Guy Duportail

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).

Monitoring Biophysical Properties of Lipid Membranes by Environment-Sensitive Fluorescent Probes

We review the main trends in the development of fluorescence probes to obtain information about the structure, dynamics, and interactions in biomembranes. These probes are efficient for studying the microscopic analogs of viscosity, polarity, and hydration, as well as the molecular order, environment relaxation, and electrostatic potentials at the sites of their location. Progress is being made in increasing the information content and spatial resolution of the probe responses. Multichannel environment-sensitive probes that can distinguish between different membrane physicochemical properties through multiple spectroscopic parameters show considerable promise.

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.

Novel Two-Band Ratiometric Fluorescence Probes with Different Location and Orientation in Phospholipid Membranes

3-hydroxyflavone (3-HF) derivatives are very attractive fluorescence sensors due to their ability to respond to small changes in their microenvironment via a dramatic alteration of the relative intensities of their two well-separated emission bands. We developed fluorescence probes with locations at different depths and orientations of 3-HF moiety in the phospholipid bilayer, which determine their fluorescence behavior. While the spectral shifts of the probes correlate with their binding site polarity, the intensity ratio is a complex parameter that is also sensitive to the local hydration. We demonstrate that even the deeply located probes sense this hydration effect, which can be modulated by the charge of the lipid heads and is anisotropic with respect to the bilayer plane. Thus the two-band ratiometric fluorescence probes can provide multiparametric information on the properties of lipid membranes at different depths.

Ultrasensitive two-color fluorescence probes for dipole potential in phospholipid membranes

The principle of electrochromic modulation of excited-state intramolecular proton-transfer reaction was applied for the design of fluorescence probes with high two-color sensitivity to dipole potential, Ψd, in phospholipid bilayers. We report on the effect of Ψd variation on excitation and fluorescence spectra of two new 3-hydroxyflavone probes, which possess opposite orientations of the fluorescent moiety in the lipid bilayer. The dipole potential in the bilayer was modulated by the addition of 6-ketocholestanol or phloretin and by substitution of dimyristoyl phosphatidylcholine lipid with its ether analog 1,2-di-o-tetradecyl-sn-glycero-3-phosphocholine, and its value was estimated by the reference styryl dye 1-(3-sulfonatopropyl)-4-{β[2-(di-n-octylamino)-6-naphthyl]vinyl}pyridinium betaine. We demonstrate that after Ψd changes, the probe orienting in the bilayer similarly to the reference dye shows similar shifts in the excitation spectra, whereas the probe with the opposite orientation shows the opposite shifts. The new observation is that the response of 3-hydroxyflavone probes to Ψd in excitation spectra is accompanied by and quantitatively correlated with dramatic changes of relative intensities of the two well separated emission bands that belong to the initial normal and the product tautomer forms of the excited-state intramolecular proton-transfer reaction. This provides a strong response to Ψd by change in emission color.

Pore formation by a two-component leukocidin from Staphyloccocus aureus within the membrane of human polymorphonuclear leukocytes

The effects of the Staphylococcus aureus leukocidin (PVL), a two-component non-hemolytic toxin, were investigated on the membrane permeability of human polymorphonuclear leukocytes (PMNs). In the absence of extracellular Ca2+, the fluorescence of ethidium bromide added to the extracellular medium increased after PVL application in a concentration-dependent manner and no variations in the free intracellular [Ca2+] of Fura2-loaded PMNs were detected. In the presence of extracellular Ca2+, the fluorescence of ethidium was not modified but the free intracellular [Ca2+] of PMNs increased after application of PVL in a concentration-dependent manner. The time lag observed before an increase in the ethidium fluorescence was longer than the time lag observed before a Fura2 fluorescence increase. Simultaneous recordings of the two probes fluorescence variations have shown the protective effect of Ca2+ and Zn2+ and the closing of the pore by 50 mM Ca2+ or 2 mM Zn2+. Moreover, the effect of Ca2+ could be reversed by the addition of EGTA. In the presence of 1 mM extracellular Ca2+ or 0.8 mM extracellular Zn2+, the pore induced by PVL had an ionic size allowing Ca2+, Mn2+, Zn2+ and Mg2+ fluxes. The addition of antibodies against either component of PVL inhibits the permeabilization provoked by the toxin even after it was initiated. It is concluded that leukocidin from S. aureus is a pore-forming toxin which, under physiological conditions ([Ca2+] = 1 to 1.5 mM), provokes the formation of an ion-sized pore inducing an increase in the free intracellular Ca2+ which may activate PMN functions.

Consequences of Isostructural Main‐Chain Modifications for the Design of Antimicrobial Foldamers: Helical Mimics of Host‐Defense Peptides Based on a Heterogeneous Amide/Urea Backbone

OTHER EXPERIENCE AND PROFESSIONAL MEMBERSHIPS : > Co-founder of ImmuPharma France http://www.immupharma.com/index.html (a small drug discovery and development company focused on developing novel, largely peptide, medicines to treat serious medical conditions). > President of the « Groupe Francais des Peptides et des Proteines » (GFPP) (2007-2009) > Organizer of the 16th GFPP Meeting (May 10-15th 2009, Albe, France). http://gfpp.free.fr/ > Management Committee Substitute Member of COST action CM0803:Functional peptidomimetic foldamers (end date 2012)

Soybean phosphatidylcholine vesicles containing plant sterols : a fluorescence anisotropy study

The typical plant sterols (sitosterol, stigmasterol and campesterol) were compared with respect to their ability to regulate membrane fluidity of soybean phosphatidylcholine (PC) vesicles. Fluidity changes were monitored by the steady-state fluorescence anisotropy with 1,6-diphenyl-1,3,5-hexatriene as a probe and assigned to a measure of the acyl chain orientational order. Sitosterol and campesterol appear to be the most suitable sterols in ordering the acyl chains of soybean lecithin bilayers, even more efficient than cholesterol, the standard of reference for sterol effects on membranes, suggesting that they play a significant role in the regulation of plant membrane properties. Stigmasterol is shown to be much less active. Cycloartenol, a biosynthetic precursor of plant sterols, increases the acyl chain order with the same efficiency as cholesterol. We also investigated the effects of two unusual sterols, 24-methylpollinastanol and 14 alpha,24-dimethylcholest-8-en-3 beta-ol, which were shown to accumulate in plants treated with fungicides belonging to two important classes, N-substituted morpholines and triazoles, respectively. These two sterols exhibit a behavior very similar to that of stigmasterol. The results are discussed in terms of sterol effects on the molecular packing of soybean PC bilayers.

Liquid Ordered and Gel Phases of Lipid Bilayers: Fluorescent Probes Reveal Close Fluidity but Different Hydration

Hydration and fluidity of lipid bilayers in different phase states were studied using fluorescent probes selectively located at the interface. The probe of hydration was a recently developed 3-hydroxyflavone derivative, which is highly sensitive to the environment, whereas the probe of fluidity was the diphenylhexatriene derivative, 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene. By variation of the cholesterol content and temperature in large unilamellar vesicles composed of sphingomyelin or dipalmitoylphosphatidlycholine, we generated different phases: gel, liquid ordered (raft), liquid crystalline, and liquid disordered (considered as liquid crystalline phase with cholesterol). For these four phases, the hydration increases in the following order: liquid ordered << gel approximately liquid disordered < liquid crystalline. The membrane fluidity shows a somewhat different trend, namely liquid ordered approximately gel < liquid disordered < liquid crystalline. Thus, gel and liquid ordered phases exhibit similar fluidity, whereas the last phase is significantly less hydrated. We expect that cholesterol due to its specific H-bonding interactions with lipids and its ability to fill the voids in lipid bilayers expels efficiently water molecules from the highly ordered gel phase to form the liquid ordered phase. In this study, the liquid ordered (raft) and gel phases are for the first time clearly distinguished by their strong difference in hydration.

Gene transfer by cationic surfactants is essentially limited by the trapping of the surfactant/DNA complexes onto the cell membrane: a fluorescence investigation

The interaction between complexes of plasmid DNA with cetyltrimethylammonium bromide (CTAB) and L929 fibroblasts was first examined using confocal microscopy. The complexes labeled with the DNA intercalator, YOYO-1, were found to be trapped onto the external face of the plasma membrane; a feature that may constitute a major limiting step in transfection. Moreover, since no cytotoxic effect appeared in these conditions, we further inferred that the CTAB molecules remained bound to the DNA. The interaction of the complexes with the membranes was best modeled with neutral vesicles. From anisotropy thermotropic curves of DPHpPC-labeled vesicles and fluorescence resonance energy transfer measurements between these vesicles and YOYO-labeled complexes, we evidenced that the binding of the complexes to the vesicle surface opened the micelle-like domains and unwound DNA. However, DNA was not released but remained stably bound via electrostatic interactions to the CTAB molecules incorporated in the external liposome leaflet. Consequently, the large diameter of the unwound plasmid DNA is likely the major factor that precludes its internalization into the cells by endocytosis. In contrast, anionic vesicles that mimic the cytoplasmic facing monolayer of the plasma membrane rapidly released DNA from the complex. This may explain the previously reported high transfection efficiency of DNA complexed with liposomes composed of neutral lipids and cationic surfactants, since the latter may destabilize the endosomal membrane and induce the release of DNA in the cytoplasm.