No abstract

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.