scholarly journals Aster Proteins Regulate the Accessible Cholesterol Pool in the Plasma Membrane

2020 ◽  
Vol 40 (19) ◽  
Author(s):  
Alessandra Ferrari ◽  
Cuiwen He ◽  
John Paul Kennelly ◽  
Jaspreet Sandhu ◽  
Xu Xiao ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Plasma Membranes ◽  
Secondary Ion Mass Spectrometry ◽  
Threshold Level ◽  
Cell Types ◽  
Cholesterol Concentration ◽  
Dependent Manner ◽  
Transport Pathway ◽  
Concentration Dependent Manner ◽  
Mouse Macrophages

ABSTRACT Recent studies have demonstrated the existence of a discrete pool of cholesterol in the plasma membranes (PM) of mammalian cells—referred to as the accessible cholesterol pool—that can be detected by the binding of modified versions of bacterial cytolysins (e.g., anthrolysin O). When the amount of accessible cholesterol in the PM exceeds a threshold level, the excess cholesterol moves to the endoplasmic reticulum (ER), where it regulates the SREBP2 pathway and undergoes esterification. We reported previously that the Aster/Gramd1 family of sterol transporters mediates nonvesicular movement of cholesterol from the PM to the ER in multiple mammalian cell types. Here, we investigated the PM pool of accessible cholesterol in cholesterol-loaded fibroblasts with a knockdown of Aster-A and in mouse macrophages from Aster-B and Aster-A/B-deficient mice. Nanoscale secondary ion mass spectrometry (NanoSIMS) analyses revealed expansion of the accessible cholesterol pool in cells lacking Aster expression. The increased accessible cholesterol pool in the PM was accompanied by reduced cholesterol movement to the ER, evidenced by increased expression of SREBP2-regulated genes. Cosedimentation experiments with liposomes revealed that the Aster-B GRAM domain binds to membranes in a cholesterol concentration-dependent manner and that the binding is facilitated by the presence of phosphatidylserine. These studies revealed that the Aster-mediated nonvesicular cholesterol transport pathway controls levels of accessible cholesterol in the PM, as well as the activity of the SREBP pathway.

Cell surface interactions in conjugation: Tetrahymena ciliary membrane vesicles

1984 ◽  
Vol 4 (4) ◽  
pp. 681-687
Author(s):  
B Love ◽  
M B Rotheim
Keyword(s):  
Cell Surface ◽  
Mating Type ◽  
Mammalian Cells ◽  
Membrane Vesicles ◽  
Surface Interactions ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ciliary Membrane ◽  
Adhesion Sites ◽  
Cell Surface Interactions

Tetrahymena ciliary membrane vesicles are shown to interact with preconjugant cells in a mating type-specific way. When cells are treated with vesicles of a different mating type before mixing for conjugation, cell pairing is enhanced, and the normal prepairing period is partially eliminated. This enhancement is mating type specific since it is not observed after pretreatment of cells with vesicles of their own mating type. In contrast, when vesicles are added at the time of mixing of two starved cultures, cell pairing is delayed in a concentration-dependent manner. By varying the conditions, we demonstrated enhancement or inhibition, or both. These results are interpreted in terms of two independent interactions of cells with vesicles. We suggest that first, vesicles substitute for another cell in cell-cell prepairing interaction and second, vesicles compete for adhesion sites produced during the prepairing period. Finally, the data presented are summarized within a speculative framework that calls attention to potential analogies with hormone-receptor signaling in mammalian cells.

scholarly journals Tracking Lysosome Migration within Chinese Hamster Ovary (CHO) Cells Following Exposure to Nanosecond Pulsed Electric Fields

2018 ◽  
Vol 5 (4) ◽  
pp. 103
Author(s):  
Gary Thompson ◽  
Hope Beier ◽  
Bennett Ibey
Keyword(s):  
Electric Field ◽  
Electric Fields ◽  
Mammalian Cells ◽  
Plasma Membranes ◽  
Chinese Hamster ◽  
Lateral Diffusion ◽  
Cell Swelling ◽  
Dependent Manner ◽  
Membrane Repair ◽  
Osmotic Swelling

Above a threshold electric field strength, 600 ns-duration pulsed electric field (nsPEF) exposure substantially porates and permeabilizes cellular plasma membranes in aqueous solution to many small ions. Repetitive exposures increase permeabilization to calcium ions (Ca2+) in a dosage-dependent manner. Such exposure conditions can create relatively long-lived pores that reseal after passive lateral diffusion of lipids should have closed the pores. One explanation for eventual pore resealing is active membrane repair, and an ubiquitous repair mechanism in mammalian cells is lysosome exocytosis. A previous study shows that intracellular lysosome movement halts upon a 16.2 kV/cm, 600-ns PEF exposure of a single train of 20 pulses at 5 Hz. In that study, lysosome stagnation qualitatively correlates with the presence of Ca2+ in the extracellular solution and with microtubule collapse. The present study tests the hypothesis that limitation of nsPEF-induced Ca2+ influx and colloid osmotic cell swelling permits unabated lysosome translocation in exposed cells. The results indicate that the efforts used herein to preclude Ca2+ influx and colloid osmotic swelling following nsPEF exposure did not prevent attenuation of lysosome translocation. Intracellular lysosome movement is inhibited by nsPEF exposure(s) in the presence of PEG 300-containing solution or by 20 pulses of nsPEF in the presence of extracellular calcium. The only cases with no significant decreases in lysosome movement are the sham and exposure to a single nsPEF in Ca2+-free solution.

Specific coupling of a cation-sensing receptor to G protein alpha-subunits in MDCK cells

1997 ◽  
Vol 273 (1) ◽  
pp. F129-F135 ◽  
Author(s):  
J. M. Arthur ◽  
G. P. Collinsworth ◽  
T. W. Gettys ◽  
L. D. Quarles ◽  
J. R. Raymond
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Plasma Membranes ◽  
Mdck Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cellular Activation ◽  
Protein Activation ◽  
A Cell ◽  
Cation Sensing

Extracellular cations such as Ca2+ stimulate a G protein-coupled, cation-sensing receptor (CaR). We used microphysiometry to determine whether an extracellular cation-sensing mechanism exists in Madin-Darby canine kidney (MDCK) cells. The CaR agonists Ca2+ and Gd3+ caused cellular activation in a concentration-dependent manner. mRNA for the CaR was identified by reverse transcription and polymerase chain reaction (PCR) using nested CaR-specific primers, identification of an appropriately located restriction site, and sequencing of the subcloned fragment obtained by PCR. G protein activation was evaluated using the GTP photoaffinity label [alpha-32P]GTP azidoanalide (AA-GTP). After stimulation with Gd3+ and cross-linking, plasma membranes were solubilized and immunoprecipitated with antisera specific for Gq/11 alpha and Gi alpha family members. Gd3+ increased incorporation of AA-GTP into Gq/11 alpha precipitates by 146 +/- 48% and into G alpha i-2 and G alpha i-3 to a lesser extent but not into G alpha i-1. Direct effects of Gd3+ on the G proteins were ruled out using partially purified mammalian G proteins expressed in Escherichia coli or Sf9 cells. We conclude that MDCK cells possess a cell-surface CaR that activates Gq/11 alpha, G alpha i-2, and G alpha i-3 but not G alpha i-1.

scholarly journals Sphingolipid Domains in the Plasma Membranes of Fibroblasts Are Not Enriched with Cholesterol

2013 ◽  
Vol 288 (23) ◽  
pp. 16855-16861 ◽  
Author(s):  
Jessica F. Frisz ◽  
Haley A. Klitzing ◽  
Kaiyan Lou ◽  
Ian D. Hutcheon ◽  
Peter K. Weber ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Plasma Membrane ◽  
High Resolution ◽  
Mammalian Cells ◽  
Plasma Membranes ◽  
Secondary Ion Mass Spectrometry ◽  
Cholesterol Depletion ◽  
Fibroblast Cells ◽  
Secondary Ion ◽  
Rule Out

The plasma membranes of mammalian cells are widely expected to contain domains that are enriched with cholesterol and sphingolipids. In this work, we have used high-resolution secondary ion mass spectrometry to directly map the distributions of isotope-labeled cholesterol and sphingolipids in the plasma membranes of intact fibroblast cells. Although acute cholesterol depletion reduced sphingolipid domain abundance, cholesterol was evenly distributed throughout the plasma membrane and was not enriched within the sphingolipid domains. Thus, we rule out favorable cholesterol-sphingolipid interactions as dictating plasma membrane organization in fibroblast cells. Because the sphingolipid domains are disrupted by drugs that depolymerize the cells actin cytoskeleton, cholesterol must instead affect the sphingolipid organization via an indirect mechanism that involves the cytoskeleton.

scholarly journals Inhibition of Orbivirus Replication by Aurintricarboxylic Acid

2020 ◽  
Vol 21 (19) ◽  
pp. 7294
Author(s):  
Celia Alonso ◽  
Sergio Utrilla-Trigo ◽  
Eva Calvo-Pinilla ◽  
Luis Jiménez-Cabello ◽  
Javier Ortego ◽  
...  
Keyword(s):  
Cell Types ◽  
Dependent Manner ◽  
Biting Midges ◽  
Lethal Challenge ◽  
Concentration Dependent Manner ◽  
Antiviral Compound ◽  
Aurintricarboxylic Acid ◽  
And Control

Bluetongue virus (BTV) and African horse sickness virus (AHSV) are vector-borne viruses belonging to the Orbivirus genus, which are transmitted between hosts primarily by biting midges of the genus Culicoides. With recent BTV and AHSV outbreaks causing epidemics and important economy losses, there is a pressing need for efficacious drugs to treat and control the spread of these infections. The polyanionic aromatic compound aurintricarboxylic acid (ATA) has been shown to have a broad-spectrum antiviral activity. Here, we evaluated ATA as a potential antiviral compound against Orbivirus infections in both mammalian and insect cells. Notably, ATA was able to prevent the replication of BTV and AHSV in both cell types in a time- and concentration-dependent manner. In addition, we evaluated the effect of ATA in vivo using a mouse model of infection. ATA did not protect mice against a lethal challenge with BTV or AHSV, most probably due to the in vivo effect of ATA on immune system regulation. Overall, these results demonstrate that ATA has inhibitory activity against Orbivirus replication in vitro, but further in vivo analysis will be required before considering it as a potential therapy for future clinical evaluation.

scholarly journals Cell-free Expression of Proton-Coupled Folate Transporter in the Presence of Nanodiscs

2021 ◽  
Author(s):  
Hoa Quynh Do ◽  
Carla M Bassil ◽  
Elizabeth I Andersen ◽  
Michaela Jansen
Keyword(s):  
Tumor Cells ◽  
Plasma Membranes ◽  
In Vitro Translation ◽  
Translation System ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
E Coli ◽  
Membrane Scaffold ◽  
The Impact

The Proton-Coupled Folate Transporter (PCFT) is a transmembrane transport protein that controls the absorption of dietary folates in the small intestine. PCFT also mediates uptake of chemotherapeutically used antifolates into tumor cells. PCFT has been identified within lipid rafts observed in phospholipid bilayers of plasma membranes, a micro environment that is altered in tumor cells. The present study aimed at investigating the impact of different lipids within Lipid-protein nanodiscs (LPNs), discoidal lipid structures stabilized by membrane scaffold proteins, to yield soluble PCFT expression in an E. coli lysate-based cell-free transcription/translation system. In the absence of detergents or lipids, we observed PCFT quantitatively as precipitate in this system. We then explored the ability of LPNs to support solubilized PCFT expression when present during in-vitro translation. LPNs consisted of either dimyristoyl phosphatidylcholine (DMPC), palmitoyl-oleoyl phosphatidylcholine (POPC), or dimyristoyl phosphatidylglycerol (DMPG). While POPC did not lead to soluble PCFT expression, both DMPG and DMPC supported PCFT translation directly into LPNs, the latter in a concentration dependent manner. The results obtained through this study provide insights into the lipid preferences of PCFT. Membrane-embedded or solubilized PCFT will enable further studies with diverse biophysical approaches to enhance the understanding of the structure and molecular mechanism of folate transport through PCFT.

scholarly journals Hydrogen sulfide upregulates acid-sensing ion channels via the MAPK-Erk1/2 signaling pathway

Function ◽  
2021 ◽  
Author(s):  
Zhong Peng ◽  
Stephan Kellenberger
Keyword(s):  
Hydrogen Sulfide ◽  
Ion Channels ◽  
Signaling Pathway ◽  
Cortical Neurons ◽  
Mammalian Cells ◽  
Mapk Signaling ◽  
Dependent Manner ◽  
Membrane Expression ◽  
Concentration Dependent Manner ◽  
Acid Sensing Ion Channels

Abstract Hydrogen sulfide (H2S) emerged recently as a new gasotransmitter and was shown to exert cellular effects by interacting with proteins, among them many ion channels. Acid-sensing ion channels (ASICs) are neuronal voltage-insensitive Na+ channels activated by extracellular protons. ASICs are involved in many physiological and pathological processes, such as fear conditioning, pain sensation and seizures. We characterize here the regulation of ASICs by H2S. In transfected mammalian cells, the H2S donor NaHS increased the acid-induced ASIC1a peak currents in a time- and concentration-dependent manner. Similarly, NaHS potentiated also the acid-induced currents of ASIC1b, ASIC2a and ASIC3. An upregulation induced by the H2S donors NaHS and GYY4137 was also observed with the endogenous ASIC currents of cultured hypothalamus neurons. In parallel with the effect on function, the total and plasma membrane expression of ASIC1a was increased by GYY4137, as determined in cultured cortical neurons. H2S also enhanced the phosphorylation of extracellular signal-regulated kinase, which belongs to the family of mitogen-activated protein kinases (MAPKs). Pharmacological blockade of the MAPK signaling pathway prevented the GYY4137-induced increase of ASIC function and expression, indicating that this pathway is required for ASIC regulation by H2S. Our study demonstrates that H2S regulates ASIC expression and function, and identifies the involved signaling mechanism. Since H2S shares several roles with ASICs, as e.g. facilitation of learning and memory, protection during seizure activity and modulation of nociception, it may be possible that H2S exerts some of these effects via a regulation of ASIC function.

scholarly journals Specific viral RNA drives the SARS CoV-2 nucleocapsid to phase separate

2020 ◽  
Author(s):  
Christiane Iserman ◽  
Christine Roden ◽  
Mark Boerneke ◽  
Rachel Sealfon ◽  
Grace McLaughlin ◽  
...  
Keyword(s):  
Small Molecules ◽  
Mammalian Cells ◽  
Upper Airway ◽  
Dependent Manner ◽  
Rna Sequences ◽  
Concentration Dependent Manner ◽  
Health Crisis ◽  
Viral Packaging ◽  
N Protein ◽  
Human Body Temperature

AbstractA mechanistic understanding of the SARS-CoV-2 viral replication cycle is essential to develop new therapies for the COVID-19 global health crisis. In this study, we show that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with the viral genome, and propose a model of viral packaging through LLPS. N-protein condenses with specific RNA sequences in the first 1000 nts (5’-End) under physiological conditions and is enhanced at human upper airway temperatures. N-protein condensates exclude non-packaged RNA sequences. We comprehensively map sites bound by N-protein in the 5’-End and find preferences for single-stranded RNA flanked by stable structured elements. Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules thus presenting screenable processes for identifying antiviral compounds effective against SARS-CoV-2.

Interaction of human lactoferrin with the rat liver

1985 ◽  
Vol 248 (4) ◽  
pp. G463-G469 ◽  
Author(s):  
M. T. Debanne ◽  
E. Regoeczi ◽  
G. D. Sweeney ◽  
F. Krestynski
Keyword(s):  
Rat Liver ◽  
Plasma Membranes ◽  
Human Lactoferrin ◽  
Dependent Manner ◽  
Ph Optimum ◽  
Concentration Dependent Manner ◽  
Isoelectric Points ◽  
Membrane Bound ◽  
Parenchymal Cells ◽  
Generalized Type

Binding of human lactoferrin (hLf) by purified rat liver plasma membranes was studied to clarify whether the liver possesses specific hLf receptors. The binding was rapid between 4 degrees and 37 degrees C, with a pH optimum close to 5.0. At 22 degrees C and in glycine-NaOH (5 mM, pH 7.4) containing 150 mM NaCl and 0.5% albumin, 1 microgram of membrane bound a maximum of 11.8 ng hLf. The dissociation constant of the interaction was 1.6 X 10(-7) M. Other proteins of high isoelectric points (lactoperoxidase, lysozyme, and particularly salmine sulfate) and a piperazine derivative inhibited hLf binding in a concentration-dependent manner. In contrast, monosaccharides (galactose, N-acetylgalactosamine, mannose, and fucose) were ineffective. By omitting NaCl from the incubation buffer, binding was increased 3.6-fold. Erythrocyte ghosts bound hLf less firmly and alveolar macrophages more firmly than hepatic plasma membranes. Liver cell fractionations performed after the intravenous injection of labeled hLf showed that approximately 88% of the hepatic radioligand was associated with parenchymal cells. When binding was expressed per unit of cell volume, however, more hLf was present in nonparenchymal than in parenchymal cells, implying that the above value was determined by the relative cell masses rather than affinities alone. It is concluded that the binding of hLf by hepatic plasma membranes is electrostatic, i.e., is mediated by the cationic nature of the ligand, and that it is explicable in terms of a "specific nonreceptor interaction" of the generalized type proposed by Cuatrecasas and Hollenberg (Adv. Protein Chem. 30: 251-451, 1976).

Maitotoxin increases inositol phosphates in rat anterior pituitary cells

1991 ◽  
Vol 6 (1) ◽  
pp. 95-99 ◽  
Author(s):  
M. A. Sortino ◽  
T. M. Delahunty ◽  
T. Yasumoto ◽  
M. J. Cronin
Keyword(s):  
Anterior Pituitary ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Cell Types ◽  
Calcium Stores ◽  
Pituitary Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Anterior Pituitary Cells ◽  
Intracellular Ca

ABSTRACT Maitotoxin is a potent marine poison that mobilizes calcium in most vertebrate cell types and accelerates secretion from anterior pituitary cells. It is not known whether voltage-sensitive calcium channels or other mechanisms initiate the effects of maitotoxin on anterior pituitary cells. Changes in intracellular Ca2+ levels may also be achieved by releasing internal calcium stores via inositol trisphosphate (InsP3). Indeed, maitotoxin rapidly increased inositol phosphate accumulation in a concentration-dependent manner. Calcium channel antagonists such as nifedipine and verapamil did not block this response nor did calcium-mobilizing agents (BAYk8644, A23187) mimic this effect. These data suggest that the mechanism by which maitotoxin acts at the pituitary may include the activation of an enzyme that produces the calcium-mobilizing signal InsP3.

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