Proton-induced endocytosis is dependent on cell membrane fluidity, lipid-phase order and the membrane resting potential

AbstractRecently it has been shown that decreasing the extracellular pH of cells stimulates the formation of inward membrane invaginations and vesicles, accompanied by an enhanced uptake of macromolecules. This type of endocytosis was coined as proton-induced uptake (PIU). Though the initial induction of inward membrane curvature was rationalized in terms of proton-based increase of charge asymmetry across the membrane, the dependence of the phenomenon on plasma membrane characteristics is still unknown. The present study shows that depolarization of the membrane resting potential elevates PIU by 25%, while hyperpolarization attenuates it by 25%. Comparison of uptake in suspended and adherent cells implicates that the resting-potential affects PIU through remodeling the actin-cytoskeleton. The pH at the external interface of the cell membrane rather than the pH gradient across it determines the extent of PIU. PIU increases linearly upon temperature increase in the range of 4–36°C, in correlation with the membrane fluidity. The plasma membrane fluidity and the lipid phase order are modulated by enriching the cell's membrane with cholesterol, tergitol, dimethylsulfoxide, 6-ketocholestanol and phloretin and by cholesterol depletion. These treatments are shown to alter the extent of PIU and are better correlated with membrane fluidity than with the lipid phase order. We suggest that the lipid phase order and fluidity influence PIU by regulating the lipid order gradient across the perimeter of the lipid-condensed microdomains (rafts) and alter the characteristic tension line that separates the higher ordered lipid-domains from the lesser ordered ones

Correlation between local cell membrane displacements and filterability of human red blood cells

AbstractLocal mechanical fluctuations of the cell membrane of human erythrocytes were shown to involve MgATP- and Mg2+-driven fast membrane displacements. We propose that these local bending deformations of the cell membrane are important for cell passage through capillaries. In order to verify this hypothesis, we examined cell membrane fluctuations and filterability of erythrocytes over a wide range of medium osmolalities (180–675 mosmol/kg H2O). The results indicate the existence of a positive correlation between the amplitude of local cell membrane displacements and cell filterability. We suggest that the occurrence of metabolically driven membrane displacements on the side surface of the red blood cell diminishes its bending stiffness and enables it to fold more efficiently upon entrance into blood capillaries. Thus, local cell membrane displacements seem to play an important role in microcirculation

Regulation of the Na+/H+ exchanger under conditions of abolished proton gradient: Isosmotic and hyperosmotic stimulation

AbstractActivation of the Na+/H+ exchanger following isosmotic and hyperosmotic stimuli was investigated in an osteoblast cell line (RCJ 1.20). The pH dependence of the transporter activity was studied under conditions of abolished proton gradient (pHi = PH*) across the membrane. The isotonic response is Na+* dependent, increases towards higher pH. values, displaying a sigmoidal dependence on pH1** (Hill coefficient ≈ 1.8) and is controlled by pH*. The greater than first order dependence of pH suggest that H+* inhibits the exchange beyond the rate expected from competition with the Na+* alone. This may be due to the existence of an external H+ regulatory site with a negative cooperative effect on the intra- or extracellular transport site. The hyperosmotic activation is Na+* independent, parallels the sigmoidal pH dependence of the isosmotic stimulus (Hill coefficient ≈ 2.0) and is mediated through an increase of the Vmax without a change in the intracellular proton sensitivity

Fast cell membrane displacements in B lymphocytes Modulation by dihydrocytochalasin B and colchicine

AbstractA novel type of cell membrane movement was characterized in B lymphocytes. Local submicron cell membrane displacements, within the frequency range 0.3–15 Hz, were registered in a murine lymphoma B cell line by a novel optical method based on point dark field microscopy. The cell membrane displacements were measured by monitoring changes in light scattering from very small illuminated areas (0.25 μm2) at the edge of the cell surface. B lymphocytes manifest a relative change in light scattering of 7.7 ± 1.3% (mean ± SD) which corresponds to cell membrane transverse displacement of 131 ± 22 nm. The confinement of cell membrane displacements to microdomains (≤0.2 μm2) emerged from the observed dependence of the displacement amplitude on the area size from which it is monitored. Colchicine (1 μM) decreased membrane fluctuations down to a value of 88 ± 14 nm, whereas dihydrocytochalasin B (2 μM) increased the amplitude of membrane displacements up to 184 ± 31 nm. These findings demonstrate the existence of a dynamic mechanical interaction between the cytoskeleton and the cell membrane in the frequency range of 0.3–15 Hz. The modulation of these interactions by the disruption of microfilaments or microtubules is explained in terms of the induced strain changes imposed on the cell membrane

Multifrequency Analysis of Single Inductive Coil Measurements Across a Gel Phantom Simulation of Internal Bleeding in the Brain

The present study is part of an ongoing effort to develop a simple diagnostic technology for detecting internal bleeding in the brain, which can be used in lieu or in support of medical imaging and thereby reduce the cost of diagnostics in general, and in particular, would make diagnostics accessible to economically disadvantaged populations. The study deals with a single coil inductive device to be used for detecting cerebral hemorrhage. It presents a first‐order experimental study that examines the predictions of our recently published theoretical study. The experimental model employs a homogeneous cylindrical phantom in which internal head bleeding was simulated by way of a fluid inclusion. We measured the changes in amplitude and phase across the coil with a network vector analyzer as a function of frequency (100–1,000 MHz), volume of blood simulating fluid, and the site of the fluid injection. We have developed a new mathematical model to statistically analyze the complex data produced in this experiment. We determined that the resolution for the fluid volume increase following fluid injection is strongly dependent on frequency as well as the location of liquid accumulation. The experimental data obtained in this study supports the predictions of our previous theoretical study, and the statistical analysis shows that the simple single coil device is sensitive enough to detect changes due to fluid volume alteration of two milliliters. Bioelectromagnetics. 2020;41:21–33. © 2019 Bioelectromagnetics SocietyThis work is based on a portion of a dissertation to be submitted by Moshe Oziel in partial fulfillment of the requirements for a PhD degree to Tel‐Aviv University

Exploiting endocytosis for transfection of mRNA for cytoplasmatic delivery using cationic gold nanoparticles

Gene therapy holds promise to cure various diseases at the fundamental level. For that, efficient carriers are needed for successful gene delivery. Synthetic 'non-viral' vectors, as cationic polymers, are quickly gaining popularity as efficient vectors for transmitting genes. However, they suffer from high toxicity associated with the permeation and poration of the cell membrane. This toxic aspect can be eliminated by nanoconjugation. Still, results suggest that optimising the oligonucleotide complexation, ultimately determined by the size and charge of the nanovector, is not the only barrier to efficient gene delivery. We herein develop a comprehensive nanovector catalogue comprising different sizes of Au NPs functionalized with two different cationic molecules and further loaded with mRNA for its delivery inside the cell. Tested nanovectors showed safe and sustained transfection efficiencies over 7 days, where 50 nm Au NPs displayed the highest transfection rates. Remarkably, protein expression was increased when nanovector transfection was performed combined with chloroquine. Cytotoxicity and risk assessment demonstrated that nanovectors are safe, ascribed to lesser cellular damage due to their internalization and delivery via endocytosis. Obtained results may pave the way to design advanced and efficient gene therapies for safely transferring oligonucleotides

Emerging methods and tools for environmental risk assessment, decision-making, and policy for nanomaterials: summary of NATO Advanced Research Workshop

Nanomaterials and their associated technologies hold promising opportunities for the development of new materials and applications in a wide variety of disciplines, including medicine, environmental remediation, waste treatment, and energy conservation. However, current information regarding the environmental effects and health risks associated with nanomaterials is limited and sometimes contradictory. This article summarizes the conclusions of a 2008 NATO workshop designed to evaluate the wide-scale implications (e.g., benefits, risks, and costs) of the use of nanomaterials on human health and the environment. A unique feature of this workshop was its interdisciplinary nature and focus on the practical needs of policy decision makers. Workshop presentations and discussion panels were structured along four main themes: technology and benefits, human health risk, environmental risk, and policy implications. Four corresponding working groups (WGs) were formed to develop detailed summaries of the state-of-the-science in their respective areas and to discuss emerging gaps and research needs. The WGs identified gaps between the rapid advances in the types and applications of nanomaterials and the slower pace of human health and environmental risk science, along with strategies to reduce the uncertainties associated with calculating these risks.Engineering and Applied Science

Emerging methods and tools for environmental risk assessment, decision-making, and policy for nanomaterials: summary of NATO Advanced Research Workshop

Nanomaterials and their associated technologies hold promising opportunities for the development of new materials and applications in a wide variety of disciplines, including medicine, environmental remediation, waste treatment, and energy conservation. However, current information regarding the environmental effects and health risks associated with nanomaterials is limited and sometimes contradictory. This article summarizes the conclusions of a 2008 NATO workshop designed to evaluate the wide-scale implications (e.g., benefits, risks, and costs) of the use of nanomaterials on human health and the environment. A unique feature of this workshop was its interdisciplinary nature and focus on the practical needs of policy decision makers. Workshop presentations and discussion panels were structured along four main themes: technology and benefits, human health risk, environmental risk, and policy implications. Four corresponding working groups (WGs) were formed to develop detailed summaries of the state-of-the-science in their respective areas and to discuss emerging gaps and research needs. The WGs identified gaps between the rapid advances in the types and applications of nanomaterials and the slower pace of human health and environmental risk science, along with strategies to reduce the uncertainties associated with calculating these risks