Detection and Quantification of Calcium Ions in the Endoplasmic Reticulum and Cytoplasm of Cultured Cells Using Fluorescent Reporter Proteins and ImageJ Software

This protocol describes a method for detecting and quantifying calcium ions in the endoplasmic reticulum (ER) and cytoplasm of cultured cells using fluorescent reporter proteins and ImageJ software. Genetically engineered fluorescent reporter proteins, such as G-CEPIA1er and GCaMP6f, localize to intracellular regions of interest (i.e., ER and cytoplasm) and emit green fluorescence upon binding to calcium ions. In this way, the fluorescence brightness of cells transfected with expression vectors for these reporters reflects the calcium ion concentration in each intracellular region. Here, we describe procedures for observing cultured cells expressing these fluorescent reporters under a fluorescence microscope, analyzing the obtained image using the free image analysis software ImageJ (https://imagej.net/ij/index.html), and determining the average fluorescence brightness of multiple cells present in the image. The current method allows us to quickly and easily quantify calcium ions on an image containing multiple cells and to determine whether there are relative differences in intracellular calcium ion concentration among experiments with different conditions

Mechanisms of productive folding and endoplasmic reticulum-associated degradation of glycoproteins and non-glycoproteins

BACKGROUND: The quality of proteins destined for the secretory pathway is ensured by two distinct mechanisms in the endoplasmic reticulum (ER): productive folding of newly synthesized proteins, which is assisted by ER-localized molecular chaperones and in most cases also by disulfide bond formation and transfer of an oligosaccharide unit; and ER-associated degradation (ERAD), in which proteins unfolded or misfolded in the ER are recognized and processed for delivery to the ER membrane complex, retrotranslocated through the complex with simultaneous ubiquitination, extracted by AAA-ATPase to the cytosol, and finally degraded by the proteasome. SCOPE OF REVIEW: We describe the mechanisms of productive folding and ERAD, with particular attention to glycoproteins versus non-glycoproteins, and to yeast versus mammalian systems. MAJOR CONCLUSION: Molecular mechanisms of the productive folding of glycoproteins and non-glycoproteins mediated by molecular chaperones and protein disulfide isomerases are well conserved from yeast to mammals. Additionally, mammals have gained an oligosaccharide structure-dependent folding cycle for glycoproteins. The molecular mechanisms of ERAD are also well conserved from yeast to mammals, but redundant expression of yeast orthologues in mammals has been encountered, particularly for components involved in recognition and processing of glycoproteins and components of the ER membrane complex involved in retrotranslocation and simultaneous ubiquitination of glycoproteins and non-glycoproteins. This may reflect an evolutionary consequence of increasing quantity or quality needs toward mammals. GENERAL SIGNIFICANCE: The introduction of innovative genome editing technology into analysis of the mechanisms of mammalian ERAD, as exemplified here, will provide new insights into the pathogenesis of various diseases

pXBP1(U) encoded in XBP1 pre-mRNA negatively regulates unfolded protein response activator pXBP1(S) in mammalian ER stress response

Upon the accumulation of unfolded proteins in the mammalian endoplasmic reticulum (ER), X-box binding protein 1 (XBP1) premessenger RNA (premRNA) is converted to mature mRNA by unconventional splicing that is mediated by the endonuclease inositol-requiring enzyme 1. The transcription factor protein (p) XBP1 spliced (S), which is translated from mature XBP1 mRNA, contains the nuclear localization signal and the transcriptional activation domain and activates the transcription of target genes, including those encoding ER chaperones in the nucleus. We show that pXBP1 unspliced (U) encoded in XBP1 pre-mRNA was constitutively expressed and markedly accumulated at the recovery phase of ER stress. pXBP1(U) contained the nuclear exclusion signal instead of the transcriptional activation domain and shuttled between the nucleus and the cytoplasm. Interestingly, pXBP1(U) formed a complex with pXBP1(S), and the pXBP1(U)–pXBP1(S) complex was sequestered from the nucleus. Moreover, the complex was rapidly degraded by proteasomes because of the degradation motif contained in pXBP1(U). Thus, pXBP1(U) is a negative feedback regulator of pXBP1(S), which shuts off the transcription of target genes during the recovery phase of ER stress

A motor neuron disease-associated mutation produces non-glycosylated Seipin that induces ER stress and apoptosis by inactivating SERCA2b

遺伝病の原因タンパク質が小胞体ストレスを引き起こすメカニズムの解明 --神経変性疾患の新規治療戦略の確立に向けて--. 京都大学プレスリリース. 2022-12-13.A causal relationship between endoplasmic reticulum (ER) stress and the development of neurodegenerative diseases remains controversial. Here, we focused on Seipinopathy, a dominant motor neuron disease, based on the finding that its causal gene product, Seipin, is a protein that spans the ER membrane twice. Gain-of-function mutations of Seipin produce non-glycosylated Seipin (ngSeipin), which was previously shown to induce ER stress and apoptosis at both cell and mouse levels albeit with no clarified mechanism. We found that aggregation-prone ngSeipin dominantly inactivated SERCA2b, the major calcium pump in the ER, and decreased the calcium concentration in the ER, leading to ER stress and apoptosis in human colorectal carcinoma-derived cells (HCT116). This inactivation required oligomerization of ngSeipin and direct interaction of the C-terminus of ngSeipin with SERCA2b, and was observed in Seipin-deficient neuroblastoma (SH-SY5Y) cells expressing ngSeipin at an endogenous protein level. Our results thus provide a new direction to the controversy noted above

XBP1: a link between the unfolded protein response, lipid biosynthesis, and biogenesis of the endoplasmic reticulum

When the protein folding capacity of the endoplasmic reticulum (ER) is challenged, the unfolded protein response (UPR) maintains ER homeostasis by regulating protein synthesis and enhancing expression of resident ER proteins that facilitate protein maturation and degradation. Here, we report that enforced expression of XBP1(S), the active form of the XBP1 transcription factor generated by UPR-mediated splicing of XBP1 mRNA, is sufficient to induce synthesis of phosphatidylcholine, the primary phospholipid of the ER membrane. Cells overexpressing XBP1(S) exhibit elevated levels of membrane phospholipids, increased surface area and volume of rough ER, and enhanced activity of the cytidine diphosphocholine pathway of phosphatidylcholine biosynthesis. These data suggest that XBP1(S) links the mammalian UPR to phospholipid biosynthesis and ER biogenesis

Linear Macropore Installation to Reduce Red-Soil Erosion in Sugarcane Fields

This study determines the cause of soil erosion in red soils in sugarcane fields, especially even with the use of subsoiling fissures, and to compare the effectiveness of a novel artificial linear-macropore with the insertion of fibrous material into the fractures. Four column treatments (tillage, subsoiling, linear-macropore with plant residue fillings, and no-tillage-with-mulching) were established. A subsoiler was used to break up hard soil layers to enhance infiltration, whereas mulching reduced the impact of raindrops on the soil. Sugarcane residue was inserted in the empty fissure to reinforce the structure, making linear macropore. Simulated rainfall with 20 mmh−1 was applied to the soil surface for 6 h per day for two days. Surface runoff, soil erosion, and drainage were measured during each run. Erosion was minimal (1/7 reduction), and bottom drainage was observed in the linear-macropore and no-tillage-with-mulching plots. Conversely, due to the formation of an impermeable layer or surface crust, high erosion (0.282 t-C ha−1 yr−1) and decreased drainage levels were detected in the subsoiling and tillage plots. Moreover, the aboveground protrusion of fibrous material at the linear-macropore maintained infiltration, even following crust formation. Field application of these four management strategies revealed the effectiveness of linear-macropore and mulching in reducing surface flow. Linear-macropore application maintains appropriate levels of infiltration, and insertion of plant residue fillings reinforces the macropore structure while also avoiding clogging. Hence, the linear-macropore scheme may represent an effective strategy for reducing surface runoff and red soil erosion

Proteomic analysis of fatty liver induced by starvation of medaka fish larvae

When medaka fish (Oryzias latipes) larvae are grown in the absence of exogenous nutrition, the liver becomes dark and positive to Oil Red O staining from 7 days post-hatch (dph). We determined the mechanism of this starvation-induced development of fatty liver by proteomic analysis using livers obtained from larvae grown in the presence or absence of 2% glucose at 5 dph. Results showed that changes in the expression levels of enzymes involved in glycolysis or the tricarboxylic acid cycle were modest, whereas the expression levels of enzymes involved in amino acid catabolism or β-oxidation of fatty acids were significantly elevated, suggesting that they become major energy sources under starvation conditions. Expression levels of enzymes for the uptake and β-oxidation of fatty acids as well as synthesis of triacylglycerol were elevated, whereas those for the synthesis of cholesterol as well as export of cholesterol and triacylglycerol were decreased under starvation conditions, which explains the accumulation of triacylglycerol in the liver. Our results provide the basis for future research to understand how gene malfunction(s) affects the development of fatty liver, which can lead to nonalcoholic steatohepatitis and then to liver cirrhosis.Key words: amino acid catabolism, β-oxidation, triacylglycerol, cholesterol, export

Experimental Study on Channel Congestion using IEEE 802.11p Communication System

International audienceVehicle to Vehicle (V2V) and Vehicle to Infrastructure (V2I) communications are expected to play an important role to improve road safety, road efficiency, and the comfort of road users. In order to support such ITS communications, IEEE standardized 802.11p amendment, which is adopted by ETSI as ITSG5 for European usages. While much expectation has been put in using the system for V2V road safety and efficiency applications, very limited knowledge is achieved regarding its characteristics especially scalability. Motivated by this, we conduct experimental study using IEEE 802.11p communication devices with GeoNetworking (Geographic addressing and routing) and investigate its channel performances affected by number of devices, packet size and traffic rate

Activation of XBP1 but not ATF6α rescues heart failure induced by persistent ER stress in medaka fish

The unfolded protein response is triggered in vertebrates by ubiquitously expressed IRE1α/β (although IRE1β is gut-specific in mice), PERK, and ATF6α/β, transmembrane-type sensor proteins in the ER, to cope with ER stress, the accumulation of unfolded and misfolded proteins in the ER. Here, we burdened medaka fish, a vertebrate model organism, with ER stress persistently from fertilization by knocking out the AXER gene encoding an ATP/ADP exchanger in the ER membrane, leading to decreased ATP concentration–mediated impairment of the activity of Hsp70- and Hsp90-type molecular chaperones in the ER lumen. ER stress and apoptosis were evoked from 4 and 6 dpf, respectively, leading to the death of all AXER-KO medaka by 12 dpf because of heart failure (medaka hatch at 7 dpf). Importantly, constitutive activation of IRE1α signaling --but not ATF6α signaling-- rescued this heart failure and allowed AXER-KO medaka to survive 3 d longer, likely because of XBP1-mediated transcriptional induction of ER-associated degradation components. Thus, activation of a specific pathway of the unfolded protein response can cure defects in a particular organ

Net-step exercise and depressive symptoms among the community-dwelling elderly in Japan

Introduction: Leisure-time physical activity (LTPA) and exercise have attracted attention as potential preventive factors against depression in the elderly. The net-step exercise (NSE) was developed in Hokkaido, Japan to assist elderly people with decreased physical functions. NSE is a non-aerobic, low-intensity, and slow balance motion LTPA. In the present study, the relationship between NSE and depressive symptoms among the community-dwelling elderly is examined. Methods: This study employed a cross-sectional design with community-dwelling elderly participants, aged 72?81 years (n = 672; mean age = 76.4 years). Participation in NSE and other LTPA, including walking, jogging, and park golf, a sport popular in Hokkaido, particularly among the elderly, was assessed. Depressive symptoms were measured using the 15-item Geriatric Depression Scale (GDS-15). Results: Univariate analysis showed that those participating in NSE more than once a month and those who reported engaging in walking or park golf more than once a week were less likely to report symptoms of depression. Multivariate analysis showed that NSE and walking had marginally significant (odds ratio (OR) = 0.53; 95% CI: 0.27?1.02) and significant (OR = 0.61; 95% CI: 0.40?0.93) inverse associations, respectively, with reports of depressive symptoms even after adjustments for age, sex, years of education, continuous walking for 15 min, experience of a fall in the past year, utilization of various long-term care services provided for the elderly, stroke, frequency of jogging, and park golf. Discussion: This study showed that participants engaging in NSE, which is a non-aerobic, low-intensity, and low frequency activity, had a marginally significantly inverse association with depressive symptoms. Longitudinal research should be conducted in the future