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 · 10−7 M–2.5 · 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.

TMA-DPH (1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene), a hydrophobic fluorescent membrane probe, interacts with living cells by instantaneous incorporation into the plasma membrane, where it becomes fluorescent. It then follows the intracellular constitutive membrane traffic and acts as a bulk membrane marker of the endocytic pathway (Illinger, D., P. Poindron, P. Fonteneau, M. Modolell, and J. G. Kuhry. 1990. Biochim. Biophys. Acta. 1030:73-81; Illinger, D., P. Poindron, and J. G. Kuhry. 1991. Biol. Cell. 73:131-138). As such, TMA-DPH displays particular properties mainly due to partition between membranes and aqueous media. From these properties, original arguments can be inferred in favor of the maturation model for the endocytic pathway, against that of pre-existing compartments, in L929 cultured mouse fibroblasts. (a) TMA-DPH labeling is seen to progress from the cell periphery to perinuclear regions during endocytosis without any noticeable loss in fluorescence intensity; with a vesicle shuttle model this evolution would be accompanied by probe dilution with a decrease in the overall intracellular fluorescence intensity, and the labeling of the inner (late) compartments could in no way become more intense than that of the peripheral (early) ones. (b) From TMA-DPH fluorescence anisotropy assays, it is concluded that membrane fluidity is the same in the successive endocytic compartments as in the plasma membrane, which probably denotes a similar phospholipidic membrane composition, as might be expected in the maturation model. (c) TMA-DPH internalization and release kinetics are more easily described with the maturation model.

The aim of this study was to examine whether or not membrane fluidity directly influences infection by enveloped viruses, and, more precisely here, the susceptibility of A/J mouse hepatocytes to Mouse Hepatitis Virus type 3 (MHV3). We therefore studied, in parallel, the effects on hepatocyte membrane fluidity and on intracellular viral titre of two treatments, i) a hypercholesterolaemic diet to increase the hepatocyte membrane cholesterol content, ii) direct phosphatidylserine incorporation into hepatocyte membrane. Membrane fluidity was monitored on isolated hepatocytes by fluorescence anisotropy with TMA-DPH, and the viral titre was determined by plaque assay. The results clearly demonstrate that membrane fluidity is not directly involved in viral infection mechanisms.

1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), a membrane fluorescence probe, interacts with living cells by instantaneous partition between the external medium and the plasma membrane, where it becomes fluorescent. The corresponding fluorescence intensity is then proportional to the cell surface. On the other hand, once incorporated into the plasma membrane, TMA-DPH follows this membrane in the constitutive intracellular traffic and behaves as a monitor for endocytosis. Using this tool on L929 synchronized cells, we showed that the endocytosis levels after 30 min uptake of the probe increased from G1 to mitosis, when they abruptly decreased. The cell surface remained constant throughout the cell cycle, except at the beginning of mitosis when it almost doubled.

Biology of the CellVolume 76, Issue 2 p. 214-214 Fluid phase endocytosis and cell cycle: A study with the fluorescent probe TMA-DPH Illinger Dominique, Illinger Dominique Laboratoire de Biophysique, URA 491 du CNRSSearch for more papers by this authorItaliano Lilliane, Italiano Lilliane Laboratoire de Biologie Cellulaire, Faculté de Pharmacie de Strasbourg et Université Louis Pasteur, B.P. 24, 67401 ILLKIRCH CEDEXSearch for more papers by this authorBeck Jean-Paul, Beck Jean-Paul Laboratoire de Biologie Cellulaire, Faculté de Pharmacie de Strasbourg et Université Louis Pasteur, B.P. 24, 67401 ILLKIRCH CEDEXSearch for more papers by this authorKuhry Jean-Georges, Kuhry Jean-Georges Laboratoire de Biophysique, URA 491 du CNRSSearch for more papers by this author Illinger Dominique, Illinger Dominique Laboratoire de Biophysique, URA 491 du CNRSSearch for more papers by this authorItaliano Lilliane, Italiano Lilliane Laboratoire de Biologie Cellulaire, Faculté de Pharmacie de Strasbourg et Université Louis Pasteur, B.P. 24, 67401 ILLKIRCH CEDEXSearch for more papers by this authorBeck Jean-Paul, Beck Jean-Paul Laboratoire de Biologie Cellulaire, Faculté de Pharmacie de Strasbourg et Université Louis Pasteur, B.P. 24, 67401 ILLKIRCH CEDEXSearch for more papers by this authorKuhry Jean-Georges, Kuhry Jean-Georges Laboratoire de Biophysique, URA 491 du CNRSSearch for more papers by this author First published: 1992 https://doi.org/10.1016/0248-4900(92)90228-SAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume76, Issue21992Pages 214-214 RelatedInformation

The fluorescent hydrophobic plasma membrane probe, trimethylamino-diphenylhexatriene (TMA-DPH) was previously shown to follow the plasma membrane throughout its internalization and recycling process and thus to behave as a marker for endo- and exocytosis in living cell systems. In this paper, we made use of these properties to investigate membrane fluidity effects associated with endocytosis in L929 cells. For that purpose we performed TMA-DPH fluorescence anisotrophy measurements which showed that endocytosis starts from particularly rigid regions of the plasma membrane (probably coated pits). The fluorescence anisotropy then continuously decreases to a lower limit corresponding to the membrane fluidity of the probe in the lysosomial membrane. Strikingly, the value of this limit is identical to the average anisotropy value in the peripheral membrane, which suggests that lysosomes and plasma membrane may have a similar phospholipidic composition and a possible common origin.

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.

The lipophilic fluorescent probe trimethylaminodiphenylhexatriene (TMA-DPH), previously used as a plasma membrane marker in membrane fluidity and exocytosis studies, was shown, to monitor the plasma-membrane internalization-recycling shuttle movement in cells. Using this approach we present here kinetic and dose-response data, which give evidence that the plasma membrane flow is enhanced in bone marrow macrophages from various mouse strains, upon in vitro activation with gamma interferon (IFN-gamma) or bacterial lipopolysaccharide (LPS), within physiological dose ranges. The effect studied evolved in line with the usual development kinetics of macrophage activation. Complementary assays on membrane fluidity, surface charge density and membrane surface indicated no related changes. From these experiments it is concluded that the observed enhancement of the plasma membrane traffic does not originate from specific limited membrane modifications, but is merely a particular feature of the overall macrophage activation.

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 fluorscence-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.

Trimethylammonium-diphenylhexatriene (TMA-DPH), a hydrophobic fluorescent probe, has been shown in earlier studies to possess a variety of particular properties in interaction with intact living cells--specific and rapid incorporation into the plasma membrane and partition equilibrium between the membranes and the buffer. These properties offer promising applications in membrane fluidity studies and in monitoring exocytosis kinetics. Furthermore, these properties offer a method described here for quantitative monitoring of phagocytosis kinetics, by means of simple fluorescence intensity measurements. This method is original in that it evaluates only the particles which have actually been internalized by phagocytosis, and not those adsorbed on the cell surface, and that it gives quantitative information on the amount of plasma membrane involved in the process. It has been tested on mouse bone marrow macrophages.

Murine resident macrophages can proliferate in vitro when they are grown in coculture on a layer of mesothelial or endothelial type feeder cells. Resident macrophages were obtained from lung explants of C57Bl/6 lpr/lpr mice and from spleen explants or peritoneal washing of Balb/c mice; the cells were seeded without further washing. After 3-4 weeks of culture, the macrophages began to proliferate on a confluent layer of feeder cells. The macrophages then could be collected in the fluid phase and reseeded for permanent culture after generation of a new feeder layer. These cells were characterized as macrophages by the following criteria: 1) their morphology, ultrastructure, and adherence properties; 2) more than 90% of the macrophages phagocytized yeasts compared with less than 1% of the feeder cells; 3) the presence of functional Fc and mannose receptors, nonspecific cytoplasmic esterases, and membrane ectoenzymes such as nicotinamide adenine dinucleotide (NAD) glycohydrolase and nucleotide pyrophosphatase; 4) by cytofluorographic phenotype analysis with monoclonal antibodies, characterizing a normal macrophage population (MAC1+, Fcrec+, H-2K+, THY1-, LYT2-, L3T4-). 5) by functional studies proving that the expanded macrophages could function as accessory cells in the induction of lymphocyte proliferation in response to concanavalin A (Con A), that they generated reactive oxygen radicals and that they were cytotoxic for tumor cells. During coculture, growth or activating factors such as macrophage colony-stimulating factor or gamma-interferon were released in the medium. Long-term cultured macrophages had chromosomal abnormalities. Our study suggests that tissue macrophages can proliferate in vitro and hence that it is possible to establish long-term cultured cell lines of macrophages of defined and reproducible characteristics.

The classical types of generalized autoimmune disease in man are systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Several murine strains which develop SLE and sometimes RA-like diseases are now available. They should help in the understanding of the etiopathology of SLE and RA. Basically two main therapeutic strategies which use solely irradiation have been tried; one being sublethal whole-body irradiation (WBI) and the other fractionated total lymphoid irradiation (TLI). Other protocols which combine lethal WBI and stem cell transplantation have often been attempted. It was regularly found that the bone marrow transplant (BMT) dictates the immune status of the recipient. This paper reviews the data published about NZB, NZB/W, BXSB and MRL mice in this context.

Une fraction préparée par précipitation éthanolique de plasma humain a été utilisée avec succès à la place du sérum total ou d'autres fractions, pour produire de l'interféron humain à partir de leucocytes spléniques. Elle est pratiquement exempte d'immunoglobulines, apyrogène, atoxique pour la souris, et donne de meilleurs rendements en interféron que le sérum précipité par le sulfate d'ammonium.

This chapter presents some results obtained on in vivo and in vitro interferon (IF) induction in the rat. Furthermore, the physicochemical characteristics and biological properties of rat IF are described. The production of rat IF in vivo presents some difficulties, when compared to those of mouse or rabbit IFs. This might indicate that (a) the virus inducer is rapidly destroyed or inactivated by the reticuloendothelial cells, (b) the rat is a poor producer of IF, or (c) native rat IF is labile (acid labile) or rapidly metabolized and excreted. In vivo, the spleen is by far the best source of IF. Sindbis virus is a convenient inducer provided it is used in large amount. The production of rat IF in cell culture is less difficult. In vitro, Newcastle disease virus (NDV) is a very convenient inducer at a multiplicity of 100 to 500 EID50. Rat IF has the physicochemical and biological properties of other mammalian IFs. It resembles in some respects mouse IF. It can be used to investigate some new properties of IF, like the modification of, or the interactions with, differentiated cell functions.

Rat interferon (IF) induced a moderate and persistent elevation of adenosine 3',5'-cyclic monophosphate (cAMP) after 6 h of contact with rat embryo fibroblasts (REFs), whereas in rat glial tumoral c6 cells IF did not enhance cAMP. Noradrenaline potentiated the antiviral state of REFs treated for 2 h or more with IF and increased cAMP in these cells, but it did not potentiate the IF-induced antiviral state of C6 cells. However, as the control cells, IF-treated C6 cells responded to catecholamines by a 100-fold or more increase in cAMP. This suggested that cAMP was not primarily involved in the establishment of the antiviral state.

FEBS LettersVolume 64, Issue 2 p. 391-395 Full-length articleFree Access Rat interferon inhibits steroid-inducible glycerol 3-phosphate dehydrogenase synthesis in a rat glial cell line Dominique Illinger, Dominique Illinger Université Louis Pasteur, U.E.R. des Sciences pharmaceutiques, Laboratoire de Virologie, 3 rue de l'Argonne, 67083 Strasbourg Cedex, FranceSearch for more papers by this authorGiliane Coupin, Giliane Coupin Université Louis Pasteur, U.E.R. des Sciences pharmaceutiques, Laboratoire de Virologie, 3 rue de l'Argonne, 67083 Strasbourg Cedex, FranceSearch for more papers by this authorMary Richards, Mary Richards Université Louis Pasteur, U.E.R. des Sciences pharmaceutiques, Laboratoire de Virologie, 3 rue de l'Argonne, 67083 Strasbourg Cedex, FranceSearch for more papers by this authorPhilippe Poindron, Corresponding Author Philippe Poindron Université Louis Pasteur, U.E.R. des Sciences pharmaceutiques, Laboratoire de Virologie, 3 rue de l'Argonne, 67083 Strasbourg Cedex, FranceFrom whom reprints should be requested.Search for more papers by this author Dominique Illinger, Dominique Illinger Université Louis Pasteur, U.E.R. des Sciences pharmaceutiques, Laboratoire de Virologie, 3 rue de l'Argonne, 67083 Strasbourg Cedex, FranceSearch for more papers by this authorGiliane Coupin, Giliane Coupin Université Louis Pasteur, U.E.R. des Sciences pharmaceutiques, Laboratoire de Virologie, 3 rue de l'Argonne, 67083 Strasbourg Cedex, FranceSearch for more papers by this authorMary Richards, Mary Richards Université Louis Pasteur, U.E.R. des Sciences pharmaceutiques, Laboratoire de Virologie, 3 rue de l'Argonne, 67083 Strasbourg Cedex, FranceSearch for more papers by this authorPhilippe Poindron, Corresponding Author Philippe Poindron Université Louis Pasteur, U.E.R. des Sciences pharmaceutiques, Laboratoire de Virologie, 3 rue de l'Argonne, 67083 Strasbourg Cedex, FranceFrom whom reprints should be requested.Search for more papers by this author First published: May 01, 1976 https://doi.org/10.1016/0014-5793(76)80334-1Citations: 17AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat References 1 C.E. Samuel, W.K. Joklik, Virology, 58, (1974), 476– 491. 2 E. Falcoff, R. Falcoff, B. Lebleu, M. Revel, J. Virol., 12, (1973), 421– 430. 3 S.L. Gupta, W.D. Graziadei III, H. Weideli, M.L. Sopori, P. Lengyel, Virology, 57, (1974), 49– 63. 4 G. Beck, P. Poindron, D. Illinger, J.P. Beck, J.P. Ebel, R. Falcoff, FEBS Lett., 48, (1974), 297– 300. 5 G. Beck, J.P. Beck, Tomkins, Colloque INSERM, Synthèse normale et pathologique des protéines chez les animaux supérieurs (1973), INSERM Paris 29– 44. 6 P. Benda, J. Lightbody, G. Sato, L. Levine, W. Sweet, Science, 161, (1968), 370– 371. 7 Vellis J. De, D. Inglish, R. Cole, J. Molson, Influence of hormones on the nervous system. D.H. Ford Proceedings of the International Society of Psychoneuroendocrinology (1970), Karger Basel 25– 39. 8 A. German, A.M. Quero, P. Poindron, Ann. Inst. Pasteur, 121, (1971), 207– 221. 9 A. Billau, C.E. Buckler, Symposia series in immunobiological standardization. International Symposium on Interferon and Interferon inducers, London 1969 (1970), Karger Basel and New York 37– 44. no. 14 10 Ginnis J.F. Mc, Vellis J. De, Biochim. Biophys. Acta, 364, (1974), 17– 27. 11 O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem., 193, (1951), 265– 275. 12 R.L. Davidson, P. Benda, Proc. Natl. Acad. Sci. US, 67, (1970), 1870– 1877. 13 I. Gresser, D. Brouty-Boye, M.T. Thomas, Coelho A. Macieira, Proc. Natl. Acad. Sci. US, 66, (1970), 1052– 1058. 14 Magnusson P. Lindahl, P. Leary, I. Gresser, Proc. Soc. Exp. Biol. Med., 138, (1971), 1044– 1050. 15 Ginnis J.F. Mc, Vellis J. De, Nature, 250, (1974), 422– 424. 16 T.P. Fondy, L. Lesin, S.J. Sollohue, G.R. Ross, J. Biol. Chem., 243, (1968), 3148– 3160. Citing Literature Volume64, Issue2May 01, 1976Pages 391-395 ReferencesRelatedInformation