Reticulum Membrane Research Articles

Protein kinase Hsl1 phosphorylates Pah1 to inhibit phosphatidate phosphatase activity and regulate lipid synthesis in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, Pah1 phosphatidate (PA) phosphatase, which catalyzes the Mg2+-dependent dephosphorylation of PA to produce diacylglycerol, plays a key role in utilizing PA for the synthesis of the neutral lipid triacylglycerol and thereby controlling the PA-derived membrane phospholipids. The enzyme function is controlled by its subcellular location as regulated by phosphorylation and dephosphorylation. Pah1 is initially inactivated in the cytosol through phosphorylation by multiple protein kinases and then activated via its recruitment and dephosphorylation by the protein phosphatase Nem1-Spo7 at the nuclear/endoplasmic reticulum membrane where the PA phosphatase reaction occurs. Many of the protein kinases that phosphorylate Pah1 have yet to be characterized with the identification of the target residues. Here, we established Pah1 as a bona fide substrate of septin-associated Hsl1, a protein kinase involved in mitotic morphogenesis checkpoint signaling. The Hsl1 activity on Pah1 was dependent on reaction time and the amounts of protein kinase, Pah1, and ATP. The Hsl1 phosphorylation of Pah1 occurred on Ser-748 and Ser-773, and the phosphorylated protein exhibited a 5-fold reduction in PA phosphatase catalytic efficiency. Analysis of cells expressing the S748A and S773A mutant forms of Pah1 indicated that Hsl1-mediated phosphorylation of Pah1 promotes membrane phospholipid synthesis at the expense of triacylglycerol, and ensures the dependence of Pah1 function on the Nem1-Spo7 protein phosphatase. This work advances understanding of how Hsl1 facilitates membrane phospholipid synthesis through the phosphorylation-mediated regulation of Pah1.

TRPV1 Channels Are New Players in the Reticulum-Mitochondria Ca2+ Coupling in a Rat Cardiomyoblast Cell Line.

The Ca2+ release in microdomains formed by intercompartmental contacts, such as mitochondria-associated endoplasmic reticulum membranes (MAMs), encodes a signal that contributes to Ca2+ homeostasis and cell fate control. However, the composition and function of MAMs remain to be fully defined. Here, we focused on the transient receptor potential vanilloid 1 (TRPV1), a Ca2+-permeable ion channel and a polymodal nociceptor. We found TRPV1 channels in the reticular membrane, including some at MAMs, in a rat cardiomyoblast cell line (SV40-transformed H9c2) by Western blotting, immunostaining, cell fractionation, and proximity ligation assay. We used chemical and genetic probes to perform Ca2+ imaging in four cellular compartments: the endoplasmic reticulum (ER), cytoplasm, mitochondrial matrix, and mitochondrial surface. Our results showed that the ER Ca2+ released through TRPV1 channels is detected at the mitochondrial outer membrane and transferred to the mitochondria. Finally, we observed that prolonged TRPV1 modulation for 30 min alters the intracellular Ca2+ equilibrium and influences the MAM structure or the hypoxia/reoxygenation-induced cell death. Thus, our study provides the first evidence that TRPV1 channels contribute to MAM Ca2+ exchanges.

Key role of three-dimensional reticular interface membranes in the formation of single polysaccharide–stabilized high internal phase emulsions

Although the interfacial activity of polysaccharides is believed to be insufficient for their applications as individual emulsifying stabilizers, four single polysaccharides examined herein, namely sodium carboxymethyl cellulose (Na-CMC), sodium alginate (SA), high-methoxyl pectin (HMP), and low-methoxyl pectin (LMP) are shown to effectively stabilize high internal phase emulsions (HIPEs) at loadings as low as 0.1 wt% under acidic conditions, that is, at pH 0.5–5.2 (Na-CMC), 0.5–4.0 (SA), 1.0–4.2 (HMP), and 1.0–3.0 (LMP). Despite their structural differences, the four studied polysaccharides feature similar HIPE stabilization mechanisms that mainly involve the formation of three-dimensional reticular interface membrane structures with fiber branching chains. Within the pH range suitable for HIPE formation, the polysaccharides mainly exist as nanofiber network structures with a grid width of 50–100 nm but adopt flake-like and other structures at higher pH. Further insights are provided by the relationship between network membrane structures formed at the interface and the fiber network structures formed by polysaccharides in the original aqueous phase. In addition, the inferred emulsification and stabilization mechanisms are shown to be significantly different from those observed for mainstream Pickering HIPE stabilizers. Thus, our work expands the understanding of the interfacial activity function of polysaccharides, provides conceptual thinking reference and design rules for the development of simple, efficient, safe, and cheap polysaccharide-based HIPEs, and supplements and furthers the theory of HIPE stabilization.

Structural and functional characterization of the cardiac mitochondrial-associated reticulum membranes in the obesogenic and diabetic ob/ob mouse model

Diabetic cardiomyopathy (DCM) strongly leads to metabolic heart failure with preserved ejection fraction (HFpEF). The involvement of mitochondria-associated reticular membranes (MAMs) in T2D-related metabolic disorders starts to be demonstrated. We recently discovered a reticulum-mitochondria Ca2 + uncoupling in a diet-induced mouse model of DCM with metabolic HFpEF. However, whether cardiac MAMs are affected by T2D and obesity specifically or by DCM with metabolic HFpEF remains unknown. We aimed to study the cardiac phenotype of another obesogenic T2D mouse model (leptin-deficient) together with the proteomic composition and function of their cardiac MAMs. Cardiac contractile function and structure were evaluated by echocardiography, electron microscopy and histology in 12 weeks old male WT and ob/ob mice. MAMs protein composition was assessed by mass spectrometry and by Uniprot and Panther softwares. Cell death in cardiomyocytes (CM) after hypoxia/reoxygenation stress was assessed using flow cytometry. Reticulum-mitochondria Ca2+ fluxes were assessed on CM using a FRET-based mitochondrial Ca2+ sensor expressed by adenoviral infection. Echocardiography analysis revealed strain rate dysfunction and a concentric hypertrophy remodeling while no change was observed in fractional shortening or diastolic function in ob/ob mice. Histological assessment showed an increased lipid deposition but similar fibrosis in the ob/ob heart compared to WT ones. Cardiac MAM length and width were similar between both groups. However, a trend towards an increased MAM protein content was measured in the ob/ob heart. Further MAM proteome analyses showed mainly increased processes in ob/ob hearts, notably the cellular response to stress, lipid metabolism, ion transport and membrane organization. Indeed, functionally, hypoxic stress induces a cell death increase in the ob/ob CM, while MAM-driven Ca2+ fluxes were unchanged. Mitochondrial respiration, CM shortening, ATP and ROS content were also similar between both groups. The T2D ob/ob mouse model does not recapitulate the main hallmarks of metabolic HFpEF and does not display any cardiac MAM-driven Ca2+ uncoupling, contrary to the high-fat-high-sucrose diet-induced obesogenic mouse model. Therefore, the alteration of the cardiac MAM-driven Ca2+ coupling seems specific to the development of DCM with metabolic HFpEF.

High-internal-phase emulsions stabilized solely by chitosan hydrochloride: Fabrication and effect of pH on stabilization mechanism

High-internal-phase emulsions (HIPEs) have numerous applications, particularly in the food industry, and are therefore extensively researched. In particular, the discovery of green, simple, and efficient HIPE stabilizers and the elucidation of their structure-activity relationships and stabilization mechanisms are of great practical significance. Herein, chitosan hydrochloride (CHC) was used to stabilize variable-transparency HIPEs under different conditions. At a CHC content of 0.08 wt.%, one-step shear dispersion at pH 6.0–6.7 produced milky white HIPEs with small droplets (∼5 μm), high stability, strong gelation, and good plasticity, whereas highly transparent HIPEs with larger droplets (∼100 μm) were formed at pH 6.8–12.7. The stabilization mechanism depended on pH, corresponding to the formation of (i) three-dimensional (3D) reticular membranes (pH 6.0–6.7) or (ii) fibrous Pickering particles (pH 6.8–12.7). This dependence was ascribed to the effects of pH on CHC structure: CHC mainly existed as a nanofiber network with grids at pH 6.0–6.7, whereas fibrous aggregates were observed at higher pH. Thus, our study presents important insights into the pH-dependent mechanism of HIPE stabilization by CHC and facilitates the development of new emulsifiers for the formulation of variable-transparency HIPEs with diverse applications.

Abstract 12618: Human And Mouse Demonstration Of The Reticulum-Mitochondria Ca 2+ Coupling As A New Therapeutic Target In Metabolic HFpEF

Context: Metabolic heart failure with preserved ejection fraction (HFpEF) is a frequent and disabling disease with yet no therapy and limited knowledge on the cardiac underlying molecular mechanisms. Ca 2+ transfer at mitochondria-associated reticular membranes (MAM) is crucial in the cardiomyocyte to tightly regulate mitochondrial Ca 2+ content and assure the excitation-energetics coupling. We aimed to specify the role of the MAM Ca 2+ coupling in both human and mouse hearts with metabolic HFpEF. Results: Mass spectrometry analysis of cardiac MAM showed an upregulation of cellular response to stress and lipid metabolism processes in two obesogenic T2D mouse models (16 weeks of high-fat high-sucrose diet, HFHSD vs standard diet, SD; 12-week old leptin-deficient ob/ob vs WT), while Ca 2+ transport was downregulated only in the HFHSD MAM. MAM Ca 2+ coupling assessment by adenoviral infection of a FRET-based mitochondrial Ca 2+ sensor, revealed a MAM Ca 2+ uncoupling in the HFHSD cardiomyocyte, leading to reduced mitochondrial Ca 2+ content and bioenergetics, while no change was measured in the ob/ob cell. HFHSD mice exhibited cardiac hypertrophy, diastolic dysfunction and mild strain rate reduction with preserved EF. Ob/ob mice only displayed strain rate reduction and concentric hypertrophy. Principal component analysis (PCA) demonstrated a divergence between the HFHSD group and the three others, as displayed by their scattering along the first PC (61% of variance) contributed mainly by strain rate, wall thickness, body weight and Ca 2+ peak amplitude. Proximity ligation assay revealed a decreased proximity between the reticular IP3-receptor and the mitochondrial porin VDAC in human left ventricle biopsies from T2D versus non-diabetic patients, obtained during valve replacement surgery: this suggests a reduced formation of the IP3R-driven Ca 2+ channeling complex. Conclusion: The ob/ob model does not recapitulate the main hallmarks of metabolic HFpEF and does not display any change in MAM Ca 2+ coupling. On the contrary, a MAM Ca 2+ uncoupling was detected both in the human T2D heart and in the heart of the HFHSD-induced metabolic HFpEF mouse, suggesting the reticulum-mitochondria Ca 2+ coupling as a critical therapeutic target in metabolic HFpEF.

Phosphatidic Acid Mediates the Nem1-Spo7/Pah1 Phosphatase Cascade in Yeast Lipid Synthesis

In the yeast Saccharomyces cerevisiae, the PAH1-encoded Mg2+-dependent phosphatidate (PA) phosphatase Pah1 regulates the bifurcation of PA to diacylglycerol (DAG) for triacylglycerol (TAG) synthesis and to CDP-DAG for phospholipid synthesis. Pah1 function is mainly regulated via control of its cellular location by phosphorylation and dephosphorylation. Pah1 phosphorylated by multiple protein kinases is sequestered in the cytosol apart from its substrate PA in the membrane. The phosphorylated Pah1 is then recruited and dephosphorylated by the protein phosphatase complex Nem1 (catalytic subunit)-Spo7 (regulatory subunit) in the endoplasmic reticulum. The dephosphorylated Pah1 hops onto and scoots along the membrane to recognize PA for its dephosphorylation to DAG. Here, we developed a proteoliposome model system that mimics the Nem1-Spo7/Pah1 phosphatase cascade to provide a tool for studying Pah1 regulation. Purified Nem1-Spo7 was reconstituted into phospholipid vesicles prepared in accordance with the phospholipid composition of the nuclear/endoplasmic reticulum membrane. The Nem1-Spo7 phosphatase reconstituted in the proteoliposomes, which were measured 60 nm in an average diameter, was catalytically active on Pah1 phosphorylated by Pho85-Pho80, and its active site was located at the external side of the phospholipid bilayer. Moreover, we determined that PA stimulated the Nem1-Spo7 activity, and the regulatory effect was governed by the nature of the phosphate headgroup but not by the fatty acyl moiety of PA. The reconstitution system for the Nem1-Spo7/Pah1 phosphatase cascade, which starts with the phosphorylation of Pah1 by Pho85-Pho80 and ends with the production of DAG, is a significant advance to understand a regulatory cascade in yeast lipid synthesis.

MICROSTRUCTURAL FEATURES OF THE VEGETATIVE ORGANS OF DELPHIUM FLEXUOSUM BIEB. COMMON IN GEORGIA

The genus Delphinium L. is rich in diterpene alkaloids, which exhibit a pronounced curare-like, anti-tumor, anti-inflammatory, analgesic, antidote and insecticidal action. The content of diterpene alkaloids in the aboveground and unde-ground vegetative organs of Delphinium flexuosum Bieb., a promising medicinal plant of the flora of Georgia, and diagnostic features of the internal structure were studied in order to determine the identity of the experimental raw material.GC/MS analysis revealed that the dominant alkaloid obtained from the vegetative organs of the plant is the pharmacologically active methyllicaconitine.  Other alkaloids have been identified as anthranoillocytonine, delcosine, lycotonine. As well, the underground organs contain also zongorine, delectine and norzongoramine. A set of diagnostic features of the internal structure of D. flexuosum was determined using the methods adopted in microtechnics. The leaf of D. flexuosum is bifacial, hypostomatic and has dorsoventral structure. It is very rarely covered with long conical trichomes, the underlying cells of the lower and upper epidermis of the leaf are non-wrinkled and curved. The differential stomatal apparatus in leaf-covering tissue is anomocytic.  Stomata are lenticular, and structure of the stomata is chaotic, in terms of the reciprocal location of the locking cells. The leaf pulp is differentiated into open, reverse-collateral conductive cones. The aboveground organs of D. flexuosum are characterized by an orderly aligned structural elements of the epidermal tissue, with linear and straight-walled stem cells and orderly arranged anomocytic stomata. Collenchymal and fibrous cells of the mechanical type and open-collateral type conducting cones with a thickening of the spiral and reticular membrane are actively reflected in the organs of the plant axis. The conductive tissue of the root system of D. flexuosum contains a number of polar rays of the wood with differentiated stepwise, spiral and ring-shaped tracheal elements.

Triaxial Mechanical Properties and Mechanism of Waterborne Polyurethane-Reinforced Road Demolition Waste as Road Bases.

The recycling and reuse of construction waste have not only effectively protected natural resources but also promoted the sustainable development of the environment. Therefore, in this article, waterborne polyurethane (WPU) as a promising new polymer reinforcement material was proposed to reinforce the road demolition waste (RDW), and the mechanical performance of WPU-reinforced RDW (named PURD) was investigated using triaxial unconsolidated and undrained shear (UU) and Scanning Electron Microscope (SEM) tests. The results showed that under the same curing time and confining pressure, the shear strength of PURD increased with the increase in WPU content. When the WPU content was 6%, the WPU presented the best reinforcement effect on RA. The failure strain of PURD increased with the increase in confining pressure, but increased first and then reduced with the increase in WPU content. The specimens with 5% WPU content showed the best ductility. At the curing time of 7 and 28 days, the internal friction angle and cohesion of PURD increased with the increase in WPU content, and they reached a maximum when the WPU content was 6%. The internal friction angle barely budged, but the cohesion increased obviously. The enhancement effect of WPU was attributed to the spatial reticular membrane structure produced by wrapping and bonding particles with the WPU film. Microscopic analysis showed that with the increase in WPU content, the internal pore and crack size of PURD gradually decreased. As the WPU content increased, the WPU film became increasingly thicker, which increased the adhesion between WPU and RA particles and made the structure of PURD become increasingly denser.

200-OR: IER3IP1 Is Critical for Maintaining Glucose Homeostasis through Regulating the Endoplasmic Reticulum Function and Survival of ß-Cells

Recessive mutations in IER3IP1 (Immediate Early Response 3 Interacting Protein 1) cause a triad of microcephaly, epilepsy, and permanent neonatal diabetes syndrome (MEDS) . IER3IP1 encodes an endoplasmic reticulum (ER) membrane protein, which is crucial for brain development, however the role of IER3IP1 in β cell function and glucose homeostasis remains unknown. Herein, we established a new β cell-specific IER3IP1 knockout (IER3IP1-βKO) mouse model and found that IER3IP1 deficiency in β cells causes severe early onset insulin deficient diabetes. Functional studies revealed a markedly dilated ER along with significantly increased proinsulin misfolding and elevated expression of the ER chaperones including PDI, ERO1, BiP, and P58IPK. Transcriptome analysis confirmed by qRT-PCR revealed a markedly decreased expression of genes associated with β cell maturation, cell cycle, and anti-apoptosis, whereas there was a significantly increased expression of anti-proliferation genes. Indeed, multiple independent approaches further demonstrated that IER3IP1-βKO impaired β cell maturation and proliferation, and promoted cell death. These data not only uncover a critical role of IER3IP1 in normal β cell function and survival, but they also provide insight into the mechanism (s) by which loss-of-function mutations of IER3IP1 lead to diabetes. Disclosure J.Yang: None. J.Sun: None. N.Li: None. T.Liu: None. S.Wang: None. X.Zhang: None. P.Arvan: n/a. M.Liu: None. J.Zhen: None. W.Feng: None. Z.Fan: None. Y.Huang: None. H.Shu: None. X.Li: None. J.Qiao: None. Y.Fan: None. Funding National Natural Science Foundation of China (81830025, 81620108004, 81870533, 81900720, 81800733, 82100865, 82070805, and 81870535)

Phosphorylation-mediated regulation of the Nem1-Spo7/Pah1 phosphatase cascade in yeast lipid synthesis.

The PAH1-encoded phosphatidate phosphatase, which catalyzes the dephosphorylation of phosphatidate to produce diacylglycerol, controls the divergence of phosphatidate into triacylglycerol synthesis and phospholipid synthesis. Pah1 is inactive in the cytosol as a phosphorylated form and becomes active on the nuclear/endoplasmic reticulum membrane as a dephosphorylated form by the Nem1-Spo7 protein phosphatase complex. The phosphorylation of Pah1 by protein kinases, which include casein kinases I and II, Pho85-Pho80, Cdc28-cyclin B, and protein kinases A and C, controls its cellular location, catalytic activity, and susceptibility to proteasomal degradation. Nem1 (catalytic subunit) and Spo7 (regulatory subunit), which form a protein phosphatase complex catalyzing the dephosphorylation of Pah1 for its activation, are phosphorylated by protein kinases A and C. In this review, we discuss the functions and interrelationships of the protein kinases in the control of the Nem1-Spo7/Pah1 phosphatase cascade and lipid synthesis.

Enhanced Orai1-mediated store-operated Ca2+ channel/calpain signaling contributes to high glucose-induced podocyte injury

Podocyte injury induced by hyperglycemia is the main cause of kidney dysfunction in diabetic nephropathy. However, the underlying mechanism is unclear. Store-operated Ca2+ entry (SOCE) regulates a diversity of cellular processes in a variety of cell types. Calpain, a Ca2+-dependent cysteine protease, was recently shown to be involved in podocyte injury. In the present study, we sought to determine whether increased SOCE contributed to high glucose (HG)–induced podocyte injury through activation of the calpain pathway. In cultured human podocytes, whole-cell patch clamp indicated the presence of functional store-operated Ca2+ channels, which are composed of Orai1 proteins and mediate SOCE. Western blots showed that HG treatment increased the protein abundance of Orai1 in a dose-dependent manner. Consistently, calcium imaging experiments revealed that SOCE was significantly enhanced in podocytes following HG treatment. Furthermore, HG treatment caused overt podocyte F-actin disorganization as well as a significant decrease in nephrin protein abundance, both of which are indications of podocyte injury. These podocyte injury responses were significantly blunted by both pharmacological inhibition of Orai1 using the small molecule inhibitor BTP2 or by genetic deletion of Orai1 using CRISPR-Cas9 lentivirus. Moreover, activation of SOCE by thapsigargin, an inhibitor of Ca2+ pump on the endoplasmic/sarcoplasmic reticulum membrane, significantly increased the activity of calpain, which was inhibited by BTP2. Finally, the calpain-1/calpain-2 inhibitor calpeptin significantly blunted the nephrin protein reduction induced by HG treatment. Taken together, our results suggest that enhanced signaling via an Orai1/SOCE/Calpain axis contributes to HG-induced podocyte injury.

Liver X receptor-agonist treatment rescues degeneration in a Drosophila model of hereditary spastic paraplegia

Hereditary spastic paraplegias (HSPs) are a group of inherited, progressive neurodegenerative conditions characterised by prominent lower-limb spasticity and weakness, caused by a length-dependent degeneration of the longest corticospinal upper motor neurons. While more than 80 spastic paraplegia genes (SPGs) have been identified, many cases arise from mutations in genes encoding proteins which generate and maintain tubular endoplasmic reticulum (ER) membrane organisation. The ER-shaping proteins are essential for the health and survival of long motor neurons, however the mechanisms by which mutations in these genes cause the axonopathy observed in HSP have not been elucidated. To further develop our understanding of the ER-shaping proteins, this study outlines the generation of novel in vivo and in vitro models, using CRISPR/Cas9-mediated gene editing to knockout the ER-shaping protein ADP-ribosylation factor-like 6 interacting protein 1 (ARL6IP1), mutations in which give rise to the HSP subtype SPG61. Loss of Arl6IP1 in Drosophila results in progressive locomotor deficits, emulating a key aspect of HSP in patients. ARL6IP1 interacts with ER-shaping proteins and is required for regulating the organisation of ER tubules, particularly within long motor neuron axons. Unexpectedly, we identified physical and functional interactions between ARL6IP1 and the phospholipid transporter oxysterol-binding protein-related protein 8 in both human and Drosophila model systems, pointing to a conserved role for ARL6IP1 in lipid homeostasis. Furthermore, loss of Arl6IP1 from Drosophila neurons results in a cell non-autonomous accumulation of lipid droplets in axonal glia. Importantly, treatment with lipid regulating liver X receptor-agonists blocked lipid droplet accumulation, restored axonal ER organisation, and improved locomotor function in Arl6IP1 knockout Drosophila. Our findings indicate that disrupted lipid homeostasis contributes to neurodegeneration in HSP, identifying a potential novel therapeutic avenue for the treatment of this disorder.

Ceramide from sphingomyelin hydrolysis induces neuronal differentiation, whereas de novo ceramide synthesis and sphingomyelin hydrolysis initiate apoptosis after NGF withdrawal in PC12 Cells

BackgroundCeramide, important for both neuronal differentiation and dedifferentiation, resides in several membranes, is synthesized in the endoplasmic reticulum, mitochondrial, and nuclear membranes, and can be further processed into glycosphingolipids or sphingomyelin. Ceramide may also be generated by hydrolysis of sphingomyelin by neutral or acidic sphingomyelinases in lysosomes and other membranes. Here we asked whether the differing functions of ceramide derived from different origins.MethodsWe added NGF to PC12 cells and to TrkA cells. These latter overexpress NGF receptors and are partially activated to differentiate, whereas NGF is required for PC12 cells to differentiate. We differentiated synthesis from hydrolysis by the use of appropriate inhibitors. Ceramide and sphingomyelin were measured by radiolabeling.ResultsWhen NGF is added, the kinetics and amounts of ceramide and sphingomyelin indicate that the ceramide comes primarily from hydrolysis but, when hydrolysis is inhibited, can also come from neosynthesis. When NGF is removed, the ceramide comes from both neosynthesis and hydrolysis.ConclusionWe conclude that the function of ceramide depends heavily on its intracellular location, and that further understanding of its function will depend on resolving its location during changes of cell status.Graphical 9jaz4dWkruTZSXMGGTBHtmVideo

High internal phase emulsions stabilized solely by carboxymethyl chitosan

The ability of carboxymethyl chitosan (CMCS) to stabilize high internal phase emulsions (HIPEs) was investigated under various conditions. Emulsions with optimal properties (ultra-high stability, good gelation, and high plasticity) were obtained using single-step shear dispersion at a CMCS content of 0.3 wt% and near-neutral pH. CMCS functioned as a “structuring agent” to form three layers of jammed networks that immobilized the oil droplets enmeshed therein, through molecular rearrangement and characteristics such as flexible molecular entanglement chains and film-forming properties. Specifically, the hierarchical structures from the innermost layer (closest to the droplets) to the outermost layer were interfacial film, branching linear/membrane structures, and outer non-interfacial membranes, respectively. The outer layers of reticular membranes (without adhering onto the droplets) improved the emulsion's bulk viscosity and steric hindrance and further pushed the first layer closer to the dispersed droplets to create more compact adsorption, stabilize a large interfacial area, and improve the protective coating. The branched structure of CMCS also enabled bridging and, hence, the tight interconnection of oil droplets. Overall, the synergistic effects of various factors allowed CMCS to effectively stabilize HIPEs. The adopted strategy is expected to facilitate the development of other emulsifiers for HIPEs.

Novel Benzo[A]Phenoxazinium Chlorides Functionalized with Sulfonamide Groups as Nir Fluorescent Probes for Vacuole, Endoplasmic Reticulum and Plasma Membrane Staining

Novel Benzo[A]Phenoxazinium Chlorides Functionalized with Sulfonamide Groups as Nir Fluorescent Probes for Vacuole, Endoplasmic Reticulum and Plasma Membrane Staining

Rice Carbohydrate-Binding Malectin-Like Protein, OsCBM1, Contributes to Drought-Stress Tolerance by Participating in NADPH Oxidase-Mediated ROS Production

Carbohydrate-binding malectin/malectin-like domain-containing proteins (CBMs) are a recently identified protein subfamily of lectins that participates various functional bioprocesses in the animal, bacterial, and plant kingdoms. However, little is known the roles of CBMs in rice development and stress response. In this study, OsCBM1, which encodes a protein containing only one malectin-like domain, was cloned and characterized. OsCBM1 is localized in both the endoplasmic reticulum and plasma membrane. Its transcripts are dominantly expressed in leaves and could be significantly stimulated by a number of phytohormone applications and abiotic stress treatments. Overexpression of OsCBM1 increased drought tolerance and reactive oxygen species production in rice, whereas the knockdown of the gene decreased them. OsCBM1 physically interacts with OsRbohA, a NADPH oxidase, and the expression of OsCBM1 in osrbohA, an OsRbohA-knockout mutant, is significantly downregulated under both normal growth and drought stress conditions. Meanwhile, OsCBM1 can also physically interacts with OsRacGEF1, a specific guanine nucleotide exchange factor for the Rop/Rac GTPase OsRac1, and transient coexpression of OsCBM1 with OaRacGEF1 significantly enhanced ROS production. Further transcriptome analysis showed that multiple signaling regulatory mechanisms are involved in the OsCBM1-mediated processes. All these results suggest that OsCBM1 participates in NADPH oxidase-mediated ROS production by interacting with OsRbohA and OsRacGEF1, contributing to drought stress tolerance of rice. Multiple signaling pathways are likely involved in the OsCBM1-mediated stress tolerance in rice.

BVES is a novel interactor of ANO5 and regulates myoblast differentiation

BackgroundAnoctamin 5 (ANO5) is a membrane protein belonging to the TMEM16/Anoctamin family and its deficiency leads to the development of limb girdle muscular dystrophy R12 (LGMDR12). However, little has been known about the interactome of ANO5 and its cellular functions.ResultsIn this study, we exploited a proximal labeling approach to identify the interacting proteins of ANO5 in C2C12 myoblasts stably expressing ANO5 tagged with BioID2. Mass spectrometry identified 41 unique proteins including BVES and POPDC3 specifically from ANO5-BioID2 samples, but not from BioID2 fused with ANO6 or MG53. The interaction between ANO5 and BVES was further confirmed by co-immunoprecipitation (Co-IP), and the N-terminus of ANO5 mediated the interaction with the C-terminus of BVES. ANO5 and BVES were co-localized in muscle cells and enriched at the endoplasmic reticulum (ER) membrane. Genome editing-mediated ANO5 or BVES disruption significantly suppressed C2C12 myoblast differentiation with little impact on proliferation.ConclusionsTaken together, these data suggest that BVES is a novel interacting protein of ANO5, involved in regulation of muscle differentiation.

ATPase Activity of the Subcellular Fractions of Colorectal Cancer Samples under the Action of Nicotinic Acid Adenine Dinucleotide Phosphate.

In tumor cells with defects in apoptosis, autophagy allows prolonged survival. Autophagy leads to an accumulation of damaged mitochondria by autophagosomes. An acidic environment is maintained in compartments of cells, such as autophagosomes, late endosomes, and lysosomes; these organelles belong to the “acid store” of the cells. Nicotinic acid adenine dinucleotide phosphate (NAADP) may affect the release of Ca2+ from these organelles and affect the activity of Ca2+ ATPases and other ion transport proteins. Recently, a growing amount of evidence has shown that the variations in the expression of calcium channels or pumps are associated with the occurrence, disease-presentation, and the prognosis of colorectal cancer. We hypothesized that activity of ATPases in cancer tissue is higher because of intensive energy metabolism of tumor cells. The aim of our study was to ascertain the effect of NAADP on ATPase activity on tissue samples of colorectal cancer patients’ and healthy individuals. We tested the effect of NAADP on the activity of Na+/K+ ATPase; Ca2+ ATPase of endoplasmic reticulum (EPR) and plasma membrane (PM) and basal ATPase activity. Patients’ colon mucus cancer samples were obtained during endoscopy from cancer and healthy areas (control) of colorectal mucosa of the same patients. Results. The mean activity of Na+/K+ pump in samples of colorectal cancer patients (n = 5) was 4.66 ± 1.20 μmol Pi/mg of protein per hour, while in control samples from healthy tissues of the same patient (n = 5) this value was 3.88 ± 2.03 μmol Pi/mg of protein per hour. The activity of Ca2+ ATPase PM in control samples was 6.42 ± 0.63 μmol Pi/mg of protein per hour and in cancer −8.50 ± 1.40 μmol Pi/mg of protein per hour (n = 5 pts). The mean activity of Ca2+ ATPase of EPR in control samples was 7.59 ± 1.21 μmol Pi/mg versus 7.76 ± 0.24 μmol Pi/mg in cancer (n = 5 pts). Basal ATPase activity was 3.19 ± 0.87 in control samples versus 4.79 ± 1.86 μmol Pi/mg in cancer (n = 5 pts). In cancer samples, NAADP reduced the activity of Na+/K+ ATPase by 9-times (p < 0.01) and the activity of Ca2+ ATPase EPR about 2-times (p < 0.05). NAADP caused a tendency to decrease the activity of Ca2+ ATPase of PM, but increased basal ATPase activity by 2-fold vs. the mean of this index in cancer samples without the addition of NAADP. In control samples NAADP caused only a tendency to decrease the activities of Na+/K+ ATPase and Ca2+ ATPase EPR, but statistically decreased the activity of Ca2+ ATPase of PM (p < 0.05). In addition, NAADP caused a strong increase in basal ATPase activity in control samples (p < 0.01). Conclusions: We found that the activity of Na+/K+ pump, Ca2+ ATPase of PM and basal ATPase activity in cancer tissues had a strong tendency to be higher than in the controls. NAADP caused a decrease in the activities of Na+/K+ ATPase and Ca2+ ATPase EPR in cancer samples and increased basal ATPase activity. In control samples, NAADP decreased Ca2+ ATPase of PM and increased basal ATPase activity. These data confirmed different roles of NAADP-sensitive “acidic store” (autophagosomes, late endosomes, and lysosomes) in control and cancer tissue, which hypothetically may be connected with autophagy role in cancer development. The effect of NAADP on decreasing the activity of Na+/K+ pump in cancer samples was the most pronounced, both numerically and statistically. Our data shows promising possibilities for the modulation of ion-transport through the membrane of cancer cells by influence on the “acidic store” (autophagosomes, late endosomes and lysosomes) as a new approach to the treatment of colorectal cancer.

Abstract 10488: Overexpression of MFN2 Alleviates Sorafenib-Induced Cardiomyocyte Necroptosis via the MAM-CaMKIIδ Pathway in vitro and in vivo

Background: The continued success of oncological therapeutics is dependent on the mitigation of treatment-related adverse events, particularly cardiovascular toxicities. As such, there is an important need to understand the basic mechanisms of drug toxicities and to explore the differences in the fundamental signaling pathways between cancer cells and non-cancer cells for the maintenance of function and survival of these cells. Our aim in this study was to elucidate the underlying mechanisms of sorafenib (Sor)-induced cardiomyocyte damage. Methods: Primary mouse cardiomyocytes were prepared and treated with Sor and various other treatments. Cardiomyocyte necroptosis was detected by flow cytometry, western blotting, and CCK8 assays. Mitochondrial Ca 2+ uptake was detected by Rhod-2 probes using confocal imaging. Morphological changes in mitochondria and mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) were imaged using transmission electron microscopy (TEM) and confocal microscopy. Results: We reported that RIP3/MLKL cascade activation, mitochondrial Ca 2+ overload, and the subsequent increase in calmodulin-dependent protein kinase II delta (CaMKIIδ) contributed to Sor-induced cardiac necroptosis. Excess MAM formation and close ER-mitochondria contact were key in the pathogenesis of Sor-induced Ca 2+ overload. Furthermore, we found that reduced mitofusin-2 (MFN2) levels corresponded to repressed MAM biogenesis and increased mitochondria-MAM tethering in cardiomyocytes. Sor-induced Mammalian Target of Rapamycin (mTOR) inactivation, followed by the activation and nuclear translocation of Transcription Factor EB (TFEB), contributed to mitophagy and MFN2 degradation. In an in vivo model, we reported that an increase in MFN2 expression specifically improved Sor-induced cardiomyocyte necroptosis by repressing the MAM-CaMKIIδ-MCU pathway. Conclusions: Sorafenib mediated cardiomyocyte necroptosis through the mTOR-MFN2-MAM-Ca 2+ -RIP3-CaMKIIδ pathway in vitro and in vivo . The overexpression of MFN2 could rescue Sor-induced cardiomyocyte necroptosis without disturbing the anti-tumor effects.