Gilbert Laustriat

TMA-DPH: A suitable fluorescence polarization probe for specific plasma membrane fluidity studies in intact living cells

Fluorescence intensity measurements and fluorescence microscopy data showed that TMA-DPH (trimethylammonium diphenylhexatriene), a cationic derivative of the fluorescence polarization probe DPH, has a considerably different behavior in L929 cultured cells than does its parent molecule. In contrast to DPH, it incorporates very rapidly in the plasma membranes of the treated cells, and remains specifically localized on the cell surface for at least 25 min. It can therefore be recommended for specific plasma membrane fluidity measurements in whole living cells. No relevant information about the localization of the probes could be obtained by other techniques used in parallel, namely: subcellular fractionation and fluorescence inhibition by trinitrobenzene sulfonate (TNBS).

Plasma membrane fluidity measurements on whole living cells by fluorescence anisotropy of trimethylammoniumdiphenylhexatriene.

Trimethylammoniumdiphenylhexatriene (TMA-DPH) is a hydrophobic fluorescent probe with a high quantum yield, which was shown earlier to have specific localization properties in the plasma membranes of whole living cells. This probe was used in aqueous suspensions of L929 mouse fibroblasts, rat mast cells and ReH6 leukemic lymphocytes for determining plasma membrane fluidity from fluorescence stationary anisotropy measurements. TMA-DPH was only partially incorporated into the membranes, most of it remained as a stable form in the buffer solution; the distribution was governed by an equilibrium. The measurements were influenced by unavoidable parasitic scattered light and an appropriate correction is described. A set of precautions for the proper use of the probe is proposed. The results indicated that the fluidity was considerably lower in whole cells than in isolated membranes from the same system.

Molecular mechanisms of photosensitization.

The first part of this article is devoted to basic concepts of photosensitization and to the primary photophysical and photochemistry processes involved in the reaction. The electronic configuration of molecular oxygen in its ground or activated states, which intervene in numerous photosensitized reactions, is reviewed. Finally, the main photosensitized reactions are reviewed and classified into three different groups: reactions due to radicals (type I), reactions due to singlet oxygen (type II) and those which do not involve oxygen (type III).

Parallel investigation of exocytosis kinetics and membrane fluidity changes in human platelets with the fluorescent probe, trimethylammonio-diphenylhexatriene

A simple, flexible and sensitive fluorescence method is described, which, from the same experiment, provides coupled quantitative informations on membrane fluidity changes and exocytosis, and reliable kinetic analyses of these effects, in intact cell suspensions. The method is based on the features peculiar to trimethylammonio-diphenylhexatriene (TMA-DPH), a fluorescent hydrophobic probe, which, in intact cells, is incorporated specifically into the plasma membranes, according to an instantaneous partition equilibrium. The method was tested on human platelets upon stimulation with various agents, such as human alpha-thrombin, adenosine diphosphate (ADP), adrenaline and ionomycin, which act through different types of mechanism. The experimental conditions were chosen to allow platelet shape change and exocytosis, but no aggregation. The kinetics and the dose-dependence of the changes in TMA-DPH fluorescence intensity and anisotropy were compared to the simultaneous physiological responses of platelets to the same stimuli, under the same conditions. Quantitative correlations were established between serotonin secretion and the increase in fluorescence intensity, whereas fluorescence anisotropy, which monitors membrane fluidity changes was associated with platelet shape change. The specificity of the effects was confirmed with appropriate antagonistic or modulating agents.

Étude par fluorescence de la phosphatase alcaline d'Escherichia coli I. Propriétés des états excités

Abstract Singlet excited state properties of alkaline phosphatase (Escherichia coli) are studied: absorption and emission spectra, fluorescence quantum yield and lifetime, quenching by potassium iodide. Results are compared with those obtained with solutions of model compounds, such as the denatured enzyme, aromatic amino acids and corresponding N-acetyl amides. Information is obtained showing that the intra-globular medium can be simulated by dioxane, and that most of the 10 tryptophyl residues (8–9) are located inside the protein; furthermore, 6 of these residues are found to be close to chemical groups inducing static quenching of fluorescence.Abstract Singlet excited state properties of alkaline phosphatase (Escherichia coli) are studied: absorption and emission spectra, fluorescence quantum yield and lifetime, quenching by potassium iodide. Results are compared with those obtained with solutions of model compounds, such as the denatured enzyme, aromatic amino acids and corresponding N-acetyl amides. Information is obtained showing that the intra-globular medium can be simulated by dioxane, and that most of the 10 tryptophyl residues (8–9) are located inside the protein; furthermore, 6 of these residues are found to be close to chemical groups inducing static quenching of fluorescence.

Evidence for early fluidity changes in the plasma membranes of interferon treated L cells, from fluorescence anisotropy data

A significant increase of the plasma membrane fluidity is observed in L cells upon treatment with mouse beta interferon, by the means of fluorescence anisotropy measurements of DPH. The effect is dose dependent and, at the difference with previously reported membrane effects of interferon, is an early one (maximum for a 30 min. treatment), and may be directly related to the initiation of antiviral activity.

Influence of 3-Substitution on Excited State Properties of Indole in Aqueous Solutions

Considerable attention has been paid to the excited states of the indole ring in the past two decades, since as, the aromatic moiety of tryptophan, it is the major near-ultraviolet chromophore of proteins where it can play the role of a fluorescent probe. Nevertheless photophysical and photochemical properties of indole are rather particular and remain largely unelucidated (Longworth, 1971).

Étude par fluorescence de la phosphatase alcaline d'Escherichia coli II. Transferts intramoleculaires de l'énergie d'excitation

Abstract The analysis of the fluorescence excitation spectrum of Escherichia coli alkaline phosphatase shows that the tyrosine-tryptophan energy transfer is negligible for this protein. On the other hand, the comparative study of the fluorescence polarisation spectra at 24 °C and −70 °C of the native and denatured enzyme, and of tryptophan, gives evidence for an intramolecular energy migration between tryptophan residues.