Extracellular Concentration Gradient Research Articles

Time-Lapse, Live-Cell Imaging of Potassium Efflux in Cell Exposed to Nanosecond Pulsed Electric Fields

Electric-field-induced cell membrane permeabilization (electroporation) is used in a wide range of applications, from cancer therapy to food processing. The mechanisms underlying this phenomenon are, however, poorly understood. Electroporation is generally thought to involve the formation of transient pores, and perhaps other conductive structures, in the cell membrane. This enables the transport of normally impermeant material across the membrane and disrupts the intracellular:extracellular ion concentration gradients (Na+, K+, Cl-) maintained by intact cells.

Multi-Channel System for Simultaneous <italic>In Situ</italic> Monitoring of Ion Flux and Membrane Potential in Plant Electrophysiology

Simultaneous detection of both ion flux and membrane potential in vivo and in situ in plants is a challenging research task. To explore the mechanisms of plant electrical activity, researchers urgently need to understand and determine the types of ions and the ion fluxes that pass in and out of cells during polarization and repolarization, but the required measurements are very difficult to perform. In this paper, we have developed a versatile system that can detect the ionic flux and the membrane potential, in vivo and in situ , simultaneously. The system uses a self-referencing ion-selective glass microelectrode and a membrane potential glass microelectrode as sensors. These sensors are linked through a preamplifier with high input impedance to a specific dynamic measurement system that can amplify small extracellular concentration gradient signals and realize simultaneous measurement of both the ion flux and the membrane potential. In addition, an interpolation fitting algorithm has been proposed to reduce the artifacts that are present in the in situ measurements during plant growth. The hydrogen ion fluxes in wheat roots were measured using the self-referencing ion-selective microelectrodes, and the proposed system was used to measure NaCl stimulation-induced changes in the membrane potentials and hydrogen ion fluxes of wheat root epidermal cells. The results demonstrate that the system can meet the ion flux and membrane potential measurement requirements.

Computational fluid dynamics simulation of an industrial P. chrysogenum fermentation with a coupled 9-pool metabolic model: Towards rational scale-down and design optimization

We assess the effect of substrate heterogeneity on the metabolic response of P. chrysogenum in industrial bioreactors via the coupling of a 9-pool metabolic model with Euler-Lagrange CFD simulations. In this work, we outline how this coupled hydrodynamic-metabolic modeling can be utilized in 5 steps. (1) A model response study with a fixed spatial extra-cellular glucose concentration gradient, which reveals a drop in penicillin production rate qp of 18–50% for the simulated reactor, depending on model setup. (2) CFD-based scale-down design, where we design a 1-vessel scale down simulator based on the organism lifelines. (3) Scale-down verification, numerically comparing the model response in the proposed scale-down simulator with large-scale CFD response. (4) Reactor design optimization, reducing the drop in penicillin production by a change of feed location. (5) Long-term fed-batch simulation, where we verify model predictions against experimental data, and discuss population heterogeneity. Overall, these steps present a coupled hydrodynamic-metabolic approach towards bioreactor evaluation, scale-down and optimization.

Visualization of Clathrin-Mediated Endocytosis During Semaphorin-Guided Axonal Growth.

Semaphorin3A (Sema3A) guides axonal growth during neuronal network development. Accumulating evidence indicates that Sema3A-induced growth cone collapse and repulsion involve endocytic membrane trafficking in the growth cone. It is now possible to visualize endocytic processes in living cells using total internal reflection fluorescence microscopy (TIRFM), a powerful tool for imaging dynamic subcellular events at the plasma membrane. In this chapter, we describe a method for TIRFM observation and analysis of clathrin-mediated endocytosis in growth cones of chicken dorsal root ganglion neurons that receive an extracellular concentration gradient of Sema3A in a culture medium.

Abstract A168: Towards an understanding of the structural basis of CXCR7 ligand binding and signaling

Abstract Chemokines are small signaling proteins that control cell migration through the formation of extracellular concentration gradients. Binding of chemokines to chemokine receptors activates intracellular signaling pathways, leading to chemotaxis of leukocytes during an inflammatory response. Chemokines and their receptors are thereby a crucial part of the immune system and regulate a number of other physiological functions including development and angiogenesis. Most chemokine receptors belong to the large family of G-protein coupled receptors that are ubiquitous in human cells and constitute ~40% of current drug targets. CXCR7 (also known as atypical chemokine receptor 3, ACKR3) is an atypical (not G-protein coupled) chemokine receptor that binds the chemokines SDF-1 (shared with CXCR4) and I-TAC (shared with CXCR3). This binding triggers β-arrestin dependent signaling pathways and regulates signaling of other receptors by controlling the extracellular chemokine concentration. CXCR7 expression is up-regulated in different forms of cancer and inhibiting CXCR7 with siRNA or a small molecule CXCR7 antagonist can reduce tumor size and slow down cancer progression. These findings make CXCR7 a therapeutic target and an antagonist of CXCR7 is currently tested in preclinical trials to treat glioblastoma multiforme. The aim of this study is to understand the mechanism of chemokine binding and signal transduction for CXCR7. This is done by determining a high resolution structure of CXCR7 in complex with SDF-1 and a small molecule antagonist using x-ray crystallography in combination with other complimentary methods. To date, the conditions for production of large amounts of stable, functional CXCR7 protein have been optimized and lipid cubic phase crystals have been obtained of CXCR7 in complex with a high affinity mutant of SDF-1. and key sites of interaction between the receptor and chemokine have been identified. Currently, crystallization conditions are being optimized to produce crystals large enough to enable structure determination. High resolution structures of CXCR7 in complex with SDF-1 and antagonists could facilitate the development of new therapeutics targeting CXCR7 with higher affinity and specificity. A structure of CXCR7 in complex with SDF-1 could also reveal the molecular details of chemokine binding, and the structural differences between the atypical CXCR7 and other chemokine receptors that lead to triggering of different signaling pathways upon chemokine binding. Citation Format: Martin Gustavsson, Yi Zheng, Ling Qin, Lauren G. Holden, Irina Kufareva, Tracy M. Handel. Towards an understanding of the structural basis of CXCR7 ligand binding and signaling. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A168.

A reason for intermittent fasting to suppress the awakening of dormant breast tumors

For their growth, dormant tumors, which lack angiogenesis may critically depend on gradients of nutrients and oxygen from the nearest blood vessel. Because for oxygen depletion the distance from the nearest blood vessel to depletion will generally be shorter than for glucose depletion, such tumors will contain anoxic living tumor cells. These cells are dangerous, because they are capable of inducing angiogenesis, which will “wake up” the tumor. Anoxic cells are dependent on anaerobic glucose breakdown for ATP generation. The local extracellular glucose concentration gradient is determined by the blood glucose concentration and by consumption by cells closer to the nearest blood vessel. The blood glucose concentration can be lowered by 20–40% during fasting. We calculated that glucose supply to the potentially hazardous anoxic cells can thereby be reduced significantly, resulting in cell death specifically of the anoxic tumor cells. We hypothesize that intermittent fasting will help to reduce the incidence of tumor relapse via reducing the number of anoxic tumor cells and tumor awakening.

Involvement of fatty acid amide hydrolase and fatty acid binding protein 5 in the uptake of anandamide by cell lines with different levels of fatty acid amide hydrolase expression: a pharmacological study.

BackgroundThe endocannabinoid ligand anandamide (AEA) is removed from the extracellular space by a process of cellular uptake followed by metabolism. In many cells, such as the RBL-2H3 cell line, inhibition of FAAH activity reduces the observed uptake, indicating that the enzyme regulates uptake by controlling the intra- : extracellular AEA concentration gradient. However, in other FAAH-expressing cells, no such effect is seen. It is not clear, however, whether these differences are methodological in nature or due to properties of the cells themselves. In consequence, we have reinvestigated the role of FAAH in gating the uptake of AEA.Methodology/Principal FindingsThe effects of FAAH inhibition upon AEA uptake were investigated in four cell lines: AT1 rat prostate cancer, RBL-2H3 rat basophilic leukaemia, rat C6 glioma and mouse P19 embryonic carcinoma cells. Semi-quantitative PCR for the cells and for a rat brain lysate confirmed the expression of FAAH. No obvious expression of a transcript with the expected molecular weight of FLAT was seen. FAAH expression differed between cells, but all four could accumulate AEA in a manner inhibitable by the selective FAAH inhibitor URB597. However, there was a difference in the sensitivities seen in the reduction of uptake for a given degree of FAAH inhibition produced by a reversible FAAH inhibitor, with C6 cells being more sensitive than RBL-2H3 cells, despite rather similar expression levels and activities of FAAH. The four cell lines all expressed FABP5, and AEA uptake was reduced in the presence of the FABP5 inhibitor SB-FI-26, suggesting that the different sensitivities to FAAH inhibition for C6 and RBL2H3 cells is not due to differences at the level of FABP-5.Conclusions/SignificanceWhen assayed using the same methodology, different FAAH-expressing cells display different sensitivities of uptake to FAAH inhibition.

Analysis of calcium signals in steering neuronal growth cones in vitro.

Calcium imaging allows us to measure the spatial and temporal changes in intracellular calcium concentration in living cells. Localized calcium elevation often functions as the polarizing signal during guided migration including axon guidance. In this chapter, we describe a protocol to quantitatively monitor the spatiotemporal dynamics of calcium signals in neuronal growth cones in the presence of an extracellular concentration gradient of axon guidance cue.

Neurotrophic factor NT-3 displays a non-canonical cell guidance signaling function for cephalic neural crest cells

Chemotactic cell migration is triggered by extracellular concentration gradients of molecules segregated by target fields. Neural crest cells (NCCs), paradigmatic as an accurately moving cell population, undergo wide dispersion along multiple pathways, invading with precision defined sites of the embryo to differentiate into many derivatives. This report addresses the involvement of NT-3 in early colonization by cephalic NCCs invading the optic vesicle region. The results of in vitro and in vivo approaches showed that NCCs migrate directionally up an NT-3 concentration gradient. We also demonstrated the expression of NT-3 in the ocular region as well as their functional TrkB, TrkC and p75 receptors on cephalic NCCs. On whole-mount embryo, a perturbed distribution of NCCs colonizing the optic vesicle target field was shown after morpholino cancelation of cephalic NT-3 or TrkC receptor on NCCs, as well as in situ blocking of TrkC receptor of mesencephalic NCCs by specific antibody released from inserted microbeads. The present results strongly suggest that, among other complementary cell guidance factor(s), the chemotactic response of NCCs toward the ocular region NT-3 gradient is essential for spatiotemporal cell orientation, amplifying the functional scope of this neurotrophic factor as a molecular guide for the embryo cells, besides its well-known canonical functions.

Hormetic Electric Field Theory of Pattern Formation

The hormetic morphogen theory of curvature (Fosslien 2009) proposes that hormetic morphogen concentration gradients modulate the synthesis of adenosine triphosphate (ATP) by cells along the gradients (field cells) and thus regulate their proliferation and induce curvature such as vascular wall curvature; however, it is unclear whether such morphogen gradients can also determine the histological pattern of the walls. Here, I propose that the ATP gradients modulate export of H(+) by vacuolar H(+)-ATPase (V-ATPase) located on the surface of field cells and generate extracellular ion concentration gradients, ion currents and electrical fields along the paths of morphogen gradients. In vitro, electrical fields can induce directional migration and elongation of vascular cells and align the cells with their long axis perpendicular to electrical field vectors (Bai et al. 2004). I suggest that likewise, in vivo vascular transmural electrical fields induced by hormetic morphogen concentration gradients can modulate cell shape i.e. cell elongation and cell curvature, and determine cell orientation. Moreover, I suggest that the electrical fields can modulate bidirectional cell migration and cell sorting via dynamic hormetic galvanotaxis analogous to in vitro isoelectric focusing in proton gradients, thus, hormetic morphogen gradients can determine the curvature of vessel walls and their histological patterns.

A tale from the Crypt: splice variants of BK channels in colonic potassium secretion

A tale from the Crypt: splice variants of BK channels in colonic potassium secretion

Engineering a scaffold-free 3D tumor model for in vitro drug penetration studies

Three-dimensional (3D) in vitro cultures are recognized for recapitulating the physiological microenvironment and exhibiting high concordance with in vivo conditions. In cancer research, the multi-cellular tumor spheroid (MCTS) model is an established 3D cancer model that exhibits microenvironmental heterogeneity close to that of tumors in vivo. However, the established process of MCTS formation is time-consuming and often uncontrolled. Here, we report a method for engineering MCTS using a transient inter-cellular linker which facilitates cell-cell interaction. Using C3A cells (a hepatocellular carcinoma cell line) as a model, we formed linker-engineered spheroids which grew to a diameter of 250 μm in 7 days, as compared to 16 days using conventional non-adherent culture. Seven-day old linker-engineered spheroids exhibited characteristics of mature MCTS, including spheroid morphology, gene expression profile, cell-cell interaction, extracellular matrix secretion, proliferation and oxygen concentration gradients, and cellular functions. Linker-engineered spheroids also displayed a resistance to drug penetration similar to mature MCTS, with dose-dependent extracellular accumulation of the drug. The linker-engineered spheroids thus provide a reliable accelerated 3D in vitro tumor model for drug penetration studies.

Acyltransferases for secreted signalling proteins (Review)

Members of the MBOAT family of multispanning transmembrane enzymes catalyze the acylation of important secreted signalling proteins of the Hedgehog, Wg/Wnt and ghrelin families. Acylation of these substrates occurs during transport through the secretory pathway and plays key roles in their biological activity and spread from producing cells, contributing to the formation of appropriate extracellular concentration gradients. Characterization of these enzymes could lead to their identification as therapeutic targets for diverse human diseases such as cancers, obesity and diabetes.

Cellular cation transport studied by 6, 7Li and 23Na NMR in a porous Mo132 Keplerate type nano-capsule as model system

Li+ ions can interplay with other cations intrinsically present in the intra- and extra-cellular space (i.e. Na+, K+, Mg2+ and Ca2+) have therapeutic effects (e.g. in the treatment of bipolar disorder) or toxic effects (at higher doses), likely because Li+ interferes with the intra-/extra-cellular concentration gradients of the mentioned physiologically relevant cations. The cellular transmembrane transport can be modelled by molybdenum-oxide-based Keplerates, i.e. nano-sized porous capsules containing 132 Mo centres, monitored through 6/7Li as well as 23Na NMR spectroscopy. The effects on the transport of Li+ cations through the 'ion channels' of these model cells, caused by variations in water amount, temperature, and by the addition of organic cationic 'plugs' and the shift reagent [Dy(PPP)2](7-) are reported. In the investigated solvent systems, water acts as a transport mediator for Li+. Likewise, the counter-transport (Li+/Na+, Li+/K+, Li+/Cs+ and Li+/Ca2+) has been investigated by 7Li NMR and, in the case of Li+/Na+ exchange, by 23Na NMR, and it has been shown that most (in the case of Na+ and K+, all (Ca2+) or almost none (Cs+) of the Li cations is extruded from the internal sites of the artificial cell to the extra-cellular medium, while Na+, K+ and Ca2+ are partially incorporated.

Asymmetric redistribution of GABA receptors during GABA gradient sensing by nerve growth cones analyzed by single quantum dot imaging

During development of the nervous system, the tip of a growing axon, the growth cone (GC), must respond accurately to stimuli that direct its growth. This axonal navigation depends on extracellular concentration gradients of numerous guidance cues, including GABA. GCs can detect even weak directional signals, yet the mechanisms underlying this sensitivity remain unclear. Past studies in other eukaryotic chemotactic systems have pointed to the role of the spatial reorganization of the transduction pathway in their sensitive response. Here we have developed a single-molecule assay to observe individual GABA(A) receptors (GABA(A)Rs) in the plasma membrane of nerve GCs subjected to directional stimuli. We report that in the presence of an external GABA gradient GABA(A)Rs redistribute asymmetrically across the GC toward the gradient source. Single-particle tracking of GABA(A)Rs shows that the redistribution results from transient interactions between the receptors and the microtubules. Moreover, the relocalization is accompanied by an enhancement in the asymmetry of intracellular calcium concentration. Altogether, our results reveal a microtubule-dependent polarized reorganization of chemoreceptors at the cell surface and suggest that this polarization serves as an amplification step in GABA gradient sensing by nerve GCs.

Experimental Generation and Computational Modeling of Intracellular pH Gradients in Cardiac Myocytes

It is often assumed that pH i is spatially uniform within cells. A double-barreled microperfusion system was used to apply solutions of weak acid (acetic acid, CO 2) or base (ammonia) to localized regions of an isolated ventricular myocyte (guinea pig). A stable, longitudinal pH i gradient (up to 1 pH i unit) was observed (using confocal imaging of SNARF-1 fluorescence). Changing the fractional exposure of the cell to weak acid/base altered the gradient, as did changing the concentration and type of weak acid/base applied. A diffusion-reaction computational model accurately simulated this behavior of pH i. The model assumes that H i + movement occurs via diffusive shuttling on mobile buffers, with little free H + diffusion. The average diffusion constant for mobile buffer was estimated as 33 × 10 −7 cm 2/s, consistent with an apparent H i + diffusion coefficient, D H app , of 14.4 × 10 −7 cm 2/s (at pH i 7.07), a value two orders of magnitude lower than for H + ions in water but similar to that estimated recently from local acid injection via a cell-attached glass micropipette. We conclude that, because H i + mobility is so low, an extracellular concentration gradient of permeant weak acid readily induces pH i nonuniformity. Similar concentration gradients for weak acid (e.g., CO 2) occur across border zones during regional myocardial ischemia, raising the possibility of steep pH i gradients within the heart under some pathophysiological conditions.

Dynamics of endocytosis and exocytosis of poly(D,L-lactide-co-glycolide) nanoparticles in vascular smooth muscle cells.

The purpose of this work was to characterize the process of endocytosis, exocytosis, and intracellular retention of poly (D,L-lactide-co-glycolide) nanoparticles in vitro using human arterial vascular smooth muscle cells (VSMCs). Nanoparticles containing bovine serum albumin (BSA) as a model protein and 6-coumarin as a fluorescent marker were formulated by a double emulsion-solvent evaporation technique. The endocytosis and exocytosis of nanoparticles in VSMCs were studied using confocal microscopy and their intracellular uptake and retention were determined quantitatively using high-performance liquid chromatography. Cellular uptake of nanoparticles (mean particle size 97 +/- 3 nm) was a concentration-, time-, and energy-dependent endocytic process. Confocal microscopy demonstrated that nanoparticles were internalized rapidly, with nanoparticles seen inside the cells as early as within 1 min after incubation. The nanoparticle uptake increased with incubation time in the presence of nanoparticles in the medium; however, once the extracellular nanoparticle concentration gradient was removed, exocytosis of nanoparticles occurred with about 65% of the internalized fraction undergoing exocytosis in 30 min. Exocytosis of nanoparticles was slower than the exocytosis of a fluid phase marker, Lucifer yellow. Furthermore, the exocytosis of nanoparticles was reduced after the treatment of cells with the combination of sodium azide and deoxyglucose, suggesting that exocytosis of nanopartides is an energy-dependent process. The nanoparticle retention increased with increasing nanoparticle dose in the medium but the effect was relatively less significant with the increase in incubation time. Interestingly, the exocytosis of nanoparticles was almost completely inhibited when the medium was depleted of serum. Further studies suggest that the addition of BSA in the serum free medium with or without platelet derived growth factor (PDGF) induced exocytosis of nanoparticles. The above result suggests that the protein in the medium is either adsorbed onto nanoparticles and/or carried along with nanoparticles inside the cells, which probably interacts with the exocytic pathway and leads to greater exocytosis of nanoparticles. The study demonstrated that endocytosis and exocytosis of nanoparticles are dynamic and energy-dependent processes. Better understanding of the mechanisms of endocytosis and exocytosis, studies determining the effect of nanoparticle formulation and composition that may affect both the processes, and characterization of intracellular distribution of nanoparticles with surface modifications would be useful in exploring nanoparticles for intracellular delivery of therapeutic agents.

Isolation of Normal Epithelial Cells Adapted to Grow at Nonphysiological Concentration of Magnesium

Extracellular magnesium (Mg) depletion inhibits the growth of the HC11 normal mammary epithelial cells. In this study we found that an acute increase in extracellular Mg generally exerts a positive effect on the growth of these cells. We also isolated and characterized two derivatives adapted to grow and proliferate at nonphysiological concentration of Mg. The growth properties of the HC-LMg cells at 25 μM Mg were comparable to those of the parental HC11 cells in the regular medium (0.5 mM Mg) despite an increased expression of the CDK inhibitor p27Kip1. They also showed a reduced dependence from serum to grow. The HC-HMg cells have been adapted to grow and proliferate at an increased (45 mM) Mg concentration. Cell total Mg content was 19.6, 9.7, and 20.1 nmol/mg protein in the HC11, HC-LMg, and HC-HMg cells, respectively. Thus, we have isolated derivatives of normal epithelial cells which are able to maintain Mg content in a physiological range in the face of different extracellular concentration gradients and will be a valuable tool for further studies on the regulation of Mg homeostasis in eukaryotic cells.

Putrescine export in Xenopuslaevis oocytes occurs against a concentration gradient: evidence for a non-diffusional export process

Putrescine export was found to occur by a non-diffusional highly regulated process using Xenopus oocytes as a model system of polyamine transport. Untreated oocytes were observed to possess high endogenous intracellular putrescine and spermidine levels with no detectable polyamine interconversion or biosynthesis over the assay intervals. The putrescine uptake process demonstrated a rapid saturation within a 5 min interval. Spermidine demonstrated a relatively larger uptake capacity with only a minimal ability to export. A kinetic analysis of the concentration-dependence of the putrescine and spermidine uptake processes indicated that the putrescine uptake process may possess two concurrent uptake components while spermidine uptake may possess a two-component process with an allosteric regulation. Elevated intracellular putrescine levels were observed to decrease against a 10-fold higher extracellular concentration gradient in a rapid and specific manner. No noticeable changes in the intracellular levels of other polyamines were observed over the same time interval. The uptake and export rates of putrescine transport also showed a concurrent, rapid and cyclical regulation. These findings support a non-diffusional putrescine export process which is highly regulated.

Thiamine homeostasis in neuroblastoma cells

We recently showed that thiamine uptake by neuroblastoma cells is mediated by two saturable transport systems: the first with high affinity for thiamine ( K m = 35 nM) is blocked by veratridine; the other, with low affinity is blocked by Ca 2+. The driving force for thiamine uptake is its phosphorylation to thiamine diphosphate (TDP) by thiamine pyrophosphokinase and subsequent binding of this cofactor to apoenzymes. Our results suggest that cells of neuronal origin possess mechanisms regulating the intracellular concentration of thiamine. At low external thiamine, the vitamin is taken up by a high-affinity transporter and pyrophosphorylated in thiamine diphosphate (TDP) : this is the TDP pool of slow turnover. An intraover extracellular concentration gradient of free thiamine is observed at low external concentration of the vitamin. At higher external thiamine concentration, TDP accumulation is limited by the binding capacitiy to the apoenzymes and unbound TDP (i.e. a small pool of fast turnover) is quickly hydrolyzed. Thiamine is slowly released by the cells by at least two different mechanisms. The first, accounting for a maximum of 50% of total thiamine release, is stimulated by external thiamine and is blocked by veratridine, suggesting that it is a self-exchange mechanism catalyzed by the high affinity thiamine transporter. The remaining thiamine efflux is neither sensitive to veratridine nor to Ca 2+ and its mechanism is unknown. About 25% of intracellular thiamine is not released, even after treatment of the cells with digitonin, thus maintaining an apparent gradient. This suggests a binding or sequestration in intracellular compartments. In neuroblastoma cells, the affinity of the transporter for thiamine is the highest reported so far, but the rate of transport is 2–3 orders of magnitude lower than in hepatocytes. In vivo, liver may be a thiamine reservoir which is replenished at high plasma thiamine concentrations, while the brain would remain able to pump thiamine (albeit slowly) and maintain steep gradients even when the external thiamine concentration becomes very low.