non-essential genes not expressed in MM cells) and in vivo for MM cells growing in a bone marrow-like scaffold system with a "humanized" mesenchymal bone marrow stromal cell compartment. Knockout of MM-preferential ER-associated genes induces markers of ER stress/unfolded protein response (e.g. BiP induction by UBE2J1 or HERPUD1 LOF); and sensitizes MM cells to proteasome inhibition. Accordingly, pharmacological inhibition (LS-102) of SYVN1 (Synovolin-1) an E3 ligase which helps remove unfolded proteins from the cytosol into the ER was more effective at a lower drug concentration in MM compared to non-MM-cell lines. Conclusions: Our results provide functional insights into the role of ERAD in MM cells and nominate specific mediators which are not broadly essential across all cancers, but are recurrent or even preferentially essential for MM. Our study indicates a previously underappreciated role for the ER-to-cytosol dislocon complex that includes HERPUD1, SEL1L, SYVN1 and UBE2J1; distinct enzymes of the N-glycan-dependent ER glycoproteins quality control system; and ER chaperones in MM. These findings provide a rationale for development of agents selectively targeting these key regulators of ER protein folding processes in MM. Disclosures Downey-Kopyscinski: Rancho BioSciences, LLC: Current Employment. Tsherniak:Tango Therapeutics: Consultancy; Cedilla Therapeutics: Consultancy. Boise:AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genetech: Membership on an entity's Board of Directors or advisory committees. Mitsiades:EMD Serono: Research Funding; Abbvie: Research Funding; Karyopharm: Research Funding; Janssen/Johnson & Johnson: Research Funding; TEVA: Research Funding; FIMECS: Consultancy, Honoraria; Takeda: Other: employment of a relative; Ionis Pharmaceuticals, Inc.: Consultancy, Honoraria; Fate Therapeutics: Consultancy, Honoraria; Sanofi: Research Funding; Arch Oncology: Research Funding.

Intracellular dynamics of Ataxin-2 in the human brains with normal and frontotemporal lobar degeneration with TDP-43 inclusions

TAR DNA-binding protein of 43 kDa (TDP-43) is a major component of intracellular aggregates formed in brains of the patients with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS), which are correctively referred to as TDP-43 proteinopathies. A link between Ataxin-2 (ATXN2) and TDP-43 proteinopathies was established when intermediate CAG repeat expansions of ATXN2 gene were found to be associated with ALS and it was shown that ATXN2 modifies TDP-43 toxicity. Although ATXN2’s contribution to TDP-43 proteinopathies has been mostly studied in ALS, recent studies have shown that intermediate repeat expansions of ATXN2 also influence the phenotype of FTLD by an unknown mechanism. To address this issue, we immunohistochemically and biochemically analyzed the intracellular dynamics of ATXN2 in brains of normal controls and FTLD-TDP cases. The immunohistochemical studies revealed that ATXN2 localized in the neuronal cytoplasm and proximal dendrites, and expressed widely and uniformly in normal human brains. A semi-quantitative immunofluorescent analysis of normal brains revealed that the cytoplasmic ATXN2 strongly associates with ribosomal protein S6 and poly-A binding protein 1 and partially overlaps with the endoplasmic reticulum marker Calnexin, suggesting a major role of ATXN2 in protein synthesis. The results of immunohistochemical and biochemical analyses of brains from FTLD-TDP cases showed the colocalization of ATXN2 and phosphorylated TDP-43 in the dystrophic neurites and the neuronal cytoplasmic inclusions in the hippocampal region, and a significant reduction of ATXN2 protein compared to controls. These results suggest that ATXN2 is involved in the pathological process of FTLD-TDP. It remains to be clarified whether reduced ATXN2 expression induces neurodegeneration by impairing protein synthesis or plays a neuroprotective role by attenuating the toxicity of TDP-43 aggregates in FTLD-TDP and other TDP-43 proteinopathies.

Early Intracellular Trafficking and Subsequent Activity of Programmed Cell Death in Channel Catfish Macrophages Infected with Edwardsiella ictaluri.

The development of Edwardsiella-containing-vacuoles (ECV) and the ability of Edwardsiella ictaluri to survive and replicate within macrophages suggests a unique process relative to normal phagosomal/lysosomal maturation and programed cell death. Developing ECV showed that endosomal membrane markers Rab5, EEA1, and Rab7 were all detected in both the wild type (WT) and an E. ictaluri type-3 secretion system (T3SS) mutant, 65ST. Co-localization with Lamp1, however, was significantly lower in the WT. The host cell endoplasmic reticulum marker, calnexin, co-localized to 65ST ECV significantly more than WT ECV, while Golgi vesicle marker, giantin, was recruited to WT ECV significantly more than 65ST. The autophagosomal marker LC3 was significantly lower in WT than in 65ST and Western blotting demonstrated significantly greater induction of the membrane localized, lipidated form, LC3-II, in 65ST ECV than in WT ECV. Activity of the apoptosis initiator caspase-8 increased post-infection in 65ST and was significantly lower in WT-infected cells. Executioner caspase-3/7 activity also increased significantly in 65ST-infected cells compared to WT-infected cells. Repression of apoptosis was further demonstrated with flow cytometry using Alexa Fluor 647-labeled Annexin V and propidium iodide. Results indicate that WT ECV fused with early and late endosomes but that phagosomal/lysosomal fusion did not occur. Additionally, WT-infected cells recruited Golgi vesicles for vacuolar size increase and bacterial growth material, and both autophagy and apoptosis were repressed in the WT. This activity was all based on the function of the E. ictaluri T3SS.

Proteasomal dysfunction and ER stress triggers 2'-hydroxy-retrochalcone-induced paraptosis in cancer cells.

Chalcones are biologically active class of compounds, known for their anticancer activities. Here we show for the first time that out of the six synthetic derivatives of chalcone tested, 2'-hydroxy-retrochalcone (HRC) was the most effective in inducing extensive cytoplasmic vacuolation mediated death called paraptosis in malignant breast and cervical cancer cells. The cell death by HRC is found to be nonapoptotic in nature due to the absence of DNA fragmentation, PARP cleavage, and phosphatidylserine externalization. It was also found to be nonautophagic as there was an increase in the levels of autophagic markers LC3I, LC3II and p62. Immunofluorescence with the endoplasmic reticulum (ER) marker protein calreticulin showed that the cytoplasmic vacuoles formed were derived from the ER. This ER dilation was due to ER stress as evidenced from the increase in polyubiquitinated proteins, Bip and CHOP. Docking studies revealed that HRC could bind to the Thr1 residue on the active site of the chymotrypsin-like subunit of the proteasome. The inhibition of proteasomal activity was further confirmed by the fluorescence based assay of the chymotrypsin-like subunit of the 26S proteasome. The cell death by HRC was also triggered by the collapse of mitochondrial membrane potential and depletion of ATP. Pretreatment with thiol antioxidants and cycloheximide were able to inhibit this programmed cell death. Thus our data suggest that HRC can effectively kill cancer cells via paraptosis, an alternative death pathway and can be a potential lead molecule for anticancer therapy.

Effects of environmental cocaine concentrations on COX and caspase-3 activity, GRP-78, ALT, CRP and blood glucose levels in the liver and kidney of the European eel (Anguilla anguilla)

Cocaine is one of the most widely used illicit drugs in the world, and as a result of incomplete removal by sewage treatment plants it is found in surface waters, where it represents a new potential risk for aquatic organisms. In this study we evaluated the influence of environmental concentrations of cocaine on the liver and the kidney of the European eel (Anguilla anguilla). The eels were exposed to 20 ng L−1 of cocaine for fifty days, after which, three and ten days after the interruption of cocaine exposure their livers and kidneys were compared to controls. The general morphology of the two organs was evaluated, as well as the following parameters: cytochrome oxidase (COX) and caspase-3 activities, as markers of oxidative metabolism and apoptosis activation, respectively; glucose-regulated protein (GRP)78 levels, as a marker of endoplasmic reticulum (ER)-stress; blood glucose level, as stress marker; serum levels of alanine aminotransferase (ALT), as a marker of liver injury and serum levels of C-reactive protein (CRP), as a marker of the inflammatory process. The liver showed morphologic alterations such as necrotic areas, karyolysis and pyknotic nuclei, while the kidneys had dilated glomeruli and the renal tubules showed pyknotic nuclei and karyolysis. In the kidney, the alterations persisted after the interruption of cocaine exposure. In the liver, COX and caspase-3 activities increased (COX: P = 0.01; caspase-3: P = 0.032); ten days after the interruption of cocaine exposure, COX activity returned to control levels (P = 0.06) whereas caspase-3 activity decreased further (P = 0.012); GRP78 expression increased only in post-exposure recovery specimens (three days: P = 0.007 and ten days: P = 0.008 after the interruption of cocaine exposure, respectively). In the kidney, COX and caspase-3 activities increased (COX: P = 0.02; caspase-3: P = 0.019); after the interruption of cocaine exposure, COX activity remained high (three days: P = 0.02 and ten days: P = 0.029 after the interruption of cocaine exposure, respectively) whereas caspase-3 activity returned to control values (three days: P = 0.69 and ten days: P = 0.67 after the interruption of cocaine exposure, respectively). Blood glucose and serum ALT and CRP levels increased (blood glucose: P = 0.01; ALT: P = 0.001; CRP: 0.015) and remained high also ten days after the interruption of cocaine exposure (blood glucose: P = 0.009; ALT: P = 0.0031; CRP: 0.036). These results suggest that environmental cocaine concentrations adversely affected liver and kidney of this species.

Intrauterine exposure to low-dose DBP in the mice induces obesity in offspring via suppression of UCP1 mediated ER stress

Dibutyl phthalate (DBP) is recognized as an environmental endocrine disruptor that has been detected in fetal and postnatal samples. Recent evidence found that in utero DBP exposure was associated with an increase of adipose tissue weight and serum lipids in offspring, but the precise mechanism is unknown. Here we aimed to study the effects of in utero DBP exposure on obesity in offspring and examine possible mechanisms. SPF C57BL/6J pregnant mice were gavaged with either DBP (5 mg /kg/day) or corn oil, from gestational day 12 until postnatal day 7. After the offspring were weaned, the mice were fed a standard diet for 21 weeks, and in the last 2 weeks 20 mice were selected for TUDCA treatment. Intrauterine exposure to low-dose DBP promoted obesity in offspring, with evidence of glucose and lipid metabolic disorders and a decreased metabolic rate. Compared to controls, the DBP exposed mice had lower expression of UCP1 and significantly higher expression of Bip and Chop, known markers of endoplasmic reticulum (ER) stress. However, TUDCA treatment of DBP exposed mice returned these parameters nearly to the levels of the controls, with increased expression of UCP1, lower expression of Bip and Chop and ameliorated obesity. Intrauterine exposure of mice to low-dose DBP appears to promote obesity in offspring by inhibiting UCP1 via ER stress, a process that was largely reversed by treatment with TUDCA.

Evaluation of the Individual and Combined Toxicity of Fumonisin Mycotoxins in Human Gastric Epithelial Cells.

Fumonisin contaminates food and feed extensively throughout the world, causing chronic and acute toxicity in human and animals. Currently, studies on the toxicology of fumonisins mainly focus on fumonisin B1 (FB1). Considering that FB1, fumonisin B2 (FB2) and fumonisin B3 (FB3) could coexist in food and feed, a study regarding a single toxin, FB1, may not completely reflect the toxicity of fumonisin. The gastrointestinal tract is usually exposed to these dietary toxins. In our study, the human gastric epithelial cell line (GES-1) was used as in vitro model to evaluate the toxicity of fumonisin. Firstly, we found that they could cause a decrease in cell viability, and increase in membrane leakage, cell death and the induction of expression of markers for endoplasmic reticulum (ER) stress. Their toxicity potency rank is FB1 > FB2 >> FB3. The results also showed that the synergistic effect appeared in the combinations of FB1 + FB2 and FB1 + FB3. Nevertheless, the combinations of FB2 + FB3 and FB1 + FB2 + FB3 showed a synergistic effect at low concentration and an antagonistic effect at high concentration. We also found that myriocin (ISP-1) could alleviate the cytotoxicity induced by fumonisin in GES-1 cells. Finally, this study may help to determine or optimize the legal limits and risk assessment method of mycotoxins in food and feed and provide a potential method to block the fumonisin toxicity.

Heterogeneous expression of plasma-membrane-localised OsOSCA1.4 complements osmotic sensing based on hyperosmolality and salt stress in Arabidopsis osca1 mutant

In plants, both hyperosmolality and salt stress induce cytosolic calcium increases within seconds, referred to as the hyperosmolality-induced [Ca2+]cyt increases, OICIcyt, and salt stress-induced [Ca2+]cyt increases, SICIcyt. Previous studies have shown that Arabidopsis reduced hyperosmolality-induced [Ca2+]i increase 1 (OSCA1.1) encodes a hyperosmolality-gated calcium-permeable channel that mediates OICIcyt in guard cells and root cells. Multiple OSCA members exist in plants; for example, Oryza sativa has 11 OsOSCAs genes, indicating that OSCAs have diverse biological functions. Here, except for OsOSCA4.1, ten full-length OsOSCAs were separately subcloned, in which OsOSCA1.4 was exclusively localised to the plasma membrane and other nine OsOSCAs-eYFP co-localised with an endoplasmic reticulum marker in Arabidopsis mesophyll protoplasts. OsOSCA1.4 was further identified as a calcium-permeable ion channel that activates an inward current after receiving an osmotic signal exerted by hyperosmolality or salt stress, and mediates OICIcyt and SICIcyt in human embryonic kidney 293 (HEK293) cells. Moreover, overexpression of OsOSCA1.4 in Arabidopsis osca1 mutant complemented osmotic Ca2+ signalling, root growth, and stomatal movement in response to hyperosmolality and salt stress. These results will facilitate further study of OsOSCA-mediated calcium signalling and its distinct roles in rice growth and development.

Essential Functions of Glycans in Human Epithelia Dissected by a CRISPR-Cas9-Engineered Human Organotypic Skin Model.

SummaryThe glycome undergoes characteristic changes during histogenesis and organogenesis, but our understanding of the importance of select glycan structures for tissue formation and homeostasis is incomplete. Here, we present a human organotypic platform that allows genetic dissection of cellular glycosylation capacities and systematic interrogation of the roles of distinct glycan types in tissue formation. We used CRISPR-Cas9 gene targeting to generate a library of 3D organotypic skin tissues that selectively differ in their capacity to produce glycan structures on the main types of N- and O-linked glycoproteins and glycolipids. This tissue library revealed distinct changes in skin formation associated with a loss of features for all tested glycoconjugates. The organotypic skin model provides phenotypic cues for the distinct functions of glycoconjugates and serves as a unique resource for further genetic dissection and identification of the specific structural features involved. The strategy is also applicable to other organotypic tissue models.

Vps13 is required for the packaging of the ER into autophagosomes during ER-phagy

Endoplasmic reticulum (ER) macroautophagy (hereafter called ER-phagy) uses autophagy receptors to selectively degrade ER domains in response to starvation or the accumulation of aggregation-prone proteins. Autophagy receptors package the ER into autophagosomes by binding to the ubiquitin-like yeast protein Atg8 (LC3 in mammals), which is needed for autophagosome formation. In budding yeast, cortical and cytoplasmic ER-phagy requires the autophagy receptor Atg40. While different ER autophagy receptors have been identified, little is known about other components of the ER-phagy machinery. In an effort to identify these components, we screened the genome-wide library of viable yeast deletion mutants for defects in the degradation of cortical ER following treatment with rapamycin, a drug that mimics starvation. Among the mutants we identified was vps13Δ. While yeast has one gene that encodes the phospholipid transporter VPS13, humans have four vacuolar protein-sorting (VPS) protein 13 isoforms. Mutations in all four human isoforms have been linked to different neurological disorders, including Parkinson's disease. Our findings have shown that Vps13 acts after Atg40 engages the autophagy machinery. Vps13 resides at contact sites between the ER and several organelles, including late endosomes. In the absence of Vps13, the cortical ER marker Rtn1 accumulated at late endosomes, and a dramatic decrease in ER packaging into autophagosomes was observed. Together, these studies suggest a role for Vps13 in the sequestration of the ER into autophagosomes at late endosomes. These observations may have important implications for understanding Parkinson's and other neurological diseases.

The Impact of Sugar-Sweetened Beverage Consumption on the Liver: A Proteomics-based Analysis.

Cardiometabolic complications such as the metabolic syndrome and Type 2 Diabetes Mellitus (T2DM) are major causes of global morbidity and mortality. As sugar-sweetened beverages (SSBs) are implicated in this process, this study aimed to obtain greater mechanistic insights. Male Wistar rats (~200 g) were gavaged with a local SSB every day for a period of six months while the control group was gavaged with an iso-volumetric amount of water. Experimental dosages were calculated according to the surface area-to-volume ratio and were equivalent to 125 mL/day (in human terms). A proteomic analysis was performed on isolated liver samples and thereafter, markers of endoplasmic reticulum (ER) stress, antioxidant/oxidant capacity, calcium regulation, and mitochondrial functionality were assessed. These data show that SSB consumption resulted in (a) the induction of mild hepatic ER stress; (b) altered hepatic mitochondrial dynamics; and (c) perturbed calcium handling across mitochondria-associated ER membranes. Despite significant changes in markers of ER stress, the antioxidant response and calcium handling (proteomics data), the liver is able to initiate adaptive responses to counteract such stressors. However, the mitochondrial data showed increased fission and decreased fusion that may put the organism at risk for developing insulin resistance and T2DM in the longer term.

Dynamic Capture and Release of Endoplasmic Reticulum Exit Sites by Golgi Stacks in Arabidopsis.

SummaryProtein transport from the endoplasmic reticulum (ER) to Golgi stacks is mediated by the coat protein complex COPII, which is assembled at an ER subdomain called ER exit site (ERES). However, the dynamic relationship between ERESs and Golgi stacks is unknown. Here, we propose a dynamic capture-and-release model of ERESs by Golgi stacks in Arabidopsis thaliana. Using variable-angle epifluorescence microscopy with high-temporal-resolution imaging, COPII-component-bound ERESs were detected as punctate structures with sizes of 300–500 nm. Some punctate ERESs are distributed on ER tubules and sheet rims, whereas others gather around a Golgi stack in an ER-network cavity to form a beaded-ring structure. Free ERESs that wander into an ER cavity are captured by a Golgi stack in a cytoskeleton-independent manner. Then, they are released by the Golgi stack for recycling. The dynamic ERES cycling might contribute to efficient transfer of de novo synthesized cargo proteins from the ER to Golgi stacks.

The Attenuating Effect of the Intraovarian Bone Morphogenetic Protein 4 on Age-Related Endoplasmic Reticulum Stress in Chicken Follicular Cells.

In the poultry, only less than 5% primordial follicles in the ovary can develop into the prehierarchical follicles (PHFs) leading to progressive development, ovulation, and egg formation. This low rate of recruitment indicates a huge potential for improvement of the laying performance. A great reduction in egg production is caused by aging with extensive follicular atresia. In this study, age-related changes in the laying performance and ovarian status were compared between the peak-lay (D280) and aged (D580) chickens. Subsequently, a cross coculture of PHFs and granulosa cells (GCs) from D280 or D580 hens was adopted to reveal the mechanism of declined follicle development. Results showed that persistent endoplasmic reticulum (ER) stress in GCs of the aged hens was accompanied with intensified apoptosis. Bone morphogenetic protein 4 (BMP4) secreted by GCs of PHFs in D280 hens was capable of relieving ER stress and improving follicular dominance for selection in D580 hens. During this action, BMP4 reduced free calreticulin (CALR, an ER marker) content and attenuated cell apoptosis in PHFs of D580 hens via the PERK-CHOP-BCL2/caspase3 or CALR-Ca2+-BCL2-caspase12 pathway. Furthermore, BMP4 prevented follicular atresia by promoting production of steroid hormones to improve survival of GCs in PHFs from the aged hens. In conclusion, intensified ER stress and apoptosis occurred in GCs of PHFs in aged chickens, while BMP4 secreted by GCs was capable of improving follicular viability by alleviating ER stress to promote follicular development.

Aquaporin-11 Contributes to TGF-β1-Induced Endoplasmic Reticulum Stress in Human Visceral Adipocytes: Role in Obesity-Associated Inflammation.

Aquaporin-11 (AQP11) is expressed in human adipocytes, but its functional role remains unknown. Since AQP11 is an endoplasmic reticulum (ER)-resident protein that transports water, glycerol, and hydrogen peroxide (H2O2), we hypothesized that this superaquaporin is involved in ER stress induced by lipotoxicity and inflammation in human obesity. AQP11 expression was assessed in 67 paired visceral and subcutaneous adipose tissue samples obtained from patients with morbid obesity and normal-weight individuals. We found that obesity and obesity-associated type 2 diabetes increased (p < 0.05) AQP11 mRNA and protein in visceral adipose tissue, but not subcutaneous fat. Accordingly, AQP11 mRNA was upregulated (p < 0.05) during adipocyte differentiation and lipolysis, two biological processes altered in the obese state. Subcellular fractionation and confocal microscopy studies confirmed its presence in the ER plasma membrane of visceral adipocytes. Proinflammatory factors TNF-α, and particularly TGF-β1, downregulated (p < 0.05) AQP11 mRNA and protein expression and reinforced its subcellular distribution surrounding lipid droplets. Importantly, the AQP11 gene knockdown increased (p < 0.05) basal and TGF-β1-induced expression of the ER markers ATF4 and CHOP. Together, the downregulation of AQP11 aggravates TGF-β1-induced ER stress in visceral adipocytes. Owing to its “peroxiporin” properties, AQP11 overexpression in visceral fat might constitute a compensatory mechanism to alleviate ER stress in obesity.

The moonlighting protein c-Fos activates lipid synthesis in neurons, an activity that is critical for cellular differentiation and cortical development

Differentiation of neuronal cells is crucial for the development and function of the nervous system. This process involves high rates of membrane expansion, during which the synthesis of membrane lipids must be tightly regulated. In this work, using a variety of molecular and biochemical assays and approaches, including immunofluorescence microscopy and FRET analyses, we demonstrate that the proto-oncogene c-Fos (c-Fos) activates cytoplasmic lipid synthesis in the central nervous system and thereby supports neuronal differentiation. Specifically, in hippocampal primary cultures, blocking c-Fos expression or its activity impairs neuronal differentiation. When examining its subcellular localization, we found that c-Fos co-localizes with endoplasmic reticulum markers and strongly interacts with lipid-synthesizing enzymes, whose activities were markedly increased in vitro in the presence of recombinant c-Fos. Of note, the expression of c-Fos dominant-negative variants capable of blocking its lipid synthesis-activating activity impaired neuronal differentiation. Moreover, using an in utero electroporation model, we observed that neurons with blocked c-Fos expression or lacking its AP-1-independent activity fail to initiate cortical development. These results highlight the importance of c-Fos-mediated activation of lipid synthesis for proper nervous system development.

Functional characterization of four ATP-binding cassette transporter A3 gene (ABCA3) variants.

ABCA3 transports phospholipids across lamellar body membranes in pulmonary alveolar type II cells and is required for surfactant assembly. Rare, biallelic, pathogenic ABCA3 variants result in lethal neonatal respiratory distress syndrome and childhood interstitial lung disease. Qualitative functional characterization of ABCA3 missense variants suggests two pathogenic classes: disrupted intracellular trafficking (type I mutant) or impaired ATPase-mediated phospholipid transport into the lamellar bodies (type II mutant). We qualitatively compared wild-type (WT-ABCA3) with four uncharacterized ABCA3 variants (c.418A>C;p.Asn140His, c.3609_3611delCTT;p.Phe1203del, c.3784A>G;p.Ser1262Gly, and c.4195G>A;p.Val1399Met) in A549 cells using protein processing, colocalization with intracellular organelles, lamellar body ultrastructure, and ATPase activity. We quantitatively measured lamellar body-like vesicle diameter and intracellular ABCA3 trafficking using fluorescence-based colocalization. Three ABCA3 variants (p.Asn140His, p.Ser1262Gly, and p.Val1399Met) were processed and trafficked normally and demonstrated well-organized lamellar body-like vesicles, but had reduced ATPase activity consistent with type II mutants. P.Phe1203del was processed normally, had reduced ATPase activity, and well-organized lamellar body-like vesicles, but quantitatively colocalized with both endoplasmic reticulum and lysosomal markers, an intermediate phenotype suggesting disruption of both intracellular trafficking and phospholipid transport. All ABCA3 mutants demonstrated mean vesicle diameters smaller than WT-ABCA3. Qualitative and quantitative functional characterization of ABCA3 variants informs mechanisms of pathogenicity.

Targeting of the membrane‐anchored Rab GAP (GTPase accelerating protein) Gyp8 to peroxisomes is regulated by the AAA ATPase Msp1

Eukaryotic cells use vesicular transport to build and maintain membrane‐bound organelles housing specific biochemical functions. Rab GTPase signaling proteins (Rabs) are key regulators of vesicular transport, controlling where and when membranes dock and fuse, ensuring lipid and protein cargoes are delivered to appropriate destinations. Rabs depend upon GTPase accelerating proteins (GAPs) to trigger GTP hydrolysis and return the signaling active, GTP‐bound Rab to its inactive, GDP‐bound state. Rab GAPs contribute to the efficiency and fidelity of transport pathways, and defective Rab GAPs are implicated in a variety of human diseases. Rab GAPs tend to be cytosolic proteins that transiently localize at membranes to survey for their client Rab. We report that the yeast Rab GAP Gyp8 is an atypical transmembrane (TM) GAP whose association with peroxisomes is regulated by the AAA ATPase Msp1, a chaperone that functions to remove tail‐anchored proteins from peroxisomes and mitochondria. Computational analysis of Gyp8 predicted a single‐pass TM domain near the carboxy terminus, characteristic of a tail‐anchored protein. Fluoresence microscopy indicated that GFP‐tagged Gyp8 co‐localized with endoplasmic reticulum (ER) and peroxisomal markers in wild type cells. In the absence of peroxisomes, GFP‐Gyp8 redistributed to the ER. Loss of Msp1 chaperone function resulted in mislocalization of GFP‐Gyp8 to mitochondrial membranes. Subcellular fractionation biochemically demonstrated that Gyp8 localized exclusively to a membrane fraction containing peroxisomes. Truncation analyses of Gyp8 indicated that the TM and luminal domains are necessary and sufficient to direct localization to peroxisomes. Growth and viability experiments of cells lacking specific Rab GAPs indicate a peroxisome‐specific carbon utilization defect in cells lacking Gyp8. These data represent the first report in any experimental system of a Rab GAP that localizes to peroxisomes.Support or Funding InformationFunded by grants from the CSUSB Office of Student Research and Associated Students Inc.

Stay in Touch-The Cortical ER of Moss Protonemata in Osmotic Stress Situations.

Plasmolysis is usually introduced to cell biology students as a tool to illustrate the plasma membrane: hypertonic solutions cause the living protoplast to shrink by osmotic water loss; hence, it detaches from the surrounding cell wall. What happens, however, with the subcellular structures in the cell cortex during this process of turgor loss? Here, we investigated the cortical endoplasmic reticulum (ER) in moss protonema cells of Physcomitrella patens in a cell line carrying a transgenic ER marker (GFP-HDEL). The plasma membrane was labelled simultaneously with the fluorescent dye FM4-64 to achieve structural separation. By placing the protonemata in a hypertonic mannitol solution (0.8 M), we were able to follow the behaviour of the cortical ER and the protoplast during plasmolysis by confocal laser scanning microscopy (CLSM). The protoplast shape and structural changes of the ER were further examined after depolymerisation of actin microfilaments with latrunculin B (1 µM). In its natural state, the cortical ER is a dynamic network of fine tubes and cisternae underneath the plasma membrane. Under acute and long-term plasmolysis (up to 45 min), changes in the protoplast form and the cortical ER, as well as the formation of Hechtian strands and Hechtian reticula, were observed. The processing of the high-resolution z-scans allowed the creation of 3D models and gave detailed insight into the ER of living protonema cells before, during and after plasmolysis.

HMGB1 Translocation in Neurons after Ischemic Insult: Subcellular Localization in Mitochondria and Peroxisomes.

High mobility group box-1 (HMGB1), a nonhistone chromatin DNA-binding protein, is released from neurons into the extracellular space under ischemic, hemorrhagic, and traumatic insults. However, the details of the time-dependent translocation of HMGB1 and the subcellular localization of HMGB1 through the release process in neurons remain unclear. In the present study, we examined the subcellular localization of HMGB1 during translocation of HMGB1 in the cytosolic compartment using a middle cerebral artery occlusion and reperfusion model in rats. Double immunofluorescence microscopy revealed that HMGB1 immunoreactivities were colocalized with MTCO1(mitochondrially encoded cytochrome c oxidase I), a marker of mitochondria, and catalase, a marker of peroxisomes, but not with Rab5/Rab7 (RAS-related GTP-binding protein), LC3A/B (microtubule-associated protein 1 light chain 3), KDEL (KDEL amino acid sequence), and LAMP1 (Lysosomal Associated Membrane Protein 1), which are endosome, phagosome, endoplasmic reticulum, and lysosome markers, respectively. Immunoelectron microscopy confirmed that immune-gold particles for HMGB1 were present inside the mitochondria and peroxisomes. Moreover, HMGB1 was found to be colocalized with Drp1 (Dynamin-related protein 1), which is involved in mitochondrial fission. These results revealed the specific subcellular localization of HMGB1 during its release process under ischemic conditions.