Direct Vpr-Vpr Interaction in Cells monitored by two Photon Fluorescence Correlation Spectroscopy and Fluorescence Lifetime Imaging

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>Results show that Vpr forms homo-oligomers at or close to the nuclear envelope. Moreover, Vpr dimers and trimers were found in the cytoplasm and in the nucleus. Point mutations in the three α helices of Vpr drastically impaired Vpr oligomerization and localization at the nuclear envelope while point mutations outside the helical regions had no effect. Theoretical structures of Vpr mutants reveal that mutations within the α-helices could perturb the leucine zipper like motifs. The ΔQ44 mutation has the most drastic effect since it likely disrupts the second helix. Finally, all Vpr point mutants caused cell apoptosis suggesting that Vpr-mediated apoptosis functions independently from Vpr oligomerization.</p> <p>Conclusion</p> <p>We report that Vpr oligomerization in HeLa cells relies on the hydrophobic core formed by the three α helices. This oligomerization is required for Vpr localization at the nuclear envelope but not for Vpr-mediated apoptosis.</p

Single-Molecule Three-Color FRET with Both Negligible Spectral Overlap and Long Observation Time

Full understanding of complex biological interactions frequently requires multi-color detection capability in doing single-molecule fluorescence resonance energy transfer (FRET) experiments. Existing single-molecule three-color FRET techniques, however, suffer from severe photobleaching of Alexa 488, or its alternative dyes, and have been limitedly used for kinetics studies. In this work, we developed a single-molecule three-color FRET technique based on the Cy3-Cy5-Cy7 dye trio, thus providing enhanced observation time and improved data quality. Because the absorption spectra of three fluorophores are well separated, real-time monitoring of three FRET efficiencies was possible by incorporating the alternating laser excitation (ALEX) technique both in confocal microscopy and in total-internal-reflection fluorescence (TIRF) microscopy

Nuclear delivery of NFκB-assisted DNA/polymer complexes: plasmid DNA quantitation by confocal laser scanning microscopy and evidence of nuclear polyplexes by FRET imaging

Quantification of a plasmid DNA (pDNA) and investigation of its polymer-associated state in the nucleus are crucial to evaluate the effectiveness of a gene-delivery system. This study was conducted with p3NF-luc-3NF, a pDNA-bearing optimized κB motif to favour NFκB-driven nuclear import. Here, a quantification of pDNA copies in the nucleus was performed by real-time confocal laser scanning microscopy in HeLa and C2C12 cells transfected with linear polyethylenimine or histidylated polylysine. Förster Resonance Energy Transfer (FRET) from the fluorescein-p3NF-luc-3NF donor to the co-localized rhodamine-polymer acceptor was carried out to investigate whether the pDNA was still condensed with the polymer in the nucleus. Upon 5 h of transfection, the nuclear amount of p3NF-luc3NF was ∼1500 copies in both cell lines whereas that of pTAL-luc, a 3NF-free counterpart pDNA, was less than 250. This quantity of p3NF-luc-3NF dropped dramatically to that of pTAL-luc in the presence of the BAY 11-7085, an inhibitor of NFκB activation. These data strongly support a nuclear import of p3NF-luc3NF mediated by NFκB. Moreover, FRET experiments clearly revealed that most of nuclear pDNA were still condensed with the polymer raising the question of their passage through the nuclear pore complex and their impact on the gene-expression efficiency

A microfluidic-FCS platform for investigation on the dissociation of Sp1-DNA complex by doxorubicin

The transcription factor (TF) Sp1 is a well-known RNA polymerase II transcription activator that binds to GC-rich recognition sites in a number of essential cellular and viral promoters. In addition, direct interference of Sp1 binding to DNA cognate sites using DNA-interacting compounds may provide promising therapies for suppression of cancer progression and viral replication. In this study, we present a rapid, sensitive and cost-effective evaluation of a GC intercalative drug, doxorubicin (DOX), in dissociating the Sp1–DNA complex using fluorescence correlation spectroscopy (FCS) in a microfluidic system. FCS allows assay miniaturization without compromising sensitivity, making it an ideal analytical method for integration of binding assays into high-throughput, microfluidic platforms. A polydimethylsiloxane (PDMS)-based microfluidic chip with a mixing network is used to achieve specific drug concentrations for drug titration experiments. Using FCS measurements, the IC(50) of DOX on the dissociation of Sp1–DNA complex is estimated to be 0.55 μM, which is comparable to that measured by the electrophoretic mobility shift assay (EMSA). However, completion of one drug titration experiment on the proposed microfluidic-FCS platform is accomplished using only picograms of protein and DNA samples and less than 1 h total assay time, demonstrating vast improvements over traditional ensemble techniques

Kinetic analysis of the nucleic acid chaperone activity of the Hepatitis C virus core protein

The multifunctional HCV core protein consists of a hydrophilic RNA interacting D1 domain and a hydrophobic D2 domain interacting with membranes and lipid droplets. The core D1 domain was found to possess nucleic acid annealing and strand transfer properties. To further understand these chaperone properties, we investigated how the D1 domain and two peptides encompassing the D1 basic clusters chaperoned the annealing of complementary canonical nucleic acids that correspond to the DNA sequences of the HIV-1 transactivation response element TAR and its complementary cTAR. The core peptides were found to augment cTAR-dTAR annealing kinetics by at least three orders of magnitude. The annealing rate was not affected by modifications of the dTAR loop but was strongly reduced by stabilization of the cTAR stem ends, suggesting that the core-directed annealing reaction is initiated through the terminal bases of cTAR and dTAR. Two kinetic pathways were identified with a fast pre-equilibrium intermediate that then slowly converts into the final extended duplex. The fast and slow pathways differed by the number of base pairs, which should be melted to nucleate the intermediates. The three peptides operate similarly, confirming that the core chaperone properties are mostly supported by its basic clusters

Enantiomeric and Diastereomeric Self-Assembled Multivalent Nanostructures : Understanding the Effects of Chirality on Binding to Polyanionic Heparin and DNA

A family of four self-assembling lipopeptides containing Ala-Lys peptides attached to a C16 aliphatic chain were synthesised. These compounds form two enantiomeric pairs that bear a diastereomeric relationship to one another (C16-l-Ala-l-Lys/C16-d-Ala-d-Lys) and (C16-d-Ala-l-Lys/C16-l-Ala-d-Lys). These diastereomeric pairs have very different critical micelle concentrations (CMCs). The self-assembled multivalent (SAMul) systems bind biological polyanions as a result of the cationic lysine groups on their surfaces. For heparin binding, there was no significant enantioselectivity, but there was a binding preference for the diastereomeric assemblies with lower CMCs. Conversely, for DNA binding, there was significant enantioselectivity for systems displaying d-lysine ligands, with a further slight preference for attachment to l-alanine, with the CMC being irrelevant

CMOS Propagation Delay Change Using Laser Die Probing

A technique has been developed to examine the propagation delay change of a CMOS inverter undergoing laser illumination. The injection current into the drain circuit of the inverter assists or opposes the discharge of a load capacitance, thereby speeding or slowing the response of the inverter. A model has been developed that uses the MOSFET large signal equations to predict the behavior of the CMOS inverter in the presence of illumination. The illumination is modeled as an equivalent current source placed across the output. SPICE simulations are conducted to verify the model above, whereas the SPICE model, and the model above, are verified by actual tests. The tests are conducted to characterize 74HCU04 inverter behavior for continuous and pulsed illumination. The pulsed illumination test used laser pulses from 10 ns to 100 ns wide positioned from 50 ns before to 50 ns after an input change to the inverter. The test data is in the form of a profile that clearly shows the propagation delay change. The SPICE model and theoretical model match well, as is expected, and both have good correspondence with the test data

Analyse quantitative des interactions moléculaires au cours de la transfection par des complexes ADN/polyéthylèneimines

Les progrès de la biologie moléculaire ont permis de concevoir l'utilisation de l'ADN en temps qu'agent thérapeutique pour le traitement de maladies génétiques héréditaires (mucoviscidose,myopathie) ou acquises (cancer). Le succès de la thérapie génique repose sur la mise au point de vecteurs capables de transférer le gène d'intérêt thérapeutique au sein de la cellule. Dans ce cadre nous avons étudié la formation, la composition et le devenir cellulaire de complexes entre l'ADN et l'un des vecteurs synthétiques de transfert de gène le plus prometteurs: la polyéthylèneimine.Dans un premier temps, les études menées par microscopie confocale ont permis d'identifier les étapes limitantes de la transfection des cellules L929 par les complexes ADN/PEI. En effet, les marquages de l'ADN par le YOYO-1, du PEI par la Rhodamine et l'utilisation d'une sonde de suivi de l'endocytose le FM4-64, ont permis de montrer qu'après internalisation par endocytose en phase fluide, les complexes étaient majoritairement retenus dans les endosomes tardifs autour du noyau réduisant ainsi le passage de l'ADN vers ce dernier et limitant de ce fait l'efficacité de transfection du PEI.Dans un deuxième temps, la mise en place d'une plate-forme de spectroscopie à corrélation de fluorescence par excitation biphotonique, nous a permis de suivre la formation et la dissociation des complexes in vitro ainsi que d'en caractériser la composition. Ainsi, nous avons montré que les complexes contenaient en moyenne 3 plasmides et que lors de leur formation 85% du PEI restait libre en solution. Ce dernier point est susceptible d'être à l'origine de la toxicité observée lors de la transfection des cellules L929. Par ailleurs, des études de FCS en milieu cellulaire ont permis de mettre en évidence la présence de PEI libre dans l'ensemble des compartiments cellulaires et une importante interaction entre le PEI les constituants intracellulaires.Progress in molecular biology made it possible to use DNA molecules as therapeutic agents for treatment of cancer or genetic diseases. The success of gene therapy depends on the development of vectors able to transfer therapeutic genes into the cell. Within this framework, we studied the formation, the composition and the cellular fate of complexes between DNA and one of the most promising non viral gene delivery vectors: polyethylenimine.The first part of this work allowed us to determine by confocal microscopy, the limiting steps of L929 cell transfection by DNA/PEI complexes. Indeed, using YOYO-1 labelled DNA, Rhodamine labelled PEI and the endocytosis marker FM4-64, we showed that after internalisation through the fluid phase endocytosis pathway, the complexes were mainly retained in the late endosomes around the nucleus. The limited release of the complexes from the endosome prevents DNA migration to the nucleus and thus reduced the transfection efficiency of PEI/DNA complexes. In the second part of the work, a two photon excitation fluorescence correlation spectroscopy setup has been developed to follow the in vitro formation and dissociation of DNA/PEI complexes and determine their composition. Thus, we showed that the complexes contained an average of 3 plasmides per complex and that after their formation 85% of the PEI remained free in solution. This high concentration of free PEI, probably explains the cytotoxicity observed during L929 transfection. In addition, intracellular FCS studies showed the presence of free PEI molecules in all cells compartments and a strong interaction between PEI and intracellular components.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Monitoring of the Formation and Dissociation of Polyethylenimine/DNA Complexes by Two Photon Fluorescence Correlation Spectroscopy

Polyethylenimines (PEI) constitute efficient nonviral vectors for gene transfer. However, because free PEI shows some cytotoxicity and because intracellular dissociation of PEI/DNA complexes seems to be required for efficient transfection, it is important to monitor the concentrations of free and bound partners in the mixtures of DNA and PEI used for transfection. To reach this objective, we used fluorescence correlation spectroscopy with two-photon excitation to characterize the complexes formed with either rhodamine-labeled 25 kDa PEI or DNA plasmid molecules. At the molar ratios of PEI nitrogen atoms to DNA phosphate usually used for transfection, we found that ∼86% of the PEI molecules were in a free form. The PEI/DNA complexes are composed on the average by 3.5 (±1) DNA plasmids and ∼30 PEI molecules. From this composition and the pK(a) of PEI, it could be inferred that in contrast to DNA condensation by small multivalent cations, only a limited neutralization of the DNA phosphate groups is required for DNA condensation by PEI. Moreover, DNA appears only poorly compacted in the PEI/DNA complexes. As an application, fluorescence correlation spectroscopy was used to monitor the purification of PEI/DNA complexes by ultrafiltration as well as the heparin-induced dissociation of the complexes