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.

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.

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.

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.

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.

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.

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.

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.

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.

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

Cystic fibrosis (CF) cells exhibit an increase in the protein expression of β-arrestin-2 (βarr2) 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 βarr2 is an important component in the mechanisms leading to cholesterol accumulation characteristic of 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.

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.

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.

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.

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.