Intracellular Sodium Ion Research Articles

Alleviation of NaCl toxicity in the cyanobacterium Synechococcus sp. PCC 7942 by exogenous calcium supplementation

Salinity (NaCl) is one of the major problems associated with irrigated agricultural lands, especially rice fields. Being the common inhabitants of rice fields, cyanobacteria frequently experience high concentration of NaCl which in turn causes cellular damage. Therefore, mitigation of NaCl stress in cyanobacteria, plant growth-promoting microorganisms, is of utmost importance. The present study was designed to investigate the role of calcium in the alleviation of NaCl stress-induced cellular in Synechococcus sp. PCC 7942. The cyanobacterium was subjected to sub-lethal concentration of NaCl (800 mM) with and without the supplementation of calcium (1 mM CaCl2) for 8 days. The results showed a drastic reduction in growth due to excess NaCl, but supplementation of CaCl2 reduced the salt stress damage and partially restored growth. Application of calcium increased pigment contents, photosynthetic efficiency, antioxidative enzyme activity, osmolyte contents and reduced the intracellular sodium ion concentration, MDA content, electrolyte leakage and free oxygen radical generation. Furthermore, proteins involved in photosynthesis, respiration, ATP synthesis and protein synthesis along with two hypothetical proteins were also observed to be upregulated in the cyanobacterium in presence of calcium. Furthermore, proteins related to oxidative stress defence, nitrogen metabolism, carbohydrate metabolism, fatty acid metabolism and secondary metabolism were found to be upregulated by several fold. Therefore, our study suggests that calcium suppresses salt toxicity in Synechococcus sp. PCC 7942 by restricting the entry of Na+ into the cell, increasing osmolyte production and upregulating defence-related proteins.

Enhanced sodium acetate tolerance in Saccharomyces cerevisiae by the Thr255Ala mutation of the ubiquitin ligase Rsp5.

Sodium and acetate inhibit cell growth and ethanol fermentation by different mechanisms in Saccharomyces cerevisiae. We identified the substitution of a conserved Thr255 to Ala (T255A) in the essential Nedd4-family ubiquitin ligase Rsp5, which enhances cellular sodium acetate tolerance. The T255A mutation selectively increased the resistance of cells against sodium acetate, suggesting that S. cerevisiae cells possess an Rsp5-mediated mechanism to cope with the composite stress of sodium and acetate. The sodium acetate tolerance was dependent on the extrusion of intracellular sodium ions by the plasma membrane-localized sodium pumps Ena1, Ena2, and Ena5 (Ena1/2/5) and two known upstream regulators: the Rim101 pH signaling pathway and the Hog1 mitogen-activated protein kinase. However, the T255A mutation affected neither the ubiquitination level of the Rsp5 adaptor protein Rim8 nor the phosphorylation level of Hog1. These data raised the possibility that Rsp5 enhances the function of Ena1/2/5 specifically in response to sodium acetate through an unknown mechanism other than ubiquitination of Rim8 and activation of Hog1-mediated signaling. Also, an industrial yeast strain that expresses the T255A variant exhibited increased initial fermentation rates in the presence of sodium acetate. Hence, this mutation has potential for the improvement of bioethanol production from lignocellulosic biomass.

Cell-structure specific necrosis by optical-trap induced intracellular nuclear oscillation

A drug-free procedure for killing malignant cells in a cell-type specific manner would represent a significant breakthrough for leukemia treatment. Here, we show that mechanically vibrating a cell in a specific oscillation condition can significantly promote necrosis. Specifically, oscillating the cell by a low-power laser trap at specific frequencies of a few Hz was found to result in increased death rate of 50% or above in different types of myelogenous leukemia cells, while normal leukocytes showed very little response to similar laser manipulations. The alteration of cell membrane permeability and cell volume, detected from ethidium bromide staining and measurement of intracellular sodium ion concentration, together with the observed membrane blebbing within 10min, suggest cell necrosis. Mechanics modelling reveals severe distortion of the cytoskeleton cortex at frequencies in the same range for peaked cell death. The disruption of cell membrane leading to cell death is therefore due to the cortex distortion, and the frequency at which this becomes significant is cell-type specific. Our findings lay down a new concept for treating leukemia based on vibration induced disruption of membrane in targeted malignant cells.

A model system using confocal fluorescence microscopy for examining real-time intracellular sodium ion regulation

The gills of euryhaline fish are the ultimate ionoregulatory tissue, achieving ion homeostasis despite rapid and significant changes in external salinity. Cellular handling of sodium is not only critical for salt and water balance but is also directly linked to other essential functions such as acid–base homeostasis and nitrogen excretion. However, although measurement of intracellular sodium ([Na+]i) is important for an understanding of gill transport function, it is challenging and subject to methodological artifacts. Using gill filaments from a model euryhaline fish, inanga (Galaxias maculatus), the suitability of the fluorescent dye CoroNa Green as a probe for measuring [Na+]i in intact ionocytes was confirmed via confocal microscopy. Cell viability was verified, optimal dye loading parameters were determined, and the dye–ion dissociation constant was measured. Application of the technique to freshwater- and 100% seawater-acclimated inanga showed salinity-dependent changes in branchial [Na+]i, whereas no significant differences in branchial [Na+]i were determined in 50% seawater-acclimated fish. This technique facilitates the examination of real-time changes in gill [Na+]i in response to environmental factors and may offer significant insight into key homeostatic functions associated with the fish gill and the principles of sodium ion transport in other tissues and organisms.

Chemical imaging of molecular changes in a hydrated single cell by dynamic secondary ion mass spectrometry and super-resolution microscopy.

Chemical imaging of single cells at the molecular level is important in capturing biological dynamics. Single cell correlative imaging is realized between super-resolution microscopy, namely, structured illumination microscopy (SIM), and time-of-flight secondary ion mass spectrometry (ToF-SIMS) using a multimodal microreactor (i.e., System for Analysis at the Liquid Vacuum Interface, SALVI). SIM characterized cells and guided subsequent ToF-SIMS analysis. Lipid fragments were identified in the cell membrane via dynamic ToF-SIMS depth profiling. Positive SIMS spectra show intracellular potassium and sodium ion transport due to exposure to nanoparticles. Spectral principal component analysis elucidates differences in chemical composition among healthy alveolar epithelial mouse lung C10 cells, cells that uptake zinc oxide nanoparticles, and various wet and dry control samples. The observation of Zn(+) gives the first direct evidence of ZnO NP uptake and dissolution by the cell membrane. Our results provide submicron chemical mapping for investigating cell dynamics at the molecular level.

Comparative characterization of the ɛ‐ and α‐subunit of the epithelial sodium channel in Xenopus laevis

The vectorial transport of sodium ions across most vertebrate epithelia is tightly linked to the activity of the epithelial sodium channel (ENaC) which allows for entry of sodium ions across the apical membrane of epithelial cells. The canonical ENaC is composed of the pore‐forming α‐subunit which assembles with the accessory β‐ and γ‐subunits to form a heterotrimeric ion channel. While α‐ENaC is crucial for proper channel functionality it may be substituted by α‐like subunits such as the delta‐subunit (δ) in humans or the epsilon‐subunit (ɛ) in Xenopus laevis. Although the inherent role of α‐containing ENaC in epithelial sodium absorption is well established, the physiological function of the ɛ‐ENaC subunit remains elusive. This study aimed to examine the expression of ɛ‐ and α‐ENaC subunits in various Xenopus laevis tissues and to comparatively characterize heterologously expressed ɛβγ‐ and αβγ‐ENaCs.Screening of ɛ‐ and α‐ENaC expression in tissue homogenates was performed by reverse transcription PCR. Transcripts for ɛ‐ENaC were predominantly detected in tissues of the urogenital tract (kidneys, urinary bladder, testis, ovary, cloaca). With the exception of the ovary these tissues also expressed α‐ENaC. Functional characterization of heterologously expressed ɛβγ‐and αβγ‐ENaC in Xenopus laevis oocytes was carried out using the two‐electrode voltage‐clamp technique. In comparison with αβγ‐ENaCs, ɛ‐containing channels produced an increased transmembrane current which was more sensitive to inhibition by extra‐ and intracellular sodium ions. Furthermore, activity of ɛβγ‐ENaC was enhanced by acidic extracellular pH but decreased under alkaline conditions, whereas α‐ENaC containing channels were activated in both cases. Additionally, ɛβγ‐ENaC was less sensitive to the ENaC blocker amiloride. A decreased amiloride‐sensitivity of transepithelial sodium transport was also detected in Ussing‐chamber recordings of urinary bladder preparations compared to lung epithelia which did not express ɛ‐ENaC. Since ɛ‐ENaC was the only difference in the ENaC‐subunit transcriptome of both tissues, its presence likely accounts for the distinct amiloride‐sensitivities.These results indicate a functional role for the ɛ‐ENaC subunit in urogenital tissues of Xenopus laevis. The presence of ɛ‐ENaC might facilitate a distinct responsiveness of these tissues to sodium and extracellular pH.

Colorimetric Assays of Na,K-ATPase.

The Na,K-ATPase is a plasma membrane enzyme that catalyzes active ion transport by the hydrolysis of ATP. Its activity in vivo is determined by many factors, particularly the concentration of intracellular sodium ions. It is the target of the cardiac glycoside class of drugs and of endogenous regulators. Its assay is often an endpoint in the investigation of physiological processes, and it is a promising drug target. As described in this unit, its enzymatic activity can be determined in extracts from tissues by test tube assay using a spectrophotometer or (32)P-ATP. The protocols in this chapter measure inorganic phosphate as the end product of hydrolysis of ATP.

A red-emitting ratiometric fluorescent probe based on a benzophosphole P-oxide scaffold for the detection of intracellular sodium ions.

We disclose the development of a ratiometric fluorescent probe based on a benzophosphole P-oxide and its application for the detection of intracellular Na(+) ions. Excitation by visible light induced red emission from this probe in water, which was subjected to a hypsochromic shift upon complexation with Na(+). Based on this change, a ratiometric analysis enabled us to visualise changes in the Na(+) concentration in living mammalian cells.

Synthetic ion transporters can induce apoptosis by facilitating chloride anion transport into cells.

Anion transporters based on small molecules have received attention as therapeutic agents because of their potential to disrupt cellular ion homeostasis. However, a direct correlation between a change in cellular chloride anion concentration and cytotoxicity has not been established for synthetic ion carriers. Here we show that two pyridine diamide-strapped calix[4]pyrroles induce coupled chloride anion and sodium cation transport in both liposomal models and cells, and promote cell death by increasing intracellular chloride and sodium ion concentrations. Removing either ion from the extracellular media or blocking natural sodium channels with amiloride prevents this effect. Cell experiments show that the ion transporters induce the sodium chloride influx, which leads to an increased concentration of reactive oxygen species, release of cytochrome c from the mitochondria and apoptosis via caspase activation. However, they do not activate the caspase-independent apoptotic pathway associated with the apoptosis-inducing factor. Ion transporters, therefore, represent an attractive approach for regulating cellular processes that are normally controlled tightly by homeostasis.

Inhibition of the Arabidopsis Salt Overly Sensitive Pathway by 14-3-3 Proteins

The Salt Overly Sensitive (SOS) pathway regulates intracellular sodium ion (Na(+)) homeostasis and salt tolerance in plants. Until recently, little was known about the mechanisms that inhibit the SOS pathway when plants are grown in the absence of salt stress. In this study, we report that the Arabidopsis thaliana 14-3-3 proteins λ and κ interact with SOS2 and repress its kinase activity. Growth in the presence of salt decreases the interaction between SOS2 and the 14-3-3 proteins, leading to kinase activation in planta. 14-3-3 λ interacts with the SOS2 junction domain, which is important for its kinase activity. A phosphorylation site (Ser-294) is identified within this domain by mass spectrometry. Mutation of Ser-294 to Ala or Asp does not affect SOS2 kinase activity in the absence of the 14-3-3 proteins. However, in the presence of 14-3-3 proteins, the inhibition of SOS2 activity is decreased by the Ser-to-Ala mutation and enhanced by the Ser-to-Asp exchange. These results identify 14-3-3 λ and κ as important regulators of salt tolerance. The inhibition of SOS2 mediated by the binding of 14-3-3 proteins represents a novel mechanism that confers basal repression of the SOS pathway in the absence of salt stress.

Corticosteroids increase intracellular free sodium ion concentration via glucocorticoid receptor pathway in cultured neonatal rat cardiomyocytes

BackgroundGlucocorticoids as well as mineralocorticoid have been shown to play essential roles in the regulation of electrical and mechanical activities in cardiomyocytes. Excess of these hormones is an independent risk factor for cardiovascular disease. Intracellular sodium ([Na+]i) kinetics are involved in cardiac diseases, including ischemia, heart failure and hypertrophy. However, intrinsic mediators that regulate [Na+]i in cardiomyocytes have not been widely discussed. Moreover, the quantitative estimation of altered [Na+]i in cultured cardiomyocytes and the association between the level of [Na+]i and the severity of pathological conditions, such as hypertrophy, have not been precisely reported. Methods and resultsWe herein demonstrate the quantitative estimation of [Na+]i in cultured neonatal rat cardiomyocytes following 24 h of treatment with corticosterone, aldosterone and dexamethasone. The physiological concentration of glucocorticoids increased [Na+]i up to approximately 2.5 mM (an almost 1.5-fold increase compared to the control) in a dose-dependent manner; this effect was blocked by a glucocorticoid receptor (GR) antagonist but not a mineralocorticoid receptor antagonist. Furthermore, glucocorticoids induced cardiac hypertrophy, and the hypertrophic gene expression was positively and significantly correlated with the level of [Na+]i. Dexamethasone induced the upregulation of Na+/Ca2 + exchanger 1 at the mRNA and protein levels. ConclusionsThe physiological concentration of glucocorticoids increases [Na+]i via GR. The dexamethasone-induced upregulation of NCX1 is partly involved in the glucocorticoid-induced alteration of [Na+]i in cardiomyocytes. These results provide new insight into the mechanisms by which glucocorticoid excess within a physiological concentration contributes to the development of cardiac pathology.

A small molecule two-photon fluorescent probe for intracellular sodium ions

We report a small-molecule two-photon fluorescent probe (ANa2) for Na(+) that shows a strong TPEF enhancement in response to Na(+) and can be easily loaded into live cells and can real time monitor the fluctuation of [Na]i in live cells and living tissue at more than 100 μm depth.

A technique for quantifying intracellular free sodium ion using a microplate reader in combination with sodium-binding benzofuran isophthalate and probenecid in cultured neonatal rat cardiomyocytes

BackgroundIntracellular sodium ([Na+]i) kinetics are involved in cardiac diseases including ischemia, heart failure, and hypertrophy. Because [Na+]i plays a crucial role in modulating the electrical and contractile activity in the heart, quantifying [Na+]i is of great interest. Using fluorescent microscopy with sodium-binding benzofuran isophthalate (SBFI) is the most commonly used method for measuring [Na+]i. However, one limitation associated with this technique is that the test cannot simultaneously evaluate the effects of several types or various concentrations of compounds on [Na+]i. Moreover, there are few reports on the long-term effects of compounds on [Na+]i in cultured cells, although rapid changes in [Na+]i during a period of seconds or several minutes have been widely discussed.FindingsWe established a novel technique for quantifying [Na+]i in cultured neonatal rat cardiomyocytes attached to a 96-well plate using a microplate reader in combination with SBFI and probenecid. We showed that probenecid is indispensable for the accurate measurement because it prevents dye leakage from the cells. We further confirmed the reliability of this system by quantifying the effects of ouabain, which is known to transiently alter [Na+]i. To illustrate the utility of the new method, we also examined the chronic effects of aldosterone on [Na+]i in cultured cardiomyocytes.ConclusionsOur technique can rapidly measure [Na+]i with accuracy and sensitivity comparable to the traditional microscopy based method. The results demonstrated that this 96-well plate based measurement has merits, especially for screening test of compounds regulating [Na+]i, and is useful to elucidate the mechanisms and consequences of altered [Na+]i handling in cardiomyocytes.

Glutamate release from astrocyte cell-line GL261 via alterations in the intracellular ion environment

Astrocytes modify and maintain neural activity and functions via gliotransmitter release such as, glutamate. They also change their properties and functions in response to alterations of ion environment resulting from neurotransmission; however, the direct evidence for whether intracellular ion alteration in astrocytes triggers gliotransmitter release is not indicated. Recent studies have reported that channelrhodopsin-2 (ChR2) is useful for alteration of intracellular ion environment in several types of cells with blue light exposure. Here, we show that ChR2-expressing GL261 (GLChR2) cells, clonal astrocytes, change their properties by photo-activation. Increased intracellular sodium and calcium ion concentrations and an altered membrane potential were observed in GLChR2 cells with blue light exposure. Alterations in the intracellular ion environment caused intracellular acidification and the inhibition of proliferation. In addition, it triggered glutamate release from GLChR2 cells. Glutamate from GLChR2 cells acted on N18 cells, clonal neuronal cells, as both a transmitter and neurotoxin depending on photo-activation. Our results show that the properties of ChR2-expressing astrocytes can be controlled by blue light exposure, and cation influx through photo-activated ChR2 might trigger functional cation influx via endogenous channels and result in the increase of glutamate release. Further, our results suggest that ChR2-expressing glial cells could become a useful tool in understanding the roles of glial cell activation and neural communication in the regulation of brain functions.

Imaging Sodium in Axons and Dendrites

This protocol describes a one-photon method to detect the time course and the spatial distribution of intracellular sodium ion concentrations [Na(+)](i), which result from action potentials and synaptic activity in different regions of neurons in brain slices. The one-photon technique has been applied to several different cell types and can likely be applied to other neurons in brain slices, particularly those that are relatively flat. Imaging of [Na(+)](i) changes is much less common than imaging of [Ca(2+)](i) changes, in part because typical signals are much smaller and more difficult to detect. However, with careful experiments, accurate and interesting results can be obtained. In this protocol, a cell is loaded with an indicator that responds to Na(+), the cell is stimulated, and the spatial and temporal characteristics of the fluorescence changes are recorded with an appropriate detector or camera.

Impairment of maize seedling photosynthesis caused by a combination of potassium deficiency and salt stress

The accumulation of intracellular sodium ions changes the ratio of K +:Na + under salt stress, which seems to affect the bioenergetic processes of photosynthesis. However, very little work has been done to evaluate the photosynthetic damage to maize resulting from a combination of salt stress and K + deficiency. Thus, our study investigated the effects of a combination of salt stress and K + deficiency on chlorophyll (Chl) fluorescence in maize seedlings by a dual-wavelength pulse-amplitude modulated fluorescence monitoring system. The results showed that the reductions in plant growth, Chl, carotenoids, K +, Mg 2+, maximum quantum efficiency (Fv/Fm), quantum yield (Y(II)), non-photochemical quenching (such as NPQ and qN) and photochemical quenching (such as qP and qL) of photosystem II (PSII), photochemical efficiency (Y(I)) of photosystem I (PSI) and non-photochemical quantum yield of PSI accepted side (Y(NA)), and electron transport rates of two photosystems of maize seedlings caused by a combination of salt stress and K + deficiency were greater than those of each individual stress. Moreover, significant increases in Na +, Cl − and the parameters of quantum yield of energy dissipation of two photosystems, including Y(NPQ), Y(NO), and Y(ND), of maize leaves subjected to the combined stresses were observed. This implied that a combination of salt stress and K + deficiency impaired the light reaction pathways of PSI and II in maize seedlings.

The Maximal Downstroke of Epicardial Potentials as an Index of Electrical Activity in Mouse Hearts

The maximal upstroke of transmembrane voltage (dV(m)/dt(max)) has been used as an indirect measure of sodium current I(Na) upon activation in cardiac myocytes. However, sodium influx generates not only the upstroke of V(m), but also the downstroke of the extracellular potentials V(e) including epicardial surface potentials V(es). The purpose of this study was to evaluate the magnitude of the maximal downstroke of V(es) (|dV(es)/dt (min)|) as a global index of electrical activation, based on the relationship of dV(m)/dt(max) to I(Na). To fulfill this purpose, we examined |dV(es)/dt(min)| experimentally using isolated perfused mouse hearts and computationally using a 3-D cardiac tissue bidomain model. In experimental studies, a custom-made cylindrical "cage" array with 64 electrodes was slipped over mouse hearts to measure V(es) during hyperkalemia, ischemia, and hypoxia, which are conditions that decrease I(Na). Values of |dV(es)/dt(min)| from each electrode were normalized (|dV(es)/dt (min)|(n)) and averaged (|dV(es)/dt(min)|(na)). Results showed that |dV(es)/dt(min)|(na) decreased during hyperkalemia by 28, 59, and 79% at 8, 10, and 12 mM [K(+)](o), respectively. |dV(es)/dt(min)| also decreased by 54 and 84% 20 min after the onset of ischemia and hypoxia, respectively. In computational studies, |dV(es)/dt(min)| was compared to dV(m)/dt(max) at different levels of the maximum sodium conductance G(Na), extracellular potassium ion concentration [K(+)](o), and intracellular sodium ion concentration [Na(+)](i), which all influence levels of I(Na). Changes in |dV(es)/dt(min)|(n) were similar to dV(m)/dt (max) during alterations of G(Na), [K(+)](o), and [Na(+)](i). Our results demonstrate that |dV(es)/dt(min)|(na) is a robust global index of electrical activation for use in mouse hearts and, similar to dV(m)/dt(max), can be used to probe electrophysiological alterations reliably. The index can be readily measured and evaluated, which makes it attractive for characterization of, for instance, genetically modified mouse hearts and drug effects on cardiac tissue.

High-content screening of drug-induced cardiotoxicity using quantitative single cell imaging cytometry on microfluidic device

Drug-induced cardiotoxicity or cytotoxicity followed by cell death in cardiac muscle is one of the major concerns in drug development. Herein, we report a high-content quantitative multicolor single cell imaging tool for automatic screening of drug-induced cardiotoxicity in an intact cell. A tunable multicolor imaging system coupled with a miniaturized sample platform was destined to elucidate drug-induced cardiotoxicity via simultaneous quantitative monitoring of intracellular sodium ion concentration, potassium ion channel permeability and apoptosis/necrosis in H9c2(2-1) cell line. Cells were treated with cisapride (a human ether-à-go-go-related gene (hERG) channel blocker), digoxin (Na(+)/K(+)-pump blocker), camptothecin (anticancer agent) and a newly synthesized anti-cancer drug candidate (SH-03). Decrease in potassium channel permeability in cisapride-treated cells indicated that it can also inhibit the trafficking of the hERG channel. Digoxin treatment resulted in an increase of intracellular [Na(+)]. However, it did not affect potassium channel permeability. Camptothecin and SH-03 did not show any cytotoxic effect at normal use (≤300 nM and 10 μM, respectively). This result clearly indicates the potential of SH-03 as a new anticancer drug candidate. The developed method was also used to correlate the cell death pathway with alterations in intracellular [Na(+)]. The developed protocol can directly depict and quantitate targeted cellular responses, subsequently enabling an automated, easy to operate tool that is applicable to drug-induced cytotoxicity monitoring with special reference to next generation drug discovery screening. This multicolor imaging based system has great potential as a complementary system to the conventional patch clamp technique and flow cytometric measurement for the screening of drug cardiotoxicity.

Intracellular ion monitoring using a gold-core polymer-shell nanosensor architecture

In this study, we describe the design of new ratiometric fluorescent nanosensors, whosearchitecture is based on a gold core surrounded by a poly(vinyl alcohol)–polyacetal shell. Tothe gold core, indicator dyes and reference dyes are attached via a cysteine linker. Thisnanosensor architecture is flexible with regards to the number and types of fluorophorelinkages possible. The robust poly(vinyl alcohol)–polyacetal shell protects the fluorophoreslinked to the core from non-specific interactions with intracellular proteins. Thenanosensors developed in this way are biocompatible and can be easily incorporatedinto mammalian cells without the use of transfection agents. Here, we show theapplication of these nanosensors for intracellular pH and sodium ion measurements.

MAb 84, a Cytotoxic Antibody that Kills Undifferentiated Human Embryonic Stem Cells via Oncosis

The monoclonal antibody mAb 84, which binds to podocalyxin-like protein-1 (PODXL) on human embryonic stem cells (hESCs), was previously reported to bind and kill undifferentiated cells in in vitro and in vivo assays. In this study, we investigate the mechanism responsible for mAb 84-induced hESCs cytotoxicity. Apoptosis was likely not the cause of mAb 84-mediated cell death because no elevation of caspase activities or increased DNA fragmentation was observed in hESCs following incubation with mAb 84. Instead, it was preceded by cell aggregation and damage to cell membranes, resulting in the uptake of propidium iodide, and the leakage of intracellular sodium ions. Furthermore, examination of the cell surface by scanning electron microscopy revealed the presence of pores on the cell surface of mAb 84-treated cells, which was absent from the isotype control. This mechanism of cell death resembles that described for oncosis, a form of cell death resulting from membrane damage. Additional data suggest that the binding of mAb 84 to hESCs initiates a sequence of events prior to membrane damage, consistent with oncosis. Degradation of actin-associated proteins, namely, alpha-actinin, paxillin, and talin, was observed. The perturbation of these actin-associated proteins consequently permits the aggregation of PODXL, thus leading to the formation of pores. To our knowledge, this is the first report of oncotic cell death with hESCs as a model.