Electrochemical measurement of membrane cholesterol correlates with CFTR function and is HDAC6-dependent.

BACKGROUND: Previous studies have demonstrated that CF epithelial cells exhibit increased cholesterol content at the plasma membrane compared to wild type controls as measured by electrochemical methods. Microtubule dysregulation that impacts intracellular transport has also been identified in CF cells and is reversible with histone deacetylase 6 (HDAC6) inhibition, a regulator of tubulin acetylation. The hypothesis of this study is that increased membrane cholesterol content in CF cells is dependent on HDAC6 regulation. METHODS: Electrochemical measurement of membrane cholesterol in mouse trachea and in primary human CF bronchial epithelial cells is used to monitor CFTR correction and manipulation of cholesterol processing by HDAC6 inhibition. RESULTS: Data demonstrate that induction of Cftr expression in an inducible CF mouse model restores tubulin acetylation levels and normalizes membrane cholesterol content. To test the relationship between tubulin acetylation, membrane cholesterol levels were measured in a CF mouse model depleted of Hdac6 expression (CF/HDA). CF/HDA mouse trachea have WT membrane cholesterol levels while CF mice have approximately two-fold increase in membrane cholesterol compared to WT consistent with previous studies. Pharmacological inhibition of HDAC6 in primary human CF bronchial epithelial cells also reduces membrane cholesterol levels. CONCLUSIONS: This study demonstrates that elevated membrane cholesterol in CF epithelium is regulated by HDAC6 function and that the electrochemical measure of membrane cholesterol correlates with both genetic and pharmacological CFTR correction.

Direct electrochemical observation of glucosidase activity in isolated single lysosomes from a living cell.

The protein activity in individual intracellular compartments in single living cells must be analyzed to obtain an understanding of protein function at subcellular locations. The current methodology for probing activity is often not resolved to the level of an individual compartment, and the results provide an extent of reaction that is averaged from a group of compartments. To address this technological limitation, a single lysosome is sorted from a living cell via electrophoresis into a nanocapillary designed to electrochemically analyze internal solution. The activity of a protein specific to lysosomes, ß-glucosidase, is determined by the electrochemical quantification of hydrogen peroxide generated from the reaction with its substrate and the associated enzymes preloaded in the nanocapillary. Sorting and assaying multiple lysosomes from the same cell shows the relative homogeneity of protein activity between different lysosomes, whereas the protein activity in single lysosomes from different cells of the same type is heterogeneous. Thus, this study for the analysis of protein activity within targeted cellular compartments allows direct study of protein function at subcellular resolution and provides unprecedented information about the homogeneity within the lysosomal population of a single cell.

Single Cell Titration-Type Assay for Plasma Membrane Cholesterol Chemical Potential.

In this paper, a titration-type assay is described that determines the minimum concentration of cholesterol in solution that is required to drive net influx of cholesterol to the plasma membrane and thus increase the cholesterol concentration. The increase in cholesterol in the plasma membrane is detected by cholesterol diffusion at the site of contact by a cholesterol oxidase-modified microelectrode. In the presented thermodynamic model, the minimum solution phase cholesterol concentration that drives influx to the plasma membrane is a close approximation of the true solution-phase equilibrium concentration of cholesterol produced from cellular cholesterol efflux and as such it is a quantitative measure of the chemical potential of cholesterol in the cellular plasma membrane. This assay provides a measure of cholesterol chemical potential in the living cellular plasma membrane through reference to a solution concentration which avoids invoking classic kinetic theory to relate a rate to a specific thermodynamic activity and which avoids uncertainty associated with mass transfer phenomena.

New Frontiers and Challenges for Single-Cell Electrochemical Analysis.

Previous measurements of cell populations might obscure many important cellular differences, and new strategies for single-cell analyses are urgently needed to re-examine these fundamental biological principles for better diagnosis and treatment of diseases. Electrochemistry is a robust technique for the analysis of single living cells that has the advantages of minor interruption of cellular activity and provides the capability of high spatiotemporal resolution. The achievements of the past 30 years have revealed significant information about the exocytotic events of single cells to elucidate the mechanisms of cellular activity. Currently, the rapid developments of micro/nanofabrication and optoelectronic technologies drive the development of multifunctional electrodes and novel electrochemical approaches with higher resolution for single cells. In this Perspective, three new frontiers in this field, namely, electrochemical microscopy, intracellular analysis, and single-cell analysis in a biological system (i.e., neocortex and retina), are reviewed. The unique features and remaining challenges of these techniques are discussed.

Cell Plasma Membrane Cholesterol as a Diagnostic.

Cholesterol is a tightly regulated major structural component of the cell plasma membrane (PM) where it forms stoichiometric complexes with phospholipids and sphingolipids. The amount of cholesterol in the PM exhibits a regulatory role in basal activity of several biomolecular processes by direct binding to proteins and by indirect local environmental effects within the PM that are also coupled to overall cellular cholesterol homeostasis. The term "active cholesterol" refers to PM cholesterol not complexed to lipids, a cholesterol state that arises above a threshold mole fraction of cholesterol in the PM. Active cholesterol level in the PM provides a control mechanism for cellular cholesterol homeostasis through its recognition by membrane bound proteins that activate genes of cholesterol synthesis enzymes. Uptake of LDL, production and release of HDL as well as reversible storage of cholesterol in the cytosol by covalent modification are also regulated and dependent on PM cholesterol (thermodynamic) activity: active cholesterol. A number of human disease states have been found to have associated alterations in PM cholesterol and thus a method for its determination is described.

Communication-Microelectrode Detection of Cholesterol Efflux from the Human Buccel Mucosa.

It has previously demonstrated that cholesterol efflux from the cell plasma membrane is increased in a mouse model of cystic fibrosis (CF) compared to a wild-type control. A noninvasive means of characterizing plasma membrane cholesterol efflux at the surface of airway tissue of CF patients is needed to extend the trends found in animal models of CF to the human disease state. Microelectrode-induced cholesterol efflux from the plasma membrane of cells at the surface of tissue is proposed as a strategy to demonstrate increased cholesterol efflux for CF in human subjects. Data demonstrating detection of cholesterol efflux from the human buccal mucosa is reported as proof-of-concept for an in vivo diagnostic assay.

Role of Exchange Protein Activated by cAMP 1 in Regulating Rates of Microtubule Formation in Cystic Fibrosis Epithelial Cells.

The regulation of microtubule dynamics in cystic fibrosis (CF) epithelial cells and the consequences of reduced rates of microtubule polymerization on downstream CF cellular events, such as cholesterol accumulation, a marker of impaired intracellular transport, are explored here. It is identified that microtubules in both CF cell models and in primary CF nasal epithelial cells repolymerize at a slower rate compared with respective controls. Previous studies suggest a role for cAMP in modulating organelle transport in CF cells, implicating a role for exchange protein activated by cAMP (EPAC) 1, a regulator of microtubule elongation, as a potential mechanism. EPAC1 activity is reduced in CF cell models and in Cftr(-/-) mouse lung compared with respective non-CF controls. Stimulation of EPAC1 activity with the selective EPAC1 agonist, 8-cpt-2-O-Me-cAMP, stimulates microtubule repolymerization to wild-type rates in CF cells. EPAC1 activation also alleviates cholesterol accumulation in CF cells, suggesting a direct link between microtubule regulation and intracellular transport. To verify the relationship between transport and microtubule regulation, expression of the protein, tubulin polymerization-promoting protein, was knocked down in non-CF human tracheal (9/HTEo(-)) cells to mimic the microtubule dysregulation in CF cells. Transduced cells with short hairpin RNA targeting tubulin polymerization-promoting protein exhibit CF-like perinuclear cholesterol accumulation and other cellular manifestations of CF cells, thus supporting a role for microtubule regulation as a mechanism linking CFTR function to downstream cellular manifestation.

Double Potential Pulse Chronocoulometry for Detection of Plasma Membrane Cholesterol Efflux at Disk Platinum Microelectrodes.

A double potential pulse scheme is reported for observation of cholesterol efflux from the plasma membrane of a single neuron cell. Capillary Pt disk microelectrodes having a thin glass insulator allow the 10 µm diameter electrode and cell to be viewed under optical magnification. The electrode, covalently functionalized with cholesterol oxidase, is positioned in contact with the cell surface resulting in enzyme catalyzed cholesterol oxidation and efflux of cholesterol from the plasma membrane at the electrode contact site. Enzymatically generated hydrogen peroxide accumulates at the electrode/cell interface during a 5 s hold-time and is oxidized during application of a potential pulse. A second, replicate potential pulse is applied 0.5 s after the first potential pulse to gauge background charge prior to significant accumulation of hydrogen peroxide. The difference in charge passed between the first and second potential pulse provides a measure of hydrogen peroxide generated by the enzyme and is an indication of the cholesterol efflux. Control experiments for bare Pt microelectrodes in contact with the cell plasma membrane show difference charge signals in the range of about 7-10 pC. Enzyme-modified electrodes in contact with the plasma membrane show signals in the range of 16-26 pC.

Reduced microtubule acetylation in cystic fibrosis epithelial cells.

Dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) leads to many cellular consequences, including perinuclear accumulation of free cholesterol due to impaired endosomal transport. The hypothesis being tested is that CF-related perinuclear cholesterol accumulation due to disrupted endocytic trafficking occurs as a result of reduced microtubule (MT) acetylation. Here, it is identified that acetylated-α-tubulin (Ac-tub) content is reduced by ∼40% compared with respective wild-type controls in both cultured CF cell models (IB3) and primary Cftr-/- mouse nasal epithelial tissue. Histone deacetylase 6 (HDAC6) has been shown to regulate MT acetylation, which provides reasonable grounds to test its impact on reduced Ac-tub content on CF cellular phenotypes. Inhibition of HDAC6, either through tubastatin treatment or HDAC6 knockdown in CF cells, increases Ac-tub content and results in redistributed free cholesterol and reduced stimulation of NF-κB activity. Mechanistically, endoplasmic reticulum stress, which is widely reported in CF and leads to aggresome formation, is identified as a regulator of MT acetylation. F508del CFTR correction with C18 in primary airway epithelial cells restores MT acetylation and cholesterol transport. A significant role for phosphatidyl inositol-3 kinase p110α is also identified as a regulator of MT acetylation.

Regulatory role of ß-arrestin-2 in cholesterol processing in cystic fibrosis epithelial cells.

Cystic fibrosis (CF) cells exhibit an increase in the protein expression of ß-arrestin-2 (ßarr2) coincident with perinuclear accumulation of free cholesterol. Arrestins are proteins that both serve as broad signaling regulators and contribute to G-protein coupled receptor internalization after agonist stimulation. The hypothesis of this study is that ßarr2 is an important component in the mechanisms leading to cholesterol accumulation characteristic of CF cells. To test this hypothesis, epithelial cells stably expressing GFP-tagged ßarr2 (ßarr2-GFP) and respective GFP-expressing control cells (cont-GFP) were analyzed by filipin staining. The ßarr2-GFP cells show a late endosomal/lysosomal cholesterol accumulation that is identical to that seen in CF cells. This ßarr2-mediated accumulation is sensitive to Rp-cAMPS treatment, and depleting ßarr2 expression in CF-model cells by shRNA alleviates cholesterol accumulation compared with controls. Cftr/ßarr2 double knockout mice also exhibit wild-type (WT) levels of cholesterol synthesis, and WT profiles of signaling protein expression have previously been shown to be altered in CF due to cholesterol-related pathways. These data indicate a significant regulatory role for ßarr2 in the development of CF-like cholesterol accumulation and give further insight into cholesterol processing mechanisms. An impact of ßarr2 expression on Niemann-Pick type C-1 (NPC1)-containing organelle movement is proposed as the mechanism of ßarr2-mediated alterations on cholesterol processing. It is concluded that ßarr2 expression contributes to altered cholesterol trafficking observed in CF cells.

Increased plasma membrane cholesterol in cystic fibrosis cells correlates with CFTR genotype and depends on de novo cholesterol synthesis.

BACKGROUND: Previous observations demonstrate that Cftr-null cells and tissues exhibit alterations in cholesterol processing including perinuclear cholesterol accumulation, increased de novo synthesis, and an increase in plasma membrane cholesterol accessibility compared to wild type controls. The hypothesis of this study is that membrane cholesterol accessibility correlates with CFTR genotype and is in part influenced by de novo cholesterol synthesis. METHODS: Electrochemical detection of cholesterol at the plasma membrane is achieved with capillary microelectrodes with a modified platinum coil that accepts covalent attachment of cholesterol oxidase. Modified electrodes absent cholesterol oxidase serves as a baseline control. Cholesterol synthesis is determined by deuterium incorporation into lipids over time. Incorporation into cholesterol specifically is determined by mass spectrometry analysis. All mice used in the study are on a C57Bl/6 background and are between 6 and 8 weeks of age. RESULTS: Membrane cholesterol measurements are elevated in both R117H and DeltaF508 mouse nasal epithelium compared to age-matched sibling wt controls demonstrating a genotype correlation to membrane cholesterol detection. Expression of wt CFTR in CF epithelial cells reverts membrane cholesterol to WT levels further demonstrating the impact of CFTR on these processes. In wt epithelial cell, the addition of the CFTR inhibitors, Gly H101 or CFTRinh-172, for 24 h surprisingly results in an initial drop in membrane cholesterol measurement followed by a rebound at 72 h suggesting a feedback mechanism may be driving the increase in membrane cholesterol. De novo cholesterol synthesis contributes to membrane cholesterol accessibility. CONCLUSIONS: The data in this study suggest that CFTR influences cholesterol trafficking to the plasma membrane, which when depleted, leads to an increase in de novo cholesterol synthesis to restore membrane content.

Observation of cellular cholesterol efflux at microcavity electrodes.

Cholesterol oxidase modified platinum microcavity electrodes are used to measure cholesterol efflux from the plasma membrane surface of a single neuron in the buccal ganglion of Aplysia at room temperature. A background subtraction analog chronocoulometry method is used to measure hydrogen peroxide accumulation in the microcavity volume resulting from cellular cholesterol efflux and enzymatic oxidation. The data are consistent with the aqueous diffusion model for cellular cholesterol efflux where plasma membrane cholesterol undergoes exchange with solution phase cholesterol.

Electrochemical analysis of cell plasma membrane cholesterol at the airway surface of mouse trachea.

Electrochemical detection of plasma membrane cholesterol at the surface of excised mouse trachea tissue is reported. Cholesterol oxidase is covalently linked to an 11-mercaptoundecanoic acid submonolayer on the platinum electrode surface. The cholesterol oxidase-modified electrodes show steady-state responses for cholesterol in solution at physiological temperatures. Experiments for direct contact between the cholesterol oxidase-modified electrode and the surface of excised trachea tissue at 37 degrees C indicate steady-state responses that are largely independent of the position of contact on the tissue surface. Tissue samples are mounted on a quartz crystal microbalance electrode to gauge contact force between the electrode and the tissue surface, and the steady-state electrode response for tissue cholesterol is shown to be largely independent of the contact force. Trachea tissue excised from a mouse model of cystic fibrosis, which is known to exhibit evaluated cholesterol in airway cells, shows an electrode response that is approximately 40% larger than the response observed at wild-type mouse trachea tissue.

Altered cholesterol homeostasis in cultured and in vivo models of cystic fibrosis.

Determining how the regulation of cellular processes is impacted in cystic fibrosis (CF) is fundamental to understanding disease pathology and to identifying new therapeutic targets. In this study, unesterified cholesterol accumulation is observed in lung and trachea sections obtained from CF patients compared with non-CF tissues, suggesting an inherent flaw in cholesterol processing. An alternate staining method utilizing a fluorescent cholesterol probe also indicates improper lysosomal storage of cholesterol in CF cells. Excess cholesterol is also manifested by a significant increase in plasma membrane cholesterol content in both cultured CF cells and in nasal tissue excised from cftr(-/-) mice. Impaired intracellular cholesterol movement is predicted to stimulate cholesterol synthesis, a hypothesis supported by the observation of increased de novo cholesterol synthesis in lung and liver of cftr(-/-) mice compared with controls. Furthermore, pharmacological inhibition of cholesterol transport is sufficient to cause CF-like elevation in cytokine production in wild-type cells in response to bacterial challenge but has no effect in CF cells. These data demonstrate via multiple methods in both cultured and in vivo models that cellular cholesterol homeostasis is inherently altered in CF. This perturbation of cholesterol homeostasis represents a potentially important process in CF pathogenesis.

Enzyme modification of platinum microelectrodes for detection of cholesterol in vesicle lipid bilayer membranes.

Platinum microelectrodes are modified with a lipid bilayer membrane incorporating cholesterol oxidase. Details for electrode surface modification are presented along with characterization studies of electrode response to cholesterol solution and to cholesterol contained in the lipid bilayer membrane of vesicles. Ferrocyanide voltammetric experiments are used to track deposition of a submonolayer of a thiol-functionalized lipid on the platinum electrode surface, vesicle fusion for bilayer formation on the thiolipid-modified surface, and incorporation of cholesterol oxidase in the electrode-supported thiolipid/lipid bilayer membrane. The data are consistent with formation of a lipid bilayer structure on the electrode surface that contains defects. Experiments for detection of cholesterol solubilized in cyclodextrin solution show steady-state current responses that correlate with cholesterol concentration. Direct contact between the electrode and a vesicle lipid bilayer membrane shows a response that correlates with vesicle membrane cholesterol content.

Steady-state detection of cholesterol contained in the plasma membrane of a single cell using lipid bilayer-modified microelectrodes incorporating cholesterol oxidase.

Platinum microelectrodes modified with a lipid bilayer membrane incorporating cholesterol oxidase are used for detection of cholesterol contained in the plasma membrane of a single cell. Amperometric responses are consistent with enzymatic catalysis being rate limiting and cholesterol diffusing laterally in the plasma membrane to the electrode contact site. Importantly, electrode response appears to correlate with the cholesterol content of the cell plasma membrane. The electrodes should be useful for characterizing cellular cholesterol tracking pathways involved in pathogenesis of disease.