The transport of carboxylic acids and important role of the Jen1p transporter during the development of yeast colonies

2013 ◽  
Vol 454 (3) ◽  
pp. 551-558 ◽  
Author(s):  
Sandra Paiva ◽  
Dita Strachotová ◽  
Helena Kučerová ◽  
Otakar Hlaváček ◽  
Sandra Mota ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lactic Acid ◽  
Cell Metabolism ◽  
Colony Development ◽  
Acid Uptake ◽  
Uptake Capacity ◽  
Solid Substrates ◽  
Lactate Uptake ◽  
Pass Through ◽  
Developmental Phases

On solid substrates, yeast colonies pass through distinct developmental phases characterized by the changes in pH of their surroundings from acidic to nearly alkaline and vice versa. At the beginning of the alkali phase colonies start to produce ammonia, which functions as a quorum-sensing molecule inducing the reprogramming of cell metabolism. Such reprogramming includes, among others, the activation of several plasma membrane transporters and is connected with colony differentiation. In the present study, we show that colony cells can use two transport mechanisms to import lactic acid: a ‘saturable’ component of the transport, which requires the presence of a functional Jen1p transporter, and a ‘non-saturable’ component (diffusion) that is independent of Jen1p. During colony development, the efficiency of both transport components changes similarly in central and outer colonial cells. Although the lactate uptake capacity of central cells gradually decreases during colony development, the lactate uptake capacity of outer cells peaks during the alkali phase and is also kept relatively high in the second acidic phase. This lactate uptake profile correlates with the localization of the Jen1p transporter to the plasma membrane of colony cells. Both lactic acid uptake mechanisms are diminished in sok2 colonies where JEN1 expression is decreased. The Sok2p transcription factor may therefore be involved in the regulation of non-saturable lactic acid uptake in yeast colonies.

scholarly journals Rapidly Developing Yeast Microcolonies Differentiate in a Similar Way to Aging Giant Colonies

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Libuše Váchová ◽  
Ladislava Hatáková ◽  
Michal Čáp ◽  
Michaela Pokorná ◽  
Zdena Palková
Keyword(s):  
Cell Metabolism ◽  
Surrounding Medium ◽  
Solid Substrates ◽  
Cell Layers ◽  
Per Se ◽  
Development And Aging ◽  
Cell Subpopulations ◽  
Pass Through ◽  
Developmental Phases ◽  
Quorum Sensing Molecule

During their development and aging on solid substrates, yeast giant colonies produce ammonia, which acts as a quorum sensing molecule. Ammonia production is connected with alkalization of the surrounding medium and with extensive reprogramming of cell metabolism. In addition, ammonia signaling is important for both horizontal (colony centre versus colony margin) and vertical (upper versus lower cell layers) colony differentiations. The centre of an aging differentiated giant colony is thus composed of two major cell subpopulations, the subpopulation of long-living, metabolically active and stress-resistant cells that form the upper layers of the colony and the subpopulation of stress-sensitive starving cells in the colony interior. Here, we show that microcolonies originating from one cell pass through similar developmental phases as giant colonies. Microcolony differentiation is linked to ammonia signaling, and cells similar to the upper and lower cells of aged giant colonies are formed even in relatively young microcolonies. A comparison of the properties of these cells revealed a number of features that are similar in microcolonies and giant colonies as well as a few that are only typical of chronologically aged giant colonies. These findings show that colony ageper seis not crucial for colony differentiation.

scholarly journals 669 Lactate uptake through MCT11, a novel monocarboxylate transporter, enforces dysfunction in terminally exhausted T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A697-A697
Author(s):  
Ronal Peralta ◽  
Greg Delgoffe
Keyword(s):  
T Cells ◽  
Lactic Acid ◽  
Tumor Burden ◽  
B16 Melanoma ◽  
T Cell Subsets ◽  
Monocarboxylate Transporter ◽  
Acid Uptake ◽  
Ribonucleoprotein Complexes ◽  
Lactate Uptake ◽  
Marker Expression

BackgroundUpon infiltration into tumors, T cells experiencing persistent antigen stimulation progressively differentiate into a state of dysfunction, known as exhaustion. Exhausted T cells are characterized by the sustained upregulation of co-inhibitory molecules and reduced effector cytokine production. Additionally, exhausted T cells exist in a state of metabolic dysfunction in the tumor microenvironment (TME), due to disrupted mitochondrial biogenesis, hypoxia and lack of metabolites. Highly glycolytic tumor and stromal cells outcompete T cells for glucose, and secrete lactate into the TME, acidifying the extracellular space. Recent studies have shown lactate can be metabolized by tumor infiltrating Tregs and macrophages. We hypothesized that CD8+ tumor-infiltrating lymphocytes (TIL) may also take up lactate as an alternative carbon source to meet their metabolic demands.MethodsFor lactate uptake experiments, B16 melanoma single cell suspensions from B6 mice were loaded with the pH sensitive dye pHrodo, then pulsed with 5µM lactic acid. MCT11 KO OT-I T cells were generated via transfection of Slc16a11 sgRNA-Cas9 ribonucleoprotein complexes, and adoptively transferred into B16-OVA bearing mice.ResultsRNA sequencing and flow cytometry data from CD8+ T cell subsets in the TME revealed MCT11 (encoded by Slc16a11), a monocarboxylate transporter (MCT) only recently discovered, to be highly and uniquely expressed in terminally exhausted T cells (Tex). As lactate is an abundant monocarboxylate in tumors, we asked whether MCT11 supports lactate uptake into Tex cells. Antibody blockade of MCT11 resulted in reduced lactic acid uptake, but whether lactic acid promoted or inhibited effector function. Intriguingly, overexpression of MCT11 in OT-I T cells adoptively transferred into B16-OVA bearing mice resulted in accelerated exhaustion: increased co-inhibitory marker expression and decreased TNFa and IFN production. Conversely, knockdown of MCT11 in the same model resulted in decreased co-inhibitory marker expression and increased TNFa and IFN production. Further, MCT11 KO OT-I T cells used therapeutically had decreased tumor burden over mice treated with control OT-I T cells. As MCT11's uptake function was blocked with an antibody, we also used the antibody therapeutically, revealing that single-agent MCT11 antibody treatment reduced tumor burden and increased survival in B16 melanoma bearing mice.ConclusionsOur data support a model where exhausted CD8+ T cells upregulate MCT11, which renders them sensitive to toxic lactic acid in the TME. Our data suggest MCT11 could be deleted on therapeutic T cells or blocked using an antibody on endogenous T cells to render exhausted T cells impervious to lactic acid such and promote tumor eradication.

Immunocytochemical localization of p65 with one-nanometer gold particles following silver development

1989 ◽  
Vol 47 ◽  
pp. 1042-1043
Author(s):  
Richard W. Burry ◽  
Diane M. Hayes
Keyword(s):  
Plasma Membrane ◽  
Bovine Serum Albumin ◽  
Calf Serum ◽  
Specific Protein ◽  
Immunocytochemical Localization ◽  
Electron Microscopic ◽  
Gold Particles ◽  
Pass Through ◽  
Label Distribution ◽  
Phosphate Buffered Saline

Electron microscopic (EM) immunocytochemistry localization of the neuron specific protein p65 could show which organelles contain this antigen. Antibodies (Ab) labeled with horseradish peroxidase (HRP) followed by chromogen development show a broad diffuse label distribution within cells and restricting identification of organelles. Particulate label (e.g. 10 nm colloidal gold) is highly desirable but not practical because penetration into cells requires destroying the plasma membrane. We report pre-embedding immunocytochemistry with a particulate marker, 1 nm gold, that will pass through membranes treated with saponin, a mild detergent.Cell cultures of the rat cerebellum were fixed in buffered 4% paraformaldehyde and 0.1% glutaraldehyde (Glut.). The buffer for all incubations and rinses was phosphate buffered saline with: 1% calf serum, 0.2% saponin, 0.1% gelatin, 50 mM glycine 1 mg/ml bovine serum albumin, and (not in the HRP labeled cultures) 0.02% sodium azide. The monoclonal #48 to p65 was used with three label systems: HRP, 1 nm avidin gold with IntenSE M development, and 1 nm avidin gold with Danscher development.

Effect of surface and intracellular pH on hepatocellular fatty acid uptake

1996 ◽  
Vol 271 (6) ◽  
pp. G1067-G1073
Author(s):  
C. Elsing ◽  
A. Kassner ◽  
W. Stremmel
Keyword(s):  
Fatty Acids ◽  
Plasma Membrane ◽  
Fatty Acid ◽  
Intracellular Ph ◽  
Outer Surface ◽  
Fatty Acid Uptake ◽  
Proton Gradient ◽  
Fatty Acid Transport ◽  
Acid Uptake ◽  
Acid Transport

Fatty acids enter hepatocytes, at least in part, by a carrier-mediated uptake mechanism. The importance of driving forces for fatty acid uptake is still controversial. To evaluate possible driving mechanisms for fatty acid transport across plasma membranes, we examined the role of transmembrane proton gradients on fatty acid influx in primary cultured rat hepatocytes. After hepatocytes were loaded with SNARF-1 acetoxymethyl ester, changes in intracellular pH (pHi) under different experimental conditions were measured and recorded by confocal laser scanning microscopy. Fatty acid transport was increased by 45% during cellular alkalosis, achieved by adding 20 mM NH4Cl to the medium, and a concomitant paracellular acidification was observed. Fatty acid uptake was decreased by 30% during cellular acidosis after withdrawal of NH4Cl from the medium. Cellular acidosis activates the Na+/H+ antiporter to export excessive protons to the outer cell surface. Inhibition of Na+/H+ antiporter activity by amiloride diminishes pHi recovery and thereby accumulation of protons at the outer surface of the plasma membrane. Under these conditions, fatty acid uptake was further inhibited by 57% of control conditions. This suggests stimulation of fatty acid influx by an inwardly directed proton gradient. The accelerating effect of protons at the outer surface of the plasma membrane was confirmed by studies in which pH of the medium was varied at constant pHi. Significantly higher fatty acid influx rates were observed at low buffer pH. Recorded differences in fatty acid uptake appeared to be independent of changes in membrane potential, because BaCl2 did not influence initial uptake velocity during cellular alkalosis and paracellular acidosis. Moreover, addition of oleate-albumin mixtures to the NH4Cl incubation buffer did not change the observed intracellular alkalinization. In contrast, after cells were acid loaded, addition of oleate-albumin solutions to the recovery buffer increased pHi recovery rates from 0.21 +/- 0.02 to 0.36 +/- 0.05 pH units/min (P < 0.05), indicating that fatty acids further stimulate Na+/H+ antiporter activity during pHi recovery from an acid load. It is concluded that carrier-mediated uptake of fatty acids in hepatocytes follows an inwardly directed transmembrane proton gradient and is stimulated by the presence of H+ at the outer surface of the plasma membrane.

scholarly journals Brucella abortusinduces a Warburg shift in host metabolism that is linked to enhanced intracellular survival of the pathogen

2017 ◽  
Author(s):  
Daniel M. Czyż ◽  
Jonathan Willett ◽  
Sean Crosson
Keyword(s):  
Lactic Acid ◽  
Host Cell ◽  
Brucella Abortus ◽  
Cell Metabolism ◽  
Lactate Production ◽  
Human Macrophage ◽  
Metabolic Shift ◽  
Intracellular Infections ◽  
Host Metabolism

ABSTRACTIntracellular bacterial pathogens exploit host cell resources to replicate and survive inside the host. Targeting these host systems is one promising approach to developing novel antimicrobials to treat intracellular infections. We show that human macrophage-like cells infected withBrucella abortusundergo a metabolic shift characterized by attenuated tricarboxylic acid cycle metabolism, reduced amino acid consumption, altered mitochondrial localization, and increased lactate production. This shift to an aerobic glycolytic state resembles the Warburg effect, a change in energy production that is well-described in cancer cells, and also occurs in activated inflammatory cells.B. abortusefficiently uses lactic acid as its sole carbon and energy source and requires the ability to metabolize lactate for normal survival in human macrophage-like cells. We demonstrate that chemical inhibitors of host glycolysis and lactate production do not affectin vitrogrowth ofB. abortusin axenic culture, but decrease its survival in the intracellular niche. Our data support a model in which infection shifts host metabolism to a Warburg-like state, andB. abortususes this change in metabolism to promote intracellular survival. Pharmacological perturbation of these features of host cell metabolism may be a useful strategy to inhibit infection by intracellular pathogens.IMPORTANCEBrucellaspp. are intracellular bacterial pathogens that cause disease in a range of mammals, including livestock. Transmission from livestock to humans is common and can lead to chronic human disease. Human macrophage-like cells infected withBrucella abortusundergo a Warburg-like metabolic shift to an aerobic glycolytic state where the host cells produce lactic acid and have reduced amino acid catabolism. We provide evidence that the pathogen can exploit this change in host metabolism to support growth and survival in the intracellular niche. Drugs that inhibit this shift in host cell metabolism inhibit intracellular replication and decrease the survival ofB. abortusin anin vitroinfection model; these drugs may be broadly useful therapeutics for intracellular infections.

Fatty acid uptake in Escherichia coli: regulation by recruitment of fatty acyl-CoA synthetase to the plasma membrane

1993 ◽  
Vol 71 (1-2) ◽  
pp. 51-56 ◽  
Author(s):  
Dev Mangroo ◽  
Gerhard E. Gerber
Keyword(s):  
Escherichia Coli ◽  
Plasma Membrane ◽  
Fatty Acid ◽  
Fatty Acid Uptake ◽  
Fatty Acyl ◽  
Acid Uptake ◽  
Bacterial Membranes ◽  
Rate Limiting Step ◽  
Triton X ◽  
Rate Limiting

Fatty acid uptake in Escherichia coli has been shown to be inhibited by starvation and to be reversed by a short preincubation of the starved cells with D- or L-lactate, succinate, and acetate; these effects on oleate uptake were due to regulation of the rate-limiting step which involves fatty acyl-CoA synthetase. Investigation into the mechanism of regulation of fatty acyl-CoA synthetase showed that D-lactate did not affect the activity of the enzyme directly. Fatty acyl-CoA synthetase was found to be activated by about 20-fold by Triton X-100 and by another 4-fold by the addition of bacterial membranes. D-Lactate treatment was shown to result in coisolation of fatty acyl-CoA synthetase with the plasma membrane; these results are consistent with the interpretation that recruitment of the enzyme to the plasma membrane by D-lactate results in its activation and consequently in the increased level of fatty acid uptake.Key words: fatty acid, uptake, regulation, recruitment, fatty acyl-CoA synthetase, Escherichia coli, plasma membrane.

scholarly journals DHHC4 and DHHC5 Facilitate Fatty Acid Uptake by Palmitoylating and Targeting CD36 to the Plasma Membrane

Cell Reports ◽  
2019 ◽  
Vol 26 (1) ◽  
pp. 209-221.e5 ◽  
Author(s):  
Juan Wang ◽  
Jian-Wei Hao ◽  
Xu Wang ◽  
Huiling Guo ◽  
Hui-Hui Sun ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fatty Acid ◽  
Fatty Acid Uptake ◽  
Acid Uptake

scholarly journals Microgravity-Induced Cell-to-Cell Junctional Contacts Are Counteracted by Antioxidant Compounds in TCam-2 Seminoma Cells

2020 ◽  
Vol 10 (22) ◽  
pp. 8289
Author(s):  
Angela Catizone ◽  
Caterina Morabito ◽  
Marcella Cammarota ◽  
Chiara Schiraldi ◽  
Katia Corano Scheri ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Line ◽  
Simulated Microgravity ◽  
Cell Metabolism ◽  
Ultrastructural Level ◽  
Antioxidant Compounds ◽  
Signal Molecules ◽  
Beta Catenin ◽  
Autophagy Induction ◽  
A Cell

The direct impact of microgravity exposure on male germ cells, as well as on their malignant counterparts, has not been largely studied. In previous works, we reported our findings on a cell line derived from a human seminoma lesion (TCam-2 cell line) showing that acute exposure to simulated microgravity altered microtubule orientation, induced autophagy, and modified cell metabolism stimulating ROS production. Moreover, we demonstrated that the antioxidant administration prevented both TCam-2 microgravity-induced microtubule disorientation and autophagy induction. Herein, expanding previous investigations, we report that simulated microgravity exposure for 24 h induced the appearance, at an ultrastructural level, of cell-to-cell junctional contacts that were not detectable in cells grown at 1 g. In line with this result, pan-cadherin immunofluorescence analyzed by confocal microscopy, revealed the clustering of this marker at the plasma membrane level on microgravity exposed TCam-2 cells. The upregulation of cadherin was confirmed by Western blot analyses. Furthermore, we demonstrated that the microgravity-induced ROS increase was responsible for the distribution of cadherin nearby the plasma membrane, together with beta-catenin since the administration of antioxidants prevented this microgravity-dependent phenomenon. These results shed new light on the microgravity-induced modifications of the cell adhesive behavior and highlight the role of ROS as microgravity activated signal molecules.

scholarly journals Modified N-linked glycosylation status predicts trafficking defective human Piezo1 channel mutations

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Jinyuan Vero Li ◽  
Chai-Ann Ng ◽  
Delfine Cheng ◽  
Zijing Zhou ◽  
Mingxi Yao ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Integral Membrane Proteins ◽  
Mechanical Stimuli ◽  
Variants Of Unknown Significance ◽  
Unknown Significance ◽  
Lymphatic Dysplasia ◽  
Ion Channel Proteins ◽  
Pass Through

AbstractMechanosensitive channels are integral membrane proteins that sense mechanical stimuli. Like most plasma membrane ion channel proteins they must pass through biosynthetic quality control in the endoplasmic reticulum that results in them reaching their destination at the plasma membrane. Here we show that N-linked glycosylation of two highly conserved asparagine residues in the ‘cap’ region of mechanosensitive Piezo1 channels are necessary for the mature protein to reach the plasma membrane. Both mutation of these asparagines (N2294Q/N2331Q) and treatment with an enzyme that hydrolyses N-linked oligosaccharides (PNGaseF) eliminates the fully glycosylated mature Piezo1 protein. The N-glycans in the cap are a pre-requisite for N-glycosylation in the ‘propeller’ regions, which are present in loops that are essential for mechanotransduction. Importantly, trafficking-defective Piezo1 variants linked to generalized lymphatic dysplasia and bicuspid aortic valve display reduced fully N-glycosylated Piezo1 protein. Thus the N-linked glycosylation status in vitro correlates with efficient membrane trafficking and will aid in determining the functional impact of Piezo1 variants of unknown significance.

scholarly journals LeNRT1.1 Improves Nitrate Uptake in Grafted Tomato Plants under High Nitrogen Demand

2018 ◽  
Vol 19 (12) ◽  
pp. 3921 ◽  
Author(s):  
Francisco Albornoz ◽  
Marlene Gebauer ◽  
Carlos Ponce ◽  
Ricardo Cabeza
Keyword(s):  
Plasma Membrane ◽  
Root Respiration ◽  
Nitrate Uptake ◽  
N Uptake ◽  
Uptake Capacity ◽  
Tomato Plants ◽  
Respiration Rates ◽  
Assimilation Capacity ◽  
N Demand ◽  
Root Plasma Membrane

Grafting has become a common practice among tomato growers to obtain vigorous plants. These plants present a substantial increase in nitrogen (N) uptake from the root zone. However, the mechanisms involved in this higher uptake capacity have not been investigated. To elucidate whether the increase in N uptake in grafted tomato plants under high N demand conditions is related to the functioning of low- (high capacity) or high-affinity (low capacity) root plasma membrane transporters, a series of experiments were conducted. Plants grafted onto a vigorous rootstock, as well as ungrafted and homograft plants, were exposed to two radiation levels (400 and 800 µmol m−2 s−1). We assessed root plasma membrane nitrate transporters (LeNRT1.1, LeNRT1.2, LeNRT2.1, LeNRT2.2 and LeNRT2.3) expression, Michaelis‒Menten kinetics parameters (Vmax and Km), root and leaf nitrate reductase activity, and root respiration rates. The majority of nitrate uptake is mediated by LeNRT1.1 and LeNRT1.2 in grafted and ungrafted plants. Under high N demand conditions, vigorous rootstocks show similar levels of expression for LeNRT1.1 and LeNRT1.2, whereas ungrafted plants present a higher expression of LeNRT1.2. No differences in the uptake capacity (evaluated as Vmax), root respiration rates, or root nitrate assimilation capacity were found among treatments.

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