Search

A macroscopic model of exciton density decays in disordered molecular systems, including contributions from molecular aggregate quenchers, is proposed. The model can be applied to ultrafast decays of dyes and for global fitting of experimental data. , Exciton density dynamics recorded in time-resolved spectroscopic measurements is a useful tool to recover information on energy transfer (ET) processes that can occur at different timescales, up to the ultrafast regime. Macroscopic models of exciton density decays, involving both direct Förster-like ET and diffusion mechanisms for exciton–exciton annihilation, are largely used to fit time-resolved experimental data but generally neglect contributions from molecular aggregates that can work as quenching species. In this work, we introduce a macroscopic model that includes contributions from molecular aggregate quenchers in a disordered molecular system. As an exemplifying case, we considered a homogenous distribution of rhodamine B dyes embedded in organic nanoparticles to set the initial parameters of the proposed model. The influence of such model parameters is systematically analysed, showing that the presence of molecular aggregate quenchers can be monitored by evaluating the exciton density long time decays. We showed that the proposed model can be applied to molecular systems with ultrafast decays, and we anticipated that it could be used in future studies for global fitting of experimental data with potential support from first-principles simulations.

We herein present a new concept to produce dual-color photoconvertible probes based on a mechanism called Directed Photooxidation Induced Conversion (DPIC). As a support of this mechanism, styryl-coumarins (SCs) bearing Aromatic Singlet Oxygen Reactive Moieties (ASORM) like furan and pyrrole have been synthesized. SCs are bright fluorophores, which undergo a hypsochromic conversion upon visible light irradiation due to directed photooxidation of the ASORM that leads to the disruption of conjugation. SC-P, a yellow emitting probe bearing a pyrrole moiety, converts to a stable blue emitting coumarin with a 68 nm shift allowing the photoconversion and tracking of lipid droplet in live cells. This new approach might pave the way to a new generation of photoconvertible dyes for advanced bioimaging applications.

Fluorescent nucleoside analogues (FNAs) are structurally diverse mimics of the natural essentially non-fluorescent nucleosides which have found numerous applications in probing the structure and dynamics of nucleic acids as well as their interactions with various biomolecules. In order to minimize disturbance in the labelled nucleic acid sequences, the FNA chromophoric groups should resemble the natural nucleobases in size and hydrogen-bonding patterns. Isomorphic and expanded FNAs are the two groups that best meet the criteria of non-perturbing fluorescent labels for DNA and RNA. Significant progress has been made over the past decades in understanding the fundamental photophysics that governs the spectroscopic and environmentally sensitive properties of these FNAs. Herein, we review recent advances in the spectroscopic and computational studies of selected isomorphic and expanded FNAs. We also show how this information can be used as a rational basis to design new FNAs, select appropriate sequences for optimal spectroscopic response and interpret fluorescence data in FNA applications.

Overexpression of EGFR drives glioblastomas (GBM) cell invasion but these tumours remain resistant to EGFR-targeted therapies such as tyrosine kinase inhibitors (TKIs). Endocytosis, an important modulator of EGFR function, is often dysregulated in glioma cells and is associated with therapy resistance. However, the impact of TKIs on EGFR endocytosis has never been examined in GBM cells. In the present study, we showed that gefitinib and other tyrosine kinase inhibitors induced EGFR accumulation in early-endosomes as a result of an increased endocytosis. Moreover, TKIs trigger early-endosome re-localization of another membrane receptor, the fibronectin receptor alpha5beta1 integrin, a promising therapeutic target in GBM that regulates physiological EGFR endocytosis and recycling in cancer cells. Super-resolution dSTORM imaging showed a close-proximity between beta1 integrin and EGFR in intracellular membrane compartments of gefitinib-treated cells, suggesting their potential interaction. Interestingly, integrin depletion delayed gefitinib-mediated EGFR endocytosis. Co-endocytosis of EGFR and alpha5beta1 integrin may alter glioma cell response to gefitinib. Using an in vitro model of glioma cell dissemination from spheroid, we showed that alpha5 integrin-depleted cells were more sensitive to TKIs than alpha5-expressing cells. This work provides evidence for the first time that EGFR TKIs can trigger massive EGFR and alpha5beta1 integrin co-endocytosis, which may modulate glioma cell invasiveness under therapeutic treatment.

One of the most characterized bioluminescent reactions involves the firefly luciferase that catalyzes the oxidation of the luciferin producing oxyluciferin in its first excited state. While relaxing to the ground state, oxyluciferin emits visible light with an emission maximum that can vary from green to red. Oxyluciferin exists under six different chemical forms resulting from a keto/enol tautomerization and the deprotonation of the phenol or enol moieties. The optical properties of each chemical form have been recently characterized by the investigations of a variety of oxyluciferin derivatives, indicating unresolved excited-state proton transfer (ESPT) reactions. In this work, femtosecond pump-probe spectroscopy and time-resolved fluorescence spectroscopy are used to investigate the picosecond kinetics of the ESPT reactions and demonstrate the excited state keto to enol conversion of oxyluciferin and its derivatives in aqueous buffer as a function of pH. A comprehensive photophysical scheme is provided describing the complex luminescence pathways of oxyluciferin in protic solution.

Time-resolved fluorescence anisotropy (TRFA) provides key information on the dynamics of biomolecules and their interaction with ligands. However, since natural nucleosides are almost non-fluorescent, its application to DNA duplexes (dsDNA) requires fluorescent labels, which can alter dsDNA stability, hinder protein binding, and complicate interpretation of TRFA experiments due to their local motion. As shown here, thienoguanosine (

Thienoguanosine (thG) is an isomorphic fluorescent guanosine (G) surrogate, which almost perfectly mimics the natural G in DNA duplexes and may therefore be used to sensitively investigate for example protein-induced local conformational changes. To fully exploit the information given by the probe, we carefully re-investigated the thG spectroscopic properties in 12-bp duplexes, when the Set and Ring Associated (SRA) domain of UHRF1 flips its 5' flanking methylcytosine (mC). The SRA-induced flipping of mC was found to strongly increase the fluorescence intensity of thG, but this increase was much larger when thG was flanked in 3' by a C residue as compared to an A residue. Surprisingly, the quantum yield and fluorescence lifetime values of thG were nearly constant, regardless of the presence of SRA and the nature of the 3' flanking residue, suggesting that the differences in fluorescence intensities might be related to changes in absorption properties. We evidenced that thG lowest energy absorption band in the duplexes can be deconvoluted into two bands peaking at ∼350 nm and ∼310 nm, respectively red-shifted and blue-shifted, compared to the spectrum of thG monomer. Using quantum mechanical calculations, we attributed the former to a nearly pure

Very few proteins are reported to bind specific lipids. Because of the high selectivity and strong binding to specific lipids, lipid-targeting pore forming toxins (PFTs) have been employed to study the distribution of lipids in cell- and model-membranes. Non-toxic and monomeric PFT-derivatives are especially useful to study living cells. In this chapter we highlight sphingomyelin (SM)-binding PFT, lysenin (Lys), its derivatives, and newly identified SM/cholesterol binding protein, nakanori. We describe the preparation of non-toxic mutant of Lys (NT-Lys) and its application in optical and super resolution microscopy. We also discuss the observation of nanometer scale lipid domains labeled with nakanori and maltose-binding protein (MBP)-Lys in electron microscopy.

Very few proteins are reported to bind specific lipids. Because of the high selectivity and strong binding to specific lipids, lipid-targeting pore forming toxins (PFTs) have been employed to study the distribution of lipids in cell- and model-membranes. Non-toxic and monomeric PFT-derivatives are especially useful to study living cells. In this chapter we highlight sphingomyelin (SM)-binding PFT, lysenin (Lys), its derivatives, and newly identified SM/cholesterol binding protein, nakanori. We describe the preparation of non-toxic mutant of Lys (NT-Lys) and its application in optical and super resolution microscopy. We also discuss the observation of nanometer scale lipid domains labeled with nakanori and maltose-binding protein (MBP)-Lys in electron microscopy.

Simple Summary γ-H2AX, a phosphorylated variant of histone H2A, is a widely used biomarker of DNA replication stress. To develop an immunological probe able to detect and track γ-H2AX in live cancer cells, we have isolated single domain antibodies (called nanobodies) that are easily expressed as functional recombinant proteins and here we report the extensive characterization of a novel nanobody that specifically recognizes γ-H2AX. The interaction of this nanobody with the C-terminal end of γ-H2AX was determined by X-ray crystallography. Moreover, the generation of a bivalent nanobody allowed us to precisely detect γ-H2AX foci in drug-treated cells as efficiently as with commercially available conventional antibodies. Furthermore, we tracked γ-H2AX foci in live cells upon intracellular delivery of the bivalent nanobody fused to the red fluorescent protein dTomato, making, consequently, this new cost-effective reagent useful for studying drug-induced replication stress in both fixed and living cancer cells. Abstract Histone H2AX phosphorylated at serine 139 (γ-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While γ-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to γ-H2AX. We solved the crystal structure of the most soluble nanobody in complex with the phosphopeptide corresponding to the C-terminus of γ-H2AX and show the atomic constituents behind its specificity. We engineered a bivalent version of this nanobody and show that bivalency is essential to quantitatively visualize γ-H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, we were able to detect γ-H2AX in a single-step assay with the same sensitivity as with validated antibodies. Moreover, we produced fluorescent nanobody-dTomato fusion proteins and applied a transduction strategy to visualize with precision γ-H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual cancer cells under a variety of conditions.

Interconversions between nucleic acid structures play an important role in transcriptional and translational regulation and also in repair and recombination. These interconversions are frequently promoted by nucleic acid chaperone proteins. To monitor their kinetics, Förster resonance energy transfer (FRET) is widely exploited using ensemble fluorescence intensity measurements in pre-steady-state stopped-flow experiments. Such experiments only provide a weighted average of the emission of all species in solution and consume large quantities of materials. Herein, we lift these limitations by combining time-resolved fluorescence (TRF) with droplet microfluidics (DmF). We validate the innovative TRF-DmF approach by investigating the well characterized annealing of the HIV-1 (+)/(–) Primer Binding Sequences (PBS) promoted by a HIV-1 nucleocapsid peptide. Upon rapid mixing of the FRET-labelled (–)PBS with its complementary (+)PBS sequence inside microdroplets, the TRF-DmF set-up enables resolving the time evolution of sub-populations of reacting species and reveals an early intermediate with a ∼50 ps donor fluorescence lifetime never identified so far. TRF-DmF also favorably compares with single molecule experiments, as it offers an accurate control of concentrations with no upper limit, no need to graft one partner on a surface and no photobleaching issues.

The thienoguanine nucleobase ((th)G(b)) is an isomorphic fluorescent analogue of guanine. In aqueous buffer at neutral pH, (th)G(b) exists as a mixture of two ground-state H1 and H3 keto-amino tautomers with distinct absorption and emission spectra and high quantum yield. In this work, we performed the first systematic photophysical characterization of (th)G(b) as a function of pH (2 to 12). Steady-state and time-resolved fluorescence spectroscopies, supplemented with theoretical calculations, enabled us to identify three additional (th)G(b) forms, resulting from pH-dependent ground-state and excited-state reactions. Moreover, a thorough analysis allowed us to retrieve their individual absorption and emission spectra as well as the equilibrium constants which govern their interconversion. From these data, the complete photoluminescence pathway of (th)G(b) in aqueous solution and its dependence as a function of pH was deduced. As the identified forms differ by their spectra and fluorescence lifetime, (th)G(b) could be used as a probe for sensing local pH changes under acidic conditions.

Here, we explore the enhancement of single-molecule emission by a polymeric nanoantenna that can harvest energy from thousands of donor dyes to a single acceptor. In this nanoantenna, the cationic dyes are brought together, in very close proximity, using bulky counterions, thus enabling ultrafast diffusion of excitation energy (≤30 fs) with minimal losses. Our 60 nm nanoparticles containing >10,000 rhodamine-based donor dyes can efficiently transfer energy to 1–2 acceptors, resulting in an antenna effect of ~1,000. Therefore, single Cy5-based acceptors become 25-fold brighter than quantum dots QD655. This unprecedented amplification of the acceptor dye emission enables observation of single molecules at illumination powers (1–10 mW cm−2) that are >10,000-fold lower than typically required in single-molecule measurements. Finally, using a basic set-up, which includes a ×20 air objective and a scalable complementary metal-oxide–semiconductor camera, we could detect single Cy5 molecules by simply shining divergent light on the sample at powers equivalent to sunlight. Donor dye nanoparticles have been used to realize structures that are 25 times brighter than quantum dots. This enabled single-molecule imaging using ambient light.

The HIV-1 Gag precursor specifically selects the unspliced viral genomic RNA (gRNA) from the bulk of cellular and spliced viral RNAsviaits nucleocapsid (NC) domain and drives gRNA encapsidation at the plasma membrane (PM). To further identify the determinants governing the intracellular trafficking of Gag-gRNA complexes and their accumulation at the PM, we compared, in living and fixed cells, the interactions between gRNA and wild-type (WT) Gag or Gag mutants carrying deletions in NC zinc fingers (ZFs), or a non-myristoylated version of Gag. Our data showed that the deletion of both ZFs simultaneously or the complete NC domain completely abolished intracytoplasmic Gag-gRNA interactions. Deletion of either ZF delayed the delivery of gRNA to the PM but did not prevent Gag-gRNA interactions in the cytoplasm, indicating that the two ZFs display redundant roles in this respect. However, ZF2 played a more prominent role than ZF1 in the accumulation of the ribonucleoprotein complexes at the PM. Finally, the myristate group which is mandatory for anchoring the complexes at the MP, was found to be dispensable for the association of Gag with the gRNA in the cytosol.STATEMENT of SIGNIFICANCEFormation of HIV-1 retroviral particles relies on specific interactions between the retroviral Gag precursor and the unspliced genomic RNA (gRNA). During the late phase of replication, Gag orchestrates the assembly of newly formed viruses at the plasma membrane (PM). It has been shown that the intracellular HIV-1 gRNA recognition is governed by the two-zinc finger (ZF) motifs of the nucleocapsid (NC) domain in Gag. Here we provided a clear picture of the role of ZFs in the cellular trafficking of Gag-gRNA complexes to the PM by showing that either ZF was sufficient to efficiently promote these interactions in the cytoplasm, while interestingly, ZF2 played a more prominent role in the relocation of these ribonucleoprotein complexes at the PM assembly sites.

International audience; The human immunodeficiency virus type 1 Gag precursor specifically selects the unspliced viral genomic RNA (gRNA) from the bulk of cellular and spliced viral RNAs via its nucleocapsid (NC) domain and drives gRNA encapsidation at the plasma membrane (PM). To further identify the determinants governing the intracellular trafficking of Gag-gRNA complexes and their accumulation at the PM, we compared, in living and fixed cells, the interactions between gRNA and wild-type Gag or Gag mutants carrying deletions in NC zinc fingers (ZFs) or a nonmyristoylated version of Gag. Our data showed that the deletion of both ZFs simultaneously or the complete NC domain completely abolished intracytoplasmic Gag-gRNA interactions. Deletion of either ZF delayed the delivery of gRNA to the PM but did not prevent Gag-gRNA interactions in the cytoplasm, indicating that the two ZFs display redundant roles in this respect. However, ZF2 played a more prominent role than ZF1 in the accumulation of the ribonucleoprotein complexes at the PM. Finally, the myristate group, which is mandatory for anchoring the complexes at the PM, was found to be dispensable for the association of Gag with the gRNA in the cytosol.

International audience; In the quest for the identication of the light emitter(s) responsible for the rey bioluminescence, the study of oxyluciferin analogues with controlled chemical and electronic structures is of particular importance. In this article, we report the results of our experimental and computational investigation of the pH-dependent absorption spectra characterizing three analogues bound into the luciferase cavity, together with adenosine-monophosphate (AMP). While the analogue microscopic pK a values don't dier much from their reference values, it turns out that AMP protonation state is analogue-dependent and never doubly-deprotonated. Careful analysis of the interactions evidences the main role of E344 glutamic acid, as well as the exibility of the cavity which can accommodate any oxyluciferin analogue. The consideration of the absorption spectra suggests that the oxyluciferin enolate form has to be excluded from the list of the bioluminescence reaction products.

Thienoguanosine ((th)G) is an isomorphic guanosine (G) surrogate that almost perfectly mimics G in nucleic acids. To exploit its full potential and lay the foundation for future applications, twenty DNA duplexes, where the bases facing and neighboring (th)G were systematically varied, were thoroughly studied using fluorescence spectroscopy, molecular dynamics simulations and mixed quantum mechanical/molecular mechanics calculations, yielding a comprehensive understanding of its photophysics in DNA. In matched duplexes, (th)G’s hypochromism was larger for flanking G/C residues but its fluorescence quantum yield (QY) and lifetime values were almost independent of the flanking bases. This was attributed to high duplex stability, which maintains a steady orientation and distance between nucleobases, so that a similar charge transfer (CT) mechanism governs the photophysics of (th)G independently of its flanking nucleobases. (th)G can therefore replace any G residue in matched duplexes, while always maintaining similar photophysical features. In contrast, the local destabilization induced by a mismatch or an abasic site restores a strong dependence of (th)G’s QY and lifetime values on its environmental context, depending on the CT route efficiency and solvent exposure of (th)G. Due to this exquisite sensitivity, (th)G appears ideal for monitoring local structural changes and single nucleotide polymorphism. Moreover, (th)G’s dominant fluorescence lifetime in DNA is unusually long (9–29 ns), facilitating its selective measurement in complex media using a lifetime-based or a time-gated detection scheme. Taken together, our data highlights (th)G as an outstanding emissive substitute for G with good QY, long fluorescence lifetimes and exquisite sensitivity to local structural changes.

International audience; The photophysical properties of oxyluciferin, the light emitter responsible for firefly bioluminescence, are pH-dependent. One of the potential proton accep-tor/donor is adenosine monophosphate (AMP). We have studied three oxylu-ciferin synthetic analogues with or without AMP, in water, in the pH=5 to 11 range, using both experimental steady-state absorption spectroscopy or the recently developed computational protocol that uses constant pH molecular dynamics and then hybrid QM/MM calculations (CpHMD-then-QM/MM). The latter features a systematic investigation of all the protonation microstates using molecular dynamics simulations coupled to thousands hybrid QM/MM vertical excitation energies. Our results demonstrate that AMP does not significantly modify the visible light absorption of the analogues, whatever the pH value. We also show that CpHMD-then-QM/MM is capable to qualitatively reproduce the pH-dependent absorption spectrum of the analogues, despite the employed low QM level of theory.

Thienoguanosine (th G) is an isomorphic analogue of guanosine with promising potentialities as fluorescent DNA label. As a free probe in protic solvents, th G exists in two tautomeric forms, identified as the H1, being the only one observed in nonprotic solvents, and H3 keto-amino tautomers. We herein investigate the photophysics of th G in solvents of different polarity, from water to dioxane, by combining time-resolved fluorescence with PCM/TD-DFT and CASSCF calculations. Fluorescence lifetimes of 14.5-20.5 and 7-13 ns were observed for the H1 and H3 tautomers, respectively, in the tested solvents. In methanol and ethanol, an additional fluorescent decay lifetime (≈3 ns) at the blue emission side (λ≈430 nm) as well as a 0.5 ns component with negative amplitude at the red edge of the spectrum, typical of an excited-state reaction, were observed. Our computational analysis explains the solvent effects observed on the tautomeric equilibrium. The main radiative and nonradiative deactivation routes have been mapped by PCM/TD-DFT calculations in solution and CASSCF in the gas phase. The most easily accessible conical intersection, involving an out-of plane motion of the sulfur atom in the five-membered ring of th G, is separated by a sizeable energy barrier (≥0.4 eV) from the minimum of the spectroscopic state, which explains the large experimental fluorescence quantum yield.

Cationic carbon dots (CDs) have been recently described as nucleic acid carriers with high in vitro and in vivo transfection efficiency and imaging properties. However, developing nanoparticles (NPs) for bio-medical applications requires assessing their safety. In the present study, we characterized the cell uptake and trafficking, as well as the cell viability loss, oxidative stress, inflammation, and mitochondrial and lysosomal perturbations evoked by cationic CDs prepared by microwave-assisted pyrolysis of citric acid and high molecular weight branched polyethyleneimine (bPEI25k), using THP-1-derived macrophages. CDs were rapidly internalized by cells and addressed to the lysosomes after their cell entry. The NPs induced a dose-and time-dependent loss in cell viability that was associated with oxidative stress and IL-8 release. The CDs triggered also a dose-dependent loss in lysosome integrity , mitochondrial dysfunction, and NLRP3 inflammasome activation. Inhibition of the lysosomal protease cathepsin B significantly reduced CD-induced mitochondrial dysfunction and NLRP3 inflammasome activation, suggesting a pivotal role of the lysosome in the toxicological effects of the NPs. Our study provides for the first time a mechanistic pathway for the toxicological effects of bPEI25k-based cationic CDs.

International audience; Firefly bioluminescence is a quite efficient process largely used for numerous applications. However, some fundamental photochemical properties of the light emitter are still to be analyzed. Indeed, the light emitter, oxyluciferin, can be in six different forms due to interexchange reactions. In this work, we present the simulation of the absorption and emission spectra of the possible natural oxyluciferin forms in water and some of their analogues considering both the solvent/oxyluciferin interactions and the dynamical effects by using MD simulations and QM/MM methods. On the one hand, the absorption band shapes have been rationalized by analyzing the electronic nature of the transitions involved. On the other hand, the simulated and experimental emission spectra have been compared. In this case, an ultrafast excited state proton transfer (ESPT) occurs in oxyluciferin and its analogues, which impairs the detection of the emission from the protonated state by steady-state fluorescence spectroscopy. Transient absorption spectroscopy was used to evidence this ultrafast ESPT and rationalize the comparison between simulated and experimental steady-state emission spectra. Finally, this work shows the suitability of the studied oxyluciferin analogues to mimic the corresponding natural forms in water solution, as an elegant way to block the desired interexchange reactions allowing the study of each oxyluciferin form separately.

Although chemical inhibition of the DNA damage response (DDR) in cancer cells triggers cell death, it is not clear if the fork blockade achieved with inhibitors that neutralise proteins of the replisome is sufficient on its own to overcome the DDR. Monoclonal antibodies to PCNA, which block the DNA elongation process in vitro, have been developed. When these antibodies were transduced into cancer cells, they are able to inhibit the incorporation of nucleoside analogues. When co-delivered with anti-PCNA siRNA, the cells were flattened and the size of their nuclei increased by up to 3-fold, prior to cell death. Analysis of these nuclei by super-resolution microscopy revealed the presence of large numbers of phosphorylated histone H2AX foci. A senescence-like phenotype of the transduced cells was also observed upon delivery of the corresponding Fab molecules or following PCNA gene disruption or when the Fab fragment of an antibody that neutralises DNA polymerase alpha was used. Primary melanoma cells and leukaemia cells that are resistant to chemical inhibitors were similarly affected by these antibody treatments. These results demonstrate that transduced antibodies can trigger a lethal DNA replication stress, which kills cancer cells by abolishing the biological activity of several constituents of the replisome.

International audience; The Pr55 Gag of human immunodeficiency virus type 1 orchestrates viral particle assembly in producer cells, which requires the genomic RNA and a lipid membrane as scaffolding platforms. The nucleocapsid (NC) domain with its two invariant CCHC zinc fingers flanked by unfolded basic sequences is thought to direct genomic RNA selection, dimerization and packaging during virus assembly. To further investigate the role of NC domain, we analyzed the assembly of Gag with deletions in the NC domain in parallel with that of wild-type Gag using fluorescence lifetime imaging microscopy combined with Förster resonance energy transfer in HeLa cells. We found that, upon binding to nucleic acids, the NC domain promotes the formation of compact Gag oligomers in the cytoplasm. Moreover, the intracellular distribution of the population of oligomers further suggests that oligomers progressively assemble during their trafficking toward the plasma membrane (PM), but with no dramatic changes in their compact arrangement. This ultimately results in the accumulation at the PM of closely packed Gag oligomers that likely arrange in hexameric lattices, as revealed by the perfect match between the experimental Förster resonance energy transfer value and the one calculated from the structural model of Gag in immature viruses. The distal finger and flanking basic sequences, but not the proximal finger, appear to be essential for Gag oligomer compaction and membrane binding. Moreover, the full NC domain was found to be instrumental in the kinetics of Gag oligomerization and intracellular trafficking. These findings further highlight the key roles played by the NC domain in virus assembly.

The HIV-1 Gag polyprotein precursor composed of the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains orchestrates virus assembly via interactions between MA and the cell plasma membrane (PM) on one hand and NC and the genomic RNA on the other hand. As the Gag precursor can adopt a bent conformation, a potential interaction of the NC domain with the PM cannot be excluded during Gag assembly at the PM. To investigate the possible interaction of NC with lipid membranes in the absence of any interference from the other domains of Gag, we quantitatively characterized by fluorescence spectroscopy the binding of the mature NC protein to large unilamellar vesicles (LUVs) used as membrane models. We found that NC, either in its free form or bound to an oligonucleotide, was binding with high affinity (∼10 7 M −1 ) to negatively charged LUVs. The number of NC binding sites, but not the binding constant, was observed to decrease with the percentage of negatively charged lipids in the LUV composition, suggesting that NC and NC/oligonucleotide complexes were able to recruit negatively charged lipids to ensure optimal binding. However, in contrast to MA, NC did not exhibit a preference for phosphatidylinositol-(4,5)-bisphosphate. These results lead us to propose a modified Gag assembly model where the NC domain contributes to the initial binding of the bent form of Gag to the PM. IMPORTANCE The NC protein is a highly conserved nucleic acid binding protein that plays numerous key roles in HIV-1 replication. While accumulating evidence shows that NC either as a mature protein or as a domain of the Gag precursor also interacts with host proteins, only a few data are available on the possible interaction of NC with lipid membranes. Interestingly, during HIV-1 assembly, the Gag precursor is thought to adopt a bent conformation where the NC domain may interact with the plasma membrane. In this context, we quantitatively characterized the binding of NC, as a free protein or as a complex with nucleic acids, to lipid membranes and showed that the latter constitute a binding platform for NC. Taken together, our data suggest that the NC domain may play a role in the initial binding events of Gag to the plasma membrane during HIV-1 assembly.

Nano-emulsions are very promising nano-carriers with high potential for loading lipophilic drugs. However, the surface of oil nano-droplets is a dynamic oil/water interface stabilized by surfactants, and its chemical modification to graft ligands is highly challenging. In this study we developed a new protocol for modification of the nano-droplets surface through a silica shell terminated by amine functions. It enabled preparation of nanocapsules of 65, 85 and 120 nm diameters with a surface coverage of ca. 2 amino groups per nm2. The nanocapsule surface was then functionalized (41% efficiency) by a model fluorescent ligand (coumarin blue) with a carboxylic function. The evidence for the successful grafting was provided by spectrofluorometry, Forster resonance energy transfer, atomic force microscopy coupled with fluorescence imaging and fluorescence correlation spectroscopy. This simple protocol for surface functionalization of the liquid/liquid interface of lipid droplets may constitute a real advance regarding potential applications that need efficient decoration of droplets with ligands.

The neuropeptide Y (NPY) plays numerous biological roles that are mediated by a family of G-protein-coupled receptors. Among the latter, the NPY Y

International audience; SAGA and ATAC are two distinct chromatin modifying co-activator complexes with distinct enzymatic activities involved in RNA polymerase II (Pol II) transcription regulation. To investigate the mobility of co-activator complexes and general transcription factors in live-cell nuclei, we performed imaging experiments based on photobleaching. SAGA and ATAC, but also two general transcription factors (TFIID and TFIIB), were highly dynamic, exhibiting mainly transient associations with chromatin, contrary to Pol II, which formed more stable chromatin interactions. Fluorescence correlation spectroscopy analyses revealed that the mobile pool of the two co-activators, as well as that of TFIID and TFIIB, can be subdivided into "fast" (free) and "slow" (chromatin-interacting) populations. Inhibiting transcription elongation decreased H3K4 trimethylation and reduced the "slow" population of SAGA, ATAC, TFIIB and TFIID In addition, inhibiting histone H3K4 trimethylation also reduced the "slow" populations of SAGA and ATAC Thus, our results demonstrate that in the nuclei of live cells the equilibrium between fast and slow population of SAGA or ATAC complexes is regulated by active transcription via changes in the abundance of H3K4me3 on chromatin.

The concept of biomineralization and encapsulation of organic molecules into inorganic matrices to alter and enhance their physical properties has been evolved and perfected in natural systems. Being inspired by the natural biomineralization of foreign components into calcite, here the inclusion of a plant virus, cowpea mosaic virus (CPMV) of 5.4% by mass into crystals of calcite is reported. The viral particles are labeled with a fluorescent tag (Alexa Fluor 532), and are observed within the calcite matrix using confocal fluorescence microscopy. Upon encapsulation, the calcite crystals exhibit an irregular and aggregated morphology, as visualized with atomic force and electron microscopy. The viral particles protected inside the calcite crystals are able to resist harsh chemical agents. While spherical viral particles such as CPMV can be easily included in calcite, viruses such as the tobacco mosaic virus are not compatible with the host, presumably due to their high aspect ratio. The results provide a simple and scalable method to incorporate viral particles into inorganic matrix, and could prove useful in thermal stabilization of sensitive viral biological agents such as vaccines in the future.

A π-extended boranil (PEB) dye has been synthesized and its photophysical properties have been recorded in a range of solvents, highlighting an intense solvatochromic fluorescence emission covering the entire visible spectrum and the near infra-red (NIR), from 534 to 764 nm, with quantum yields up to 69%. A beneficial embedment of PEB dye in the amphiphilic block copolymer Cremophor EL® leads to a strong emission (λem = 617nm, Φ = 0.33) in PBS buffer. Moreover, two-photon absorption (TPA) spectroscopy evidenced a large two photon (2P) cross section (420 GM), enabling the use of PEB dye in 2P-excitation fluorescence microscopy. The cellular uptake and distribution of PEB in HeLa cells was monitored using fluorescence lifetime imaging microscopy (FLIM) and real-time widefield imaging, showing a strong influence of the local environment on the emission along with a preferential internalization in acidic vesicles.

Intrabodies, when expressed in cells after genetic fusion to fluorescent proteins, are powerful tools to study endogenous protein dynamics inside cells. However, it remains challenging to determine the conditions for specific imaging and precise labelling of the target antigen with such intracellularly expressed antibody fragments. Here, we show that single-chain Fv (scFv) antibody fragments can be generated that specifically recognize proliferating cell nuclear antigen (PCNA) when expressed in living cancer cells. After selection by phage display, the anti-PCNA scFvs were screened in vitro after being tagged with dimeric glutathione-S-transferase. Anti-PCNA scFvs of increased avidity were further engineered by mutagenesis with sodium bisulfite and error-prone PCR, such that they were almost equivalent to conventional antibodies in in vitro assays. These intrabodies were then rendered bifunctional by fusion to a C-terminal fragment of p21 protein and could thereby readily detect PCNA bound to chromatin in cells. Finally, by linking these optimized peptide-conjugated scFvs to an enhanced green fluorescent protein, fluorescent intrabody-based reagents were obtained that allowed the fate of PCNA in living cells to be examined. The approach described may be applicable to other scFvs that can be solubly expressed in cells, and it provides a unique means to recognize endogenous proteins in living cells with high accuracy. Copyright © 2014 John Wiley & Sons, Ltd.

Introduction Overexpression of epidermal growth factor (EGFR) drives glioblastoma (GBM) cell invasion and tumour progression. EGFR has been a major therapeutic target in GBM. However, clinical trials were disappointing, and we are still missing molecular basis to explain these poor results. In other solid tumours, extracellular matrix proteins found in the tumour microenvironment and their heterodimeric integrin receptors trigger resistance to EGFR targeted therapy. We recently established that the fibronectin receptor, α5β1 integrin is a pertinent therapeutic target in GBM. Therefore, we seek to evaluate whether α5β1 integrin is involved in therapy targeting EGFR in GBM. Material and methods We used GBM cell line (U87) overexpressing or down expressing α5 integrin subunit. EGFR was inhibited by clinically approved tyrosine kinase inhibitors (TKIs) (gefitinib, erlotinib, lapatinib). For cell motility assays, we performed spheroid dissemination assays, in 2D and 3D environment and chemotaxis assays through Boyden chambers. We also evaluated the impact of integrin expression on cell clonogenicity and cell grow th (2D, spheroids) in presence of EGFR inhibitors. Confocal microscopy confirmed integrin, EGFR and endosomes markers localization. Protein colocalization in early-endosomes was confirmed by confocal image analysis and dSTORM super-resolution. Results and discussions We showed that loss of integrin α5 expression increased U87 cell sensitivity to TKIs in spheroid cell evasion assays but did not affect TKIs efficiency on chemotaxis, cell growth (2D or 3D) or cell clonogenicity. EGFR is thus critical for cell dissemination from GBM tumour spheroids and overexpression of α5 integrin can circumvent EGFR inhibition by TKIs. Using confocal imaging, we showed that TKIs provoked a massive relocalization of integrin and EGFR in Rab5, EEA1 or LRP1 positive endosomes. Dynamic studies confirmed that TKIs increased EGFR receptor endocytosis. By colocalization studies and super-resolution dSTORM imaging, we clearly showed that in intracellular vesicules integrin and EGFR are in close proximity, suggesting a potential interaction. Conclusion We showed in GBM cells that α5β1 integrin expression triggers resistance to EGFR-targeting TKIs and that TKIs alter EGFR and integrin membrane trafficking. These data point out the potential importance of integrin and EGFR endocytosis and trafficking in GBM resistance to TKIs.

Phosphorylated histone H2AX (&gamma, H2AX), a central player in the DNA damage response (DDR), serves as a biomarker of DNA double-strand break repair. Although DNA damage is generally visualized by the formation of &gamma, H2AX foci in injured nuclei, it is unclear whether the widespread uniform nuclear &gamma, H2AX (called pan-nuclear) pattern occurring upon intense replication stress (RS) is linked to DDR. Using a novel monoclonal antibody that binds exclusively to the phosphorylated C-terminus of H2AX, we demonstrate that H2AX phosphorylation is systematically pan-nuclear in cancer cells stressed with RS-inducing drugs just before they die. The pan-nuclear &gamma, H2AX pattern is abolished by inhibition of the DNA-PK kinase. Cell death induction of cancer cells treated with increasing combinations of replication and kinase (ATR and Chk1) inhibitory drugs was proportional to the appearance of pan-nuclear &gamma, H2AX pattern. Delivery of labeled anti-&gamma, H2AX Fabs in stressed cells demonstrated at a single cell level that pan-nuclear &gamma, H2AX formation precedes irreversible cell death. Moreover, we show that H2AX is not required for RS-induced cell death in HeLa cells. Thus, the nuclear-wide formation of &gamma, H2AX is an incident of RS-induced cell death and, thus, the pan nuclear H2AX pattern should be regarded as an indicator of lethal RS-inducing drug efficacy.

The ability of a nonviral nucleic acid carrier to deliver its cargo to cells with low associated toxicity is a critical issue for clinical applications of gene therapy. We describe biodegradable cationic DOPC-C12 E4 conjugates in which transfection efficiency is based on a Trojan horse strategy. In situ production of the detergent compound C12 E4 through conjugate hydrolysis within the acidic endosome compartment was expected to promote endosome membrane destabilization and subsequent release of the lipoplexes into cytosol. The transfection efficiency of the conjugates has been assessed in vitro, and associated cytotoxicity was determined. Cellular uptake and intracellular distribution of the lipoplexes have been investigated. The results show that direct conjugation of DOPC with C12 E4 produces a versatile carrier that can deliver both DNA and siRNA to cells in vitro with high efficiency and low cytotoxicity. SAR studies suggest that this compound might represent a reasonable compromise between the membrane activity of the released detergent and susceptibility of the conjugate to degradation enzymes in vitro. Although biodegradability of the conjugates had low impact on carrier efficiency in vitro, it proved critical in vivo. Significant improvement of transgene expression was obtained in the mouse lung tuning biodegradability of the carrier. Importantly, this also allowed reduction of the inflammatory response that invariably characterizes cationic-lipid-mediated gene transfer in animals.

Addition of Vpr C-terminus to various cell types provokes cell apoptosis. This property was recently shown useful to develop inhibitors of cell proliferation. In that context, we investigated the cellular uptake of rhodamine- and fluorescein-labeled Vpr(52–96) peptides to understand the mechanism of Vpr C-terminus entry into cells. Dynamic light scattering data indicated that this peptide spontaneously formed polydispersed aggregates in cell culture medium. The fluorescently labeled Vpr(52–96) peptide was efficiently internalized, appearing either as large fluorescent patches in the cytoplasm or in a more diffuse form throughout the cell. Using isothermal titration calorimetry, we demonstrated that Vpr(52–96) can tightly associate with heparin, a glycosaminoglycan analog of heparan sulphate, suggesting a central role of the ubiquitous cell surface-associated heparan sulphate proteoglycans for the internalization of Vpr C-terminus. Fluorescently-labeled transferrin and methyl-β-cyclodextrin showed that the Vpr C-terminus was mediated through clathrin- and caveolae/raft-dependent endocytosis. We found that Vpr C-terminus uptake was partly blocked at 4 °C suggesting the importance of membrane fluidity for Vpr C-terminus entry. In fact, atomic force microscopy and liposome leakage further indicated that the Vpr peptide can destabilize and disrupt model membrane bilayers, suggesting that this mechanism may contribute to the passive entry of the peptide. Finally, using fluorescence lifetime imaging, we found that the Vpr(52–96) peptide was stable in cells for at least 48 h, probably as a consequence of the poor accessibility of the peptide to proteolytic enzymes in aggregates.

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.

In a search for environmentally sensitive (solvatochromic) dyes with superior properties, we extended the electronic conjugation of one of the best solvatochromic dyes, Prodan, by substituting its naphthalene core with fluorene. The newly synthesized fluorene derivatives bearing strong electron-donor (dialkylamino) and -acceptor (carbonyl) groups at the 2 and 7 positions showed red-shifted absorption (close to 400 nm), twice as large of a absorption coefficient (43 000 M−1 cm−1), and a manifold larger two-photon absorption cross section (∼400 GM) compared to Prodan. Studies in solvents revealed much stronger fluorescence solvatochromism of the new dyes, which is connected with their twice as large transition dipole moment (14.0 D). Similarly to Prodan, they exhibit high fluorescence quantum yields, while their photostability is largely improved. Thus, substitution of the naphthalene core in Prodan with fluorene resulted in new fluorophores with superior spectroscopic and solvatochromic properties. We expe...

We describe a simple fluorescent protein-based method to investigate interactions with a viral movement protein in living cells that relies on the in vivo re-localization of proteins in the presence of their interaction partners. We apply this method in combination with fluorescence lifetime imaging microscopy (FLIM) to demonstrate that a domain of the Tobacco mosaic virus (TMV) movement protein (MP) previously predicted to mediate protein:protein interactions is dispensable for these contacts. We suggest that this method can be generalized for analysis of other protein interactions in planta.