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.

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.

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.

Cell surface membrane homeostasis and intracellular membrane traffic balance in mouse L929 cells.

We have developed a simple method for synchronizing L929 mouse fibroblasts. Cultured as monolayers, these cells stop growing at confluency and arrest at the end of the G1 phase. Upon seeding at low density, they enter the S phase simultaneously. Using these cells we then looked at the evolution of the surface membrane area during the cell cycle using the fluorescence membrane probe TMA-DPH. In contact with cells, this probe partitions between the membrane (probe fluorescent) and the external medium (non-fluorescent), delivering a signal proportional to the membrane area. This area was constant until just before mitosis, when it increased at once. With the same probe as an endocytic marker, we examined how this membrane homeostasis could be consistent with intracellular membrane trafficking. The study was limited to one selected period of the cell cycle (6-9 hours). We observed that 14% of the membrane endocytosed was not recycled, but was replaced at the cell surface by newly formed membrane from biosynthetic pathways. Brefeldin A modified the membrane traffic, but not the overall membrane homeostasis. The results are discussed in the framework of a maturation model.

Differentiation between clathrin-dependent and clathrin-independent endocytosis by means of membrane fluidity measurements.

The fluorescence probe [1-(4-trimethylammonium]-6-phenyl-1,3,5-hexatriene (TMA-DPH) displays properties relevant for both monitoring endocytosis kinetics and assessing membrane fluidity by fluorescence-anisotropy measurements (1). Thus, it is, possible with this probe to follow the evolution of membrane fluidity during endocytosis, from the very beginning of the process, i.e., the formation of endocytic vesicles. In most cases, endocytosis is known to start with clathrin-coated vesicles. Still, there are more and more arguments in favor of a complementary endocytic pathway without clathrin. In this article we present membrane-fluidity data for very early endocytosis, which allow an upper limit to be determined for the contribution of a putative nonclathrin pathway. We show that this limit is markedly higher for bone marrow-derived macrophages than for mouse fibroblasts of the L929 cell line.

CFTR is involved in membrane endocytosis but not in fluid-phase and receptor-mediated endocytosis in human respiratory epithelial cells.

Cystic fibrosis transmembrane conductance regulator (CFTR) protein has been reported to be a cAMP-regulator of plasma membrane recycling in epithelial cells overexpressing CFTR. To assess its role in the different endocytic processes in human respiratory epithelial cells, the rates of internalization of membrane, fluid-phase and receptor-mediator tracers were compared, under control conditions and after treatment with the cAMP agonist forskolin in normal and cystic fibrosis (CF) cells. In both control and treated-cells, CFTR was only present in the plasma membrane of normal but not in CF cells. Similarly, activation of Cl- efflux only occurred in normal and not in CF-treated cells. The rate of membrane endocytosis was significantly decreased by 35% in normal treated-cells, whereas it was not significantly decreased (12%) in CF-treated cells. Upon forskolin treatment, the decrease of the rate of both fluid-phase and receptor-mediated endocytosis was not significantly different between normal and CF cells. These results demonstrate that CFTR is involved in membrane endocytosis but not in fluid-phase and receptor-mediated endocytosis in human respiratory epithelial cells.

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.

Contrary results on mouse hepatitis virus type 3 susceptibility in A/J mouse hepatocytes of phosphatidylserine treatment and of a hypercholesterolaemic diet: no correlation with membrane fluidity levels.

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.

Influence of the clathrin coat on the membrane lipidic organization of endocytic vesicles: a fluorescence study.

Endocytic coated vesicles (CV) were purified from bovine brain, and uncoated vesicles (UV) were obtained from the latter by dialysis against 1 M Tris. Membrane dynamics were explored in both vesicle populations using two complementary fluorescence approaches: diphenylhexatriene fluorescence anisotropy to account for rotational lipid movements, and pyrene excimerization with a phosphatidylcholine pyrene derivative for translational motion. It was concluded that membrane fluidity was considerably higher in UV than in CV, and that adding bulk coat proteins (adaptors+clathrin) to uncoated vesicles re-established the low fluidity found in coated vesicles. However, adding coat protein constituents separately had no effect.

The kinetic aspects of intracellular fluorescence labeling with TMA-DPH support the maturation model for endocytosis in L929 cells.

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.

Effects of Clostridium difficile toxin B on human monocytes and macrophages: possible relationship with cytoskeletal rearrangement.

Toxin B from Clostridium difficile is cytopathic in vitro for various types of cells, including polymorphonuclear cells, lymphocytes, and monocytes. Since intestine lamina propria is rich in macrophages, we studied the effect of toxin B on human monocytes and on human macrophages generated in vitro by long-term culture of purified circulating blood monocytes. Upon addition of toxin B, human monocytes exhibited few modifications whereas macrophages adopted a stellate morphology, with rounding up of the perikaryon. Toxin B made microfilaments of actin disappear and induced an important reorganization of vimentin and a redistribution of tubulin. Membrane area increased by approximately 16%. Toxin B did not affect the viability of human mononuclear phagocytes and did not exert any significant lytic effect. It profoundly altered the phagocytic function of macrophages. When activated by gamma interferon in the presence of toxin B, monocytes were more cytotoxic for U-937 target cells than control monocytes activated in absence of toxin. Finally, the combined treatment of monocytes with gamma interferon and toxin B increased significantly the secretion of tumor necrosis factor alpha, whereas toxin B alone was unable to induce tumor necrosis factor production. These results suggest that morphological and functional alterations induced in human mononuclear phagocytes by toxin B from C. difficile are due to the disorganization of the cytoskeleton and the resulting impairment of the membrane traffic equilibrium.

Comparative evolution of endocytosis levels and of the cell surface area during the L929 cell cycle: a fluorescence study with TMA-DPH.

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.

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

The temperature-dependence of fluid phase endocytosis was investigated in L929 cells, using a recently described fluorescence approach with trimethylamino-diphenylhexatriene (TMA-DPH). 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 anisotropy 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 degrees C, but only reduced in a quasi-linear way with temperature. Conversely the further fusion events between the vesicles and large vacuolar bodies (endosomes, lysosomes) were strongly and discontinuously influenced: they were almost totally suppressed below 15 degrees 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, vinblastine) 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 slowed down by cooling.

Membrane fluidity aspects in endocytosis; a study with the fluorescent probe trimethylamino-diphenylhexatriene in L929 cells.

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.

The plasma-membrane internalization and recycling is enhanced in macrophages upon activation with gamma-interferon and lipopolysaccharide; a study using the fluorescent probe trimethylaminodiphenylhexatriene.

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.

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.

TMA-DPH a fluorescent probe of membrane dynamics in living cells. How to use it in phagocytosis.

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.

Effects of random copolymers of lysine on the thermotropic behaviour of dipalmitoylphosphatidylglycerol vesicles. A fluorescence anisotropy study.

The effects of some random copolymers of lysine on the thermotropic behaviour of phospholipid vesicles, mainly dipalmitoylphosphatidylglycerol (DPPG), were studied by monitoring the steady-state fluorescence anisotropy with 1,6-diphenyl-1,3,5-hexatriene (DPH) as a membrane probe. A characteristic effect of the polylysine-tyrosine 4:1 copolymer was observed: DPPG vesicles are strongly stabilized, the gel-to-fluid transition going from 40 degrees C in the absence to 52 degrees C with an excess of copolymer (corresponding to a lipid/amino-acid ratio R = 0.5). For R greater than 0.5, the gel-to-fluid transition shows a three-step profile. This triphasic transition, which appears with a much better resolution than with polylysine, demonstrates the formation of stable distinct domains of reasonable size, coexisting from 41 to 51 degrees C. Such stability, over a temperature range of 10 degrees C, is quite unusual and unexpected for a phase separation of mixed lipid phases. The peculiarity of this copolymer was proved by investigating the interactions of DPPG vesicles with polylysine-tryptophan 4:1, polyornithine-lysine 4:1 and polylysine-tyrosine 1:1. We hypothesize that the observed effects are correlated with the ability of these copolymers to adopt/not adopt an alpha-helical or beta-sheet conformation upon interacting with anionic vesicles.

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.

A fluorescent hydrophobic probe used for monitoring the kinetics of exocytosis phenomena.

A fluorescence method is presented for quantitatively analyzing exocytosis phenomena and monitoring their kinetics. The method is based on the particular properties of a hydrophobic fluorescent probe, 1-[4-(trimethylammonio)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) [Prendergast, F.G., Haugland, R.P., & Callahan, P.J. (1981) Biochemistry 20, 7333-7338; Kuhry, J.G., Fonteneau, P., Duportail, G., Maechling, C., & Laustriat, G. (1983) Cell Biophys. 5, 129-140; Kuhry, J.G., Duportail, G., Bronner, C., & Laustriat, G. (1985) Biochim. Biophys. Acta 845, 60-67]. When this probe is interacted with intact resting cells in aqueous suspensions, it labels solely the membranes that are in contact with the external medium and is incorporated into them according to a partition equilibrium; i.e., the amount of the probe incorporated is proportional to the available membrane surface. TMA-DPH is highly fluorescent in membranes and not at all in water. Thus, a measurement of the TMA-DPH fluorescence intensity provides a signal proportional to the membrane surface. In secretory cells, the membrane surface available for the probe is increased upon fusion of the membrane of the secretory granules with the cell plasma membranes, directly or via intergranule fusion. Thus, when these cells are stimulated, more TMA-DPH is incorporated than in resting cells since the probe is allowed to also interact with the granule membranes now connected with the external medium by pores. This process results in a proportional increase in the TMA-DPH fluorescence intensity. The response was found to be very rapid and able to follow accurately the exocytosis kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)