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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 molecular ruler: A three‐color single‐molecule fluorescence resonance energy transfer (FRET) method is presented (see Figure inset), which can be used to simultaneously measure multiple molecular distance changes during molecular conformational changes and binding. The ability to directly study conformational subpopulations in a mixture of molecules with different interdye distances is highlighted by the well‐separated peaks in the two‐ dimensional histogram in the Figure.

Background: The human immunodeficiency virus type 1 (HIV-1) encodes several regulatory proteins, notably Vpr which influences the survival of the infected cells by causing a G2/M arrest and apoptosis. Such an important role of Vpr in HIV-1 disease progression has fuelled a large number of studies, from its 3D structure to the characterization of specific cellular partners. However, no direct imaging and quantification of Vpr-Vpr interaction in living cells has yet been reported. To address this issue, eGFP-and mCherry proteins were tagged by Vpr, expressed in HeLa cells and their interaction was studied by two photon fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy.

Though polyethylenimine (PEI) is one of the most efficient nonviral vectors, one concern is the significant cytotoxicity of free PEI that represents about 80% of the PEI molecules in PEI/DNA mixtures used for transfection. In this respect, the aim of this work was to further investigate the intracellular fate of PEI during transfection of L929 fibroblasts. To this end, we analyzed by fluorescence correlation spectroscopy (FCS) using two-photon excitation the intracellular concentration and diffusion properties of labeled PEI and PEI/DNA complexes in various compartments of L929 cells. High initial fluorescence intensity, rapid photobleaching and the absence of measurable autocorrelation curves in most selected locations in cytoplasm suggest that PEI/DNA complexes and PEI accumulate (up to 30 times the concentration in the extracellular medium) in late endosomes bound to the inner membrane face. This feature, together with membrane destabilizing properties of PEI, may explain the release of PEI into cytoplasm and subsequent diffusion into the nucleus. In the nucleus, the concentration of PEI was found to be about 2.5- to 3.5-fold higher than the one in the incubation medium. Moreover, autocorrelation curves obtained in the nuclear compartment can be analyzed with either a two-component model (with the major fraction undergoing free Brownian diffusion) or an anomalous diffusion model. Both the endosomal disruption and the large intranuclear PEI concentration may contribute to PEI cytotoxicity.

Ligand binding to G protein-coupled receptors is a complex process that involves sequential receptor conformational changes, ligand translocation, and possibly ligand-induced receptor oligomerization. Binding events at muscarinic acetylcholine receptors are usually interpreted from radioligand binding studies in terms of two-step ligand-induced receptor isomerization. We report here, using a combination of fluorescence approaches, on the molecular mechanisms for Bodipypirenzepine binding to enhanced green fluorescent protein (EGFP)-fused muscarinic M1 receptors in living cells. Real time monitoring, under steady-state conditions, of the strong fluorescence energy transfer signal elicited by this interaction permitted a fine kinetic description of the binding process. Timeresolved fluorescence measurements allowed us to identify discrete EGFP lifetime species and to follow their redistribution upon ligand binding. Fluorescence correlation spectroscopy, with EGFP brightness analysis, showed that EGFP-fused muscarinic M1 receptors predominate as monomers in the absence of ligand and dimerize upon pirenzepine binding. Finally, all these experimental data could be quantitatively reconciled into a three-step mechanism, with four identified receptor conformational states. Fast ligand binding to a peripheral receptor site initiates a sequence of conformational changes that allows the ligand to access to inner regions of the protein and drives ligandreceptor complexes toward a high affinity dimeric state. G protein-coupled receptors (GPCRs)3 trigger a wide palette of signaling pathways (1, 2), including G protein-independent responses (3). These receptors display multiple conformational and functional states, dependent on the cellular context, differentially selected and stabilized by ligands, and discriminated by downstream protein partners (4 -9). The occurrence of distinct receptor conformational species is supported by structural arguments provided by metal ion site engineering (10) or in situ disulfide cross-linking (11) and by direct monitoring of receptor intramolecular rearrangements through fluorescencebased methods (for reviews see Refs. 8,9,12).Few studies focused on the initial ligand binding step, its kinetic description, and its relationship with functionally relevant receptor conformational states. These aspects were addressed by monitoring intermolecular fluorescence resonance energy transfer (FRET) between a GFP-tagged receptor (donor) and a fluorescent ligand (acceptor). Both neurokinin A binding to class A tachykinin NK2 receptors (4, 13) and parathyroid hormone binding to class B parathyroid hormone receptors (14) proceeded in two steps, featuring two kinetically distinguishable conformational states. Whether such biphasic binding reactions are a general feature of GPCRs, independent on the pharmacological nature of the ligand, and whether they reflect different receptor functional states or sequential binding steps remain important questions to be elucidated.Muscarinic cholinergic receptors (15) disp...

No abstract

The stability of the complexes between DNA and nonviral vectors is a crucial parameter for efficient gene delivery into target cells. The stability must be high enough to prevent any dissociation during interaction with the plasma membrane but low enough to allow the dissociation that is required for efficient internalization into the nucleus. In this report, we investigated the stability of complexes of DNA with two cysteine surfactants (guanidinocysteine N-decylamide, C10-C G+ , and ornithinyl-cysteinyl-tetradecylamide, C14-CO), able to convert themselves, via oxidative dimerization, into cationic cystine lipids. To this end, we determined the critical aggregation concentration (cac) and the binding constants of the surfactants for DNA by using the fluorescence quenching of the DNA bis-intercalating agent, YOYO-1, that results from the dye clustering induced by the collapse of DNA. The cac's of C 10-C G+ and C14-CO monomeric forms are 2.5 and 1 µM, respectively, and are slightly less than the 5 µM value for CTAB, taken as a model of nondimerizable surfactant. Dimerization of C10-C G+ and C14-CO reduces the cac to 400 and 1 nM, respectively. The strong stabilization induced by oxidation of C14-CO is further confirmed by the increase in the rigidity of the micellelike domains in the complexes, as deduced from the rotational correlation time of the hydrophobic probe 1,6-diphenylhexatriene. In keeping with the stability data, no dissociation of the (C 14-CO)2/DNA complexes occurs in the presence of neutral vesicles (that mimic the external leaflet of the plasma membrane), while a significant dissociation was observed with (C10-C G+ )2/DNA complexes and an even larger one with CTAB/DNA complexes. Similarly, (C14-CO)2/DNA complexes do not dissociate in the presence of anionic vesicles (that mimic the cytoplasmic leaflet of the plasma membrane), while a complete dissociation and DNA release occurs with both (C 10-C G+ )2/DNA and CTAB/DNA complexes. Both the initial interaction with the plasma membrane and the release of DNA in the cytoplasm are strongly dependent on the stability of the complexes obtained with this new class of nonviral vectors.