Jean-Georges Kuhry

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.

Role of endocytosis in the transfection of L929 fibroblasts by polyethylenimine/DNA complexes

Polyethylenimine (PEI) is one of the most efficient nonviral vectors for gene therapy. The aim of this study was to investigate the role of endocytosis in the transfection of synchronized L929 fibroblasts by PEI/DNA complexes. This was performed by confocal microscopy and flow cytometry, using the endocytosis marker FM4-64 and PEI/DNA complexes labeled either with the DNA intercalator YOYO-1, or with fluorescein covalently linked to PEI. Endocytosis appeared as the major if not the sole mode of entry of the PEI/DNA complexes into the L929 cells. The complexes followed a typical fluid phase endocytosis pathway and were efficiently taken up in less than 10 min in endosomes that did not exceed 200 nm in diameter. Later, the localization of the complexes became perinuclear and fusion between late endosomes was shown to occur. Comparison with the intracellular trafficking of the same complexes in EA.hy 926 cells (W.T. Godbey, K. Wu, A.G. Mikos, Proc. Natl. Acad. Sci. USA 96 (1999)) revealed that endocytosis of PEI/DNA complexes is strongly cell-dependent. In L929 cells, escape of the complexes from the endosomes is a major barrier for transfection. This limited the number of transfected cells to a few percent, even though an internalization of PEI/DNA complexes was observed in most cells. In addition, the entry of the complexes into the nucleus apparently required a mitosis and did not involve the lipids of the endosome membrane. This entry seems to be a short-lived event that involves only a few complexes.

A comparison of the fluorescence properties of TMA-DPH as a probe for plasma membrane and for endocytic membrane

In earlier studies, the fluorescence probe 1-(4-(trimethylamino)phenyl)-6-phenylhexa-1,3,5-triene (TMA-DPH) was shown to interact with living cells by instantaneous incorporation into the plasma membrane, according to a water (probe not fluorescent)/membrane (probe highly fluorescent) partition equilibrium. This made it interesting both as a fluorescence anisotropy probe for plasma membrane fluidity determinations and as a quantitative tracer for endocytosis and intracellular membrane traffic. In order to ascertain the limiting concentrations for its use in these applications, we performed a systematic study of its fluorescence properties (intensity, lifetime, anisotropy) in the plasma membrane and in endocytic membranes of intact L929 mouse fibroblasts. Some of the experiments were repeated on mouse-bone-marrow-derived macrophages and on phospholipidic LUV to confirm the results. Rather unexpectedly, it was observed that: (i) the incorporation of TMA-DPH into the membranes, monitored by UV absorption measurements, remained proportional to the probe concentration over the wide range explored (5 x 10(-7) M-2.5 x 10(-5) M); (ii) however, concerning fluorescence, quenching effects occurred in the membranes above certain critical concentrations. These effects were shown to result from Förster-type resonance auto-transfer; (iii) strikingly, the critical concentrations were considerably higher in early-endocytic-vesicle membranes than in the bulk plasma membrane. It was established that membrane fluidity was involved and this was confirmed by the parallel study on phospholipidic vesicles. Potential applications of these properties as a novel approach for evaluating membrane fluidity are suggested.

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.

Internalization of the lipophilic fluorescent probe trimethylamino-diphenylhexatriene follows the endocytosis and recycling of the plasma membrane in cells

The lipophilic fluorescent probe trimethylamino-diphenylhexatriene (TMA-DPH) has been shown previously to behave as a marker of plasma membrane in living cell systems, and it has therefore been widely used in membrane fluidity studies via fluorescence anisotropy measurements. However, progressive internalization of this probe in cells could lead to unsuitable interferences, when long incubations times were required. The mechanism of this internalization had not yet been elucidated. We present here fluorescence-intensity kinetic results and fluorescence micrographic data on L929 cells and on mouse bone-marrow macrophages, which allow us to identify the mechanism as fluid-phase pinocytosis: the probe remains associated with the plasma membrane throughout its internalization-recycling flow and it is finally concentrated in lysosomes. The study was facilitated by the partition equilibrium property of TMA-DPH between plasma membranes and the external aqueous medium, which allowed to immediately distinguish the internalized fraction of the probe from the peripheral labelling, by simply washing cells. This conclusion is confirmed by the features of the influence of temperature on TMA-DPH internalization.

Fluid phase endocytosis investigated by fluorescence with trimethylamino-diphenylhexatriene in L929 cells; the influence of temperature and of cytoskeleton depolymerizing drugs

Summry— The temperature‐dependence of fluid phase endocytosis was investigated in L929 cells, using a recently described fluorescence approach with trimethylamino‐diphenylhexatriene (TMA‐DPH) [7, 9]. In interaction with cells, this probe is rapidly incorporated into the plasma membrane and follows its intracellular traffic of internalization‐recycling, thus behaving as a suitable marker for fluid phase endocytosis. The kinetics of the process may be followed accurately by simple fluorescence intensity measurements, while complementary fluorescence anistropy and micrographic data may be obtained in parallel with the same probe. It was shown that the formation of endocytic vesicles was not inhibited by cooling the cells, even down to 4°C, but only reduced in a quasi‐linear way with temperature. Conversely the further fusion events between the vesicle and large vacuular bodies (endosomes, lysosomes) were strongly and discontinuously influenced: they were almost totally suppressed below 15°C. The evolution of the membrane fluidity during endocytosis, which was monitored by fluorescence anisotropy measurements, indicated that the fusion inhibition was probably correlated with the inability of the endocytic vesicles to shed their initial clathrin coat at low temperature. Moreover, microscopic observations showed that at low temperature the endocytic vesicles hardly moved from the place of their formation. Pretreatment of the cells with microtubule and microfilament depolymerizing drugs (cytochalasin B, vinblastin) led to the conclusion that the cytoskeleton played little role in the vesicle movements. Altogether, the results suggested that the progression of the vesicles towards the cell core resulted from successive fusion events, which explained why they were considerably showed down by cooling.

Interactions of the monomeric and dimeric flavones apigenin and amentoflavone with the plasma membrane of L929 cells; a fluorescence study.

Flavonoids are ubiquitous polyphenolic compounds, found in vascular plants, which are endowed with a large variety of biological effects. Some of these effects have been assumed to result from interactions with the cell plasma membrane. In order to investigate the nature of these interactions a fluorescence study was performed with two flavonoids, currently used in one of the laboratories: apigenin and its homologous dimer amentoflavone. After preliminary assays with DPH in several types of phospholipid liposomes, the effects of these flavonoids on the membrane of mouse L929 fibroblasts were compared, using the non-permeant probe TMA-DPH. Amentoflavone, unlike apigenin, induced a static quenching effect, which denoted an important, but reversible, association of the molecule with the plasma membrane. In addition, amentoflavone treatment induced a dose-dependent increase in TMA-DPH fluorescence anisotropy, which could be interpreted as an increase in membrane lipidic order. For apigenin, the effect was much less important. Moreover, exploiting the capacity of TMA-DPH to label endocytic compartments, it was shown that, after association with the membrane, amentoflavone is not internalized into the cell. Possible correlations of these membrane effects with other biological properties are discussed.

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.

The influence of microtubule integrity on plasma membrane fluidity in L929 cells

The aim of this work was to examine the possible influence of the integrity of the microtubule network on the plasma membrane fluidity of L929 mouse fibroblasts. The L929 cell line was selected for the ease of culture and the stability of its characteristics. The cells were treated with colchicine, nocodazole and vinblastine, three microtubule-depolymerizing drugs, at various concentrations and for various times. Membrane fluidity was assessed from fluorescence depolarization measurements with the plasma membrane probe TMA-DPH. Each of the drugs induced a significant, dose-dependent decrease in fluorescence anisotropy. The effect levelled off (5-7% decrease) after approximately 90 min of treatment, and could be unambiguously interpreted as resulting from an increase in membrane fluidity. The cumulative action of the drugs did not significantly increase the effect. The effects of colchicine and nocodazole could be reversed by incubation in drug-free medium, but not that of vinblastine. The results are discussed in correlation with the kinetics of the three drugs interaction with tubulin or microtubules. It is concluded that the microtubule integrity contributed to the high plasma membrane lipidic order, but less than other factors, like the lipid composition and the cholesterol content.The aim of this work was to examine the possible influence of the integrity of the microtubule network on the plasma membrane fluidity of L929 mouse fibroblasts. The L929 cell line was selected for the ease of culture and the stability of its characteristics. The cells were treated with colchicine, nocodazole and vinblastine, three microtubule-depolymerizing drugs, at various concentrations and for various times. Membrane fluidity was assessed from fluorescence depolarization measurements with the plasma membrane probe TMA-DPH. Each of the drugs induced a significant, dose-dependent decrease in fluorescence anisotropy. The effect levelled off (5-7% decrease) after ~ 90 min of treatment, and could be unambiguously interpreted as resulting from an increase in membrane fluidity. The cumulative action of the drugs did not significantly increase the effect. The effects of colchicine and nocodazole could be reversed by incubation in drug-free medium, but not that of vinblastine. The results are discussed in correlation with the kinetics of the three drugs interaction with tubulin or microtubules. It is concluded that the microtubule integrity contributed to the high plasma membrane lipidic order, but less than other factors, like the lipid composition and the cholesterol content.