Fluorescence Resonance Energy Transfer Technique Research Articles

DNA aptamers against the DUX4 protein reveal novel therapeutic implications for FSHD.

Aberrant expression of the transcription factor double homeobox protein 4 (DUX4) can lead to a number of diseases including facio‐scapulo‐humeral muscular dystrophy (FSHD), acute lymphoblastic leukemia, and sarcomas. Inhibition of DUX4 may represent a therapeutic strategy for these diseases. By applying Systematic Evolution of Ligands by EXponential Enrichment (SELEX), we identified aptamers against DUX4 with specific secondary structural elements conveying high affinity to DUX4 as assessed by fluorescence resonance energy transfer and fluorescence polarization techniques. Sequences analysis of these aptamers revealed the presence of two consensus DUX4 motifs in a reverse complementary fashion forming hairpins interspersed with bulge loops at distinct positions that enlarged the binding surface with the DUX4 protein, as determined by crystal structure analysis. We demonstrate that insertion of specific structural elements into transcription factor binding oligonucleotides can enhance specificity and affinity.

P158 LOSS OF AUTOTAXIN ALTERS INNATE AND ADAPTIVE IMMUNE MECHANISM AND PROMOTES SPONTANEOUS COLITIS IN IL10-KO MICE

Abstract Background & Aims Autotaxin (Atx) is a secreted enzyme converting lysophosphatidylcholine (LPC) and sphingosyl-phosphorylcholine into lysophosphatidic acid and sphingosine 1-phosphate, respectively. Given the high affinity of LPC to cholesterol and the enrichment of cholesterol and sphingolipids in lipid rafts wherein LPS sensor Toll-like receptor 4 (TLR4) and its co-receptor CD14 reside, we hypothesized that Atx deficiency inhibits TLR4-mediated innate and adaptive immunity; thereby, accelerating the susceptibility to microbe-induced intestinal inflammation. Method We generated myeloid cell lineage-restricted Atx-knockout (ko) mice (AtxdME/dME) to study TLR4-mediated immune and inflammatory responses and investigated LPS-receptor complex formation through fluorescence resonance energy transfer technique, confocal microscopy, and flow cytometry. Lamina propria CD4+ T cells and bacterial load in the intestinal mucosa were examined. An impact of Atx deficiency in inflammatory bowel diseases (IBD) was investigated using AtxdME/dME;Il10-/- mice that have both Atx-deletion in myeloid cells and a global Il10 deletion. We examined the serum samples from IBD patients. Results With peritoneal macrophages from AtxdME/dME mice, we identified that Atx-ko disrupted the integrity of lipid rafts at the plasma membrane, resulting in the inhibition of TLR4 complex formation. Accordingly, the recruitment of adaptor molecules to TLR4 was suppressed, and TLR4-mediated responses were substantially reduced in Atx-ko macrophages. TLR4-induced innate immunity such as phagocytosis was attenuated in Atx-ko macrophages. The activation of CD4+ effector T cells and regulatory T cells was diminished in the lamina propria lymphocytes of AtxdME/dME mice compared to that of Atx+/+ littermates. Consequently, AtxdME/dME mice had a higher bacterial prevalence in the intestinal mucosa compared to controls. Just like the notion that commensal microbes translocated from the lumen into the intestinal mucosa can elicit spontaneous colitis in Il10-/- mice, combining AtxdME/dME with Il10-/- mice (AtxdME/dME; Il10-/-) did accelerate spontaneous colitis development. AtxdME/dME; Il10-/- mice had gross inflammation occurring throughout the colon, massive neutrophil infiltration and necrosis in the colonic mucosa, and increased mortality (Log-rank P=0.0046), while Atx+/+;Il10-/- littermates are normal. Notably, ATX serum protein level was lower in UC (n=26) and CD (n=34) patients compared to the level in normal subjects (n=26) (P<0.001), indicating an association of reduced ATX levels with the intestinal inflammation. Conclusions Collectively, we found that Atx deficiency suppresses TLR4-mediated innate and adaptive immune mechanisms; thereby accelerating the susceptibility to microbe-induced gut inflammation.

Single-Molecule Analysis and Engineering of DNA Motors.

Molecular motors are diverse enzymes that transduce chemical energy into mechanical work and, in doing so, perform critical cellular functions such as DNA replication and transcription, DNA supercoiling, intracellular transport, and ATP synthesis. Single-molecule techniques have been extensively used to identify structural intermediates in the reaction cycles of molecular motors and to understand how substeps in energy consumption drive transitions between the intermediates. Here, we review a broad spectrum of single-molecule tools and techniques such as optical and magnetic tweezers, atomic force microscopy (AFM), single-molecule fluorescence resonance energy transfer (smFRET), nanopore tweezers, and hybrid techniques that increase the number of observables. These methods enable the manipulation of individual biomolecules via the application of forces and torques and the observation of dynamic conformational changes in single motor complexes. We also review how these techniques have been applied to study various motors such as helicases, DNA and RNA polymerases, topoisomerases, nucleosome remodelers, and motors involved in the condensation, segregation, and digestion of DNA. In-depth analysis of mechanochemical coupling in molecular motors has made the development of artificially engineered motors possible. We review techniques such as mutagenesis, chemical modifications, and optogenetics that have been used to re-engineer existing molecular motors to have, for instance, altered speed, processivity, or functionality. We also discuss how single-molecule analysis of engineered motors allows us to challenge our fundamental understanding of how molecular motors transduce energy.

Observation of Continuous Contraction and a Metastable Misfolded State during the Collapse and Folding of a Small Protein

To obtain proper insight into how structure develops during a protein folding reaction, it is necessary to understand the nature and mechanism of the polypeptide chain collapse reaction, which marks the initiation of folding. Here, the time-resolved fluorescence resonance energy transfer technique, in which the decay of the fluorescence light intensity with time is used to determine the time evolution of the distribution of intra-molecular distances, has been utilized to study the folding of the small protein, monellin. It is seen that when folding begins, about one-third of the protein molecules collapse into a molten globule state (IMG), from which they relax by continuous further contraction to transit to the native state. The larger fraction gets trapped into a metastable misfolded state. Exit from this metastable state occurs via collapse to the lower free energy IMG state. This exit is slow, on a time scale of seconds, because of activation energy barriers. The trapped misfolded molecules as well as the IMG molecules contract continuously and slowly as structure develops. A phenomenological model of Markovian evolution of the polymer chain undergoing folding, incorporating these features, has been developed, which fits well the experimentally observed time evolution of distance distributions. The observation that the “wrong turn” to the misfolded state has not been eliminated by evolution belies the common belief that the folding of functional protein sequences is very different from that of a random heteropolymer, and the former have been selected by evolution to fold quickly.

Abstract LB-024: Inactivation of the 25-hydroxyvitamin D(3)-1(alpha)-hydroxylase gene (CYP27B1): evidence for impaired vitamin D signaling in an MMTV-PYMT mouse model of breast cancer

Abstract Purpose : The hormonally active form of vitamin D [1,25(OH)2D3], has several antitumor effects including antiproliferative, prodifferentiative and proapoptotic functions in tissues expressing and binding the vitamin D receptor. 1,25(OH)2D3 is synthesized from its precursor 25-hydroxyvitamin D [25(OH)D] via the catalytic action of the mitochondria cytochrome P450 enzyme 25(OH)D-1α-hydroxylase (CYP27B1). In the current study we investigated the role of CYP27B1 ablation on vitamin D signaling using the mouse mammary tumor virus promoter-driven polyoma middle T oncoprotein (MMTV-PyMT) mouse, an oncogene-driven model of highly aggressive spontaneous mammary tumors that closely mimics the estrogen receptor negative breast cancer in human disease. Methods: Tumors from animals were digested and primary cells were obtained from both CYP27B1 ablated AOH93Cre+ cells and wild type MT1107 Cre- control cells. The cells were next grown in complete DMEM medium, transfected with either a GFP-tagged human VDR or RXRα and treated with 1,25(OH)2D3 (10−7 mol/L) or 25(OH)D3 (10−7 mol/L) before data acquisition. Results: Using live and fixed cell fluorescence imaging, in situ proximity ligation assay and immunoblotting techniques, we show that the nuclear localization of both endogenous and exogenously tagged VDR and RXR were impaired in CYP27B1 ablated cells when compared to non-ablated cells. Furthermore, the intranuclear shuttling of VDR and its interaction with RXR were significantly reduced in CYP27B1 ablated cells as shown by both Florescence Recovery After Photobleaching and Fluorescence Resonance Energy Transfer techniques. Our results also show that CYP27B1 ablation disrupts VDR function by significantly impairing its nuclear localization, intranuclear transport, interaction with adaptor proteins, hVDR/hRXR, and this correlated with impaired binding to DNA and chromatin. Lastly, we demonstrate impaired induction of 53BP1, p65 and γ-H2AX DNA double-strand breaks in the CYP27B1 ablated cells compared to non-ablated cells. Conclusion: These results suggest that ablation of CYP27B1 results in dysregulation in vitamin D signaling which may impair its anticancer functions. Citation Format: Sylvester Jusu, John Presley, Bertrand Jean-Claude, Ursula Stochaj, Richard Kremer. Inactivation of the 25-hydroxyvitamin D(3)-1(alpha)-hydroxylase gene (CYP27B1): evidence for impaired vitamin D signaling in an MMTV-PYMT mouse model of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-024.

Gated Mesoporous Silica Nanocarriers for Hypoxia-Responsive Cargo Release.

Mesoporous silica nanocarriers (MSNs) are appealing in terms of their large cavity surface area and high loading capacity, but they have been suffering from premature cargo release. Herein, we report a gated smart MSN that is sensitive to low oxygen concentration (i.e., hypoxia) via taking advantage of the superior electron-accepting ability of the azobenzene moiety. The azobenzene polymer was employed as the responsive gate-keeper that was deposited on the MSN surface, followed by coating with amphiphilic Pluronic F68 for steric stabilization. The obtained nanocarriers were less than 200 nm. The in vitro polymer degradation was spectrophotometrically witnessed via the employment of a reducing agent, namely, sodium dithionite, with astrong electron-donating ability. The hypoxia-responsive cargo release from the gated MSN was quantitatively demonstrated in breast cancer cells (MCF-7) using the fluorescence resonance energy transfer (FRET) technique where coumarin 6 and rhodamine B was selected as the FRET donor and acceptor, respectively. The FRET ratio was used as the index and decreased linearly over time under hypoxia, whereas it almost remained steady under normoxia. In addition, a model photosensitizer, namely, chlorin e6, was also loaded in the gated MSN whose toxicity under hypoxia was verified. This study developed a hypoxia-responsive MSN with the azobenzene polymer as the removable gate-keeper, which would expand the application of MSNs in pharmaceutical and biomedical areas since thelow oxygen concentration is a unique trigger in many pathological conditions.

SmFRET Probing Reveals Substrate-Dependent Conformational Dynamics of E. coli Multidrug MdfA

Bacterial multidrug-resistance transporters of the major facilitator superfamily are distinguished by their extraordinary ability to bind structurally diverse substrates, thus serving as a highly efficient tool to protect cells from multiple toxic substances present in their environment, including antibiotic drugs. However, details of the dynamic conformational changes of the transport cycle involved remain to be elucidated. Here, we used the single-molecule fluorescence resonance energy transfer technique to investigate the conformational behavior of the Escherichia coli multidrug transporter MdfA under conditions of different substrates, pH, and alkali metal ions. Our data show that different substrates exhibit distinct effects on both the conformational distribution and transition rate between two major conformations. Although the cationic substrate tetraphenylphosphonium favors the outward-facing conformation, it has less effect on the transition rate. In contrast, binding of the electroneutral substrate chloramphenicol tends to stabilize the inward-facing conformation and decreases the transition rate. Therefore, our study supports the notion that the MdfA transporter uses distinct mechanisms to transport electroneutral and cationic substrates.

Light-Controlled in Vitro Gene Delivery Using Polymer-Tethered Spiropyran as a Photoswitchable Photosensitizer.

A gene delivery system using spiropyran as a photoswitchable photosensitizer for the controlled photochemical internalization effect was developed by engineering the outer coating of a polyethylenimine/DNA complex with a small amount of spiropyran-containing cationic copolymers. The successful binding of cationic polymers by the polyethylenimine coating was detected by the distance-sensitive fluorescence resonance energy-transfer technique that evidenced the occurrence of energy transfer between fluorescein-labeled cationic copolymers and polyethylenimine-condensed rhodamine-labeled DNA. The ternary polyplexes feature reversible controllability of singlet oxygen generation based on the dual effect of spiropyrans in photochromism and aggregation-induced enhanced photosensitization, allowing significant light-induced amplification of bPEI-mediated in vitro transgene efficiency (from original 15% to final 91%) at a low DNA dose, with the integrity of supercoiled DNA structure unaffected. The use of spiropyran without the need of other photosensitizers circumvents the issue of uncontrolled long-lasting photocytotoxicity in gene delivery.

Metastasis‐inducing protein S100A4 interacts with p53 in the nuclei of living cells

AbstractObjectiveS100A4 can bind to p53 in vitro. However, it is unclear whether the proteins interact in the nuclei of living cells.MethodsInitially, using an optical biosensor, the ability of CFP‐S100A4 to bind with p53‐YFP was examined in vitro. The fluorescence resonance energy transfer (FRET) technique using laser confocal microscopy was applied to detect the interaction of S100A4 with p53 in living cells.ResultsImaging FRET in HeLa cells expressing S100A4 and p53 showed an increase in cyan fluorescent protein fluorescence intensity, after photobleaching yellow fluorescent protein in cell nuclei. In comparison, the imaging of cell nuclei expressing mutant S100A4‐C and p53 or S100A4 and yellow fluorescent protein as the negative control showed the occurrence of FRET, and demonstrated that the interaction between S100A4 and p53 occurred in the nuclei of living HeLa cells. However, FRET did not occur in the cytoplasm of cells, showing that S100A4 did not interact with p53 in the cytoplasm.ConclusionS100A4 can interact directly with p53 in the nuclei of living cells. This might be a molecular basis for metastasis‐inducing S100A4 in vivo. The results also suggest that the inhibition of the calcium‐binding site of S100A4 might be a possible anticancer therapy.

Core–shell CdSeTe/ZnS quantum dots for the detection of microRNA-155 based on the fluorescence resonance energy transfer technique via the formation of a network structure

A fluorescence sensor was designed for miR155. When target hybridized with HP, numerous H1-AuNPs@SiO2 could be produced. The FL of A1-QDs was quenched by H1-AuNPs@SiO2via FRET function. The decrease of FL was proportional with miR155 concentration.

Chemoselective Acylation of Hydrazinopeptides to Access Fluorescent Probes for Time-Resolved FRET Assays on GPCRs.

Fluorescence techniques represent a powerful tool to investigate dynamic and functional architecture of GPCRs. Thus, fluorescent GPCR ligands have found various applications in cellular imaging, in the development of binding assays as replacements for radioligands in the study of ligand-receptor but also in receptor-receptor interactions at the cell surface or in native tissues. To extend the applicability of these techniques, the design and the synthesis of fluorescent probes are critical steps. As there are numerous peptide receptors in the GPCR family, fluorescent peptide-based probes are of importance. Herein, we present a convenient method to facilitate the solution-phase fluorescent labeling of peptides which is based on the chemoselective acylation of α-hydrazinopeptides. This approach combines the advantages to use commercially available amine-reactive dyes and very mild conditions that are fully compatible with the chemical sensitivity of the dyes. It gives a rapid access to fluorescent peptidic probes compatible with the time-resolved fluorescence resonance energy transfer (TR-FRET) techniques.

Aripiprazole and haloperidol protect neurite lesions via reducing excessive D2R-DISC1 complex formation

Dopamine D2 receptor (D2R) hyperactivity causes altered brain development and later produces onset of symptoms mimicking schizophrenia. It is known that D2R interacts with disrupted in schizophrenia 1 (DISC1); however, the effect of D2R-DISC1 interaction in intracellular signalling and neurite growth has not been studied. This study investigated the effect of D2R over-activation on Akt-GSK3β signalling and neurite morphology in cortical neurons. Over-activation of D2Rs caused neurite lesions, which were associated with decreased protein kinase B (Akt) and glycogen synthase kinase 3 beta (GSK3β) phosphorylation in cortical neurons. The antipsychotic drug aripiprazole was more effective in the prevention of neurite lesions than haloperidol. Unlike haloperidol, aripiprazole prevented downregulation of phospho (p) Akt-pGSK3β induced by D2R hyperactivity, indicating involvement of different pathways. D2Rs were hyperactive in cortical neurons of mice with DISC1 mutation, which caused more severe neurite lesions in cortical neurons treated with quinpirole. Immunofluorescent staining for Ca2+/calmodulin-dependent protein kinase II (CaMKII) confirmed that cortical pyramidal neurons were involved in the D2R hyperactivity-induced neurite lesions. Using the fluorescence resonance energy transfer (FRET) technique, we provide direct evidence that D2R hyperactivity led to D2R-DISC1 complex formation, which altered pGSK3β signalling. This study showed that D2R hyperactivity-induced D2R-DISC1 complex formation is associated with decreased pAkt-pGSK3β signalling and in turn, caused neurite impairment. Aripiprazole and haloperidol prevented the impairment of neurite growth but appeared to do so via different intracellular signalling pathways.

A unique FRET approach toward detection of single-base mismatch DNA in BRCA1 gene

Early detection of mutation carriers in predisposing genes such as BRCA1 plays an important role in disease prevention. This work developed a quantum dots-based (QDs-based) fluorescence resonance energy transfer (FRET) technique for the detection of single-base mismatch DNA in BRCA1 gene. The FRET between QDs as the donor and silver nanocluster (AgNCs) as the acceptor was designed by the strong interaction between CdTe QDs with appropriate size and dsDNA through binding to its major groove. The dsDNA was formed by the hybridization of ssDNA labeled to AgNCs with target DNA, which introduced CdTe QDs into the major grooves to place the AgNCs in close proximity to the QDs. The complementary and single-base mismatch DNA led to obviously different FRET signals. The FRET signal linearly correlated to the concentration of single-base mismatch DNA in the range of 1.5 × 10−10–1.0 × 10−6 mol L−1. The proposed method showed a detection limit of 80 pmol L−1 and the sensitivity comparable to the previously reported assays, indicating promising potential for single nucleotide polymorphisms diagnosis in clinical application.

Fluorescence Resonance Energy Transfer Visualization of Molecular Delivery from Polymeric Micelles.

Polymeric micelles are important carriers for anticancer drug delivery. However, rare papers focused on the dynamic of drug in the core of micelles. In this paper, we used fluorescence resonance energy transfer (FRET) technique to investigate the dynamic diffusion of drug from polymeric micelles. mPEG-PCL diblock copolymers were used as carriers. A pair of fluorescence molecules Cy3 and Cy5 was selected to evoke the FRET phenomenon. Cy5 was immobilized on the terminal group of PCL segments, Cy3 was encapsulated in the Cy5 modified polymeric micelles as the model drug. The drug loaded polymeric micelles were incubated with 4T1 breast cancer cells. The FRET was observed to explore the dynamic of Cy3 in the micelles. The results showed that the Cy3 loaded micelles were stable in aqueous solution as the energy-transfer efficiency (ETE, I660/I565) rarely decreased even when the time was as long as 120 h. The ETE increased with the content of encapsulated Cy3. The FRET spectra showed that the ETE of the Cy3 loaded polymeric micelles lowered with the release of Cy3 in PBS. The intracellular tracking of the Cy3 loaded micelles found more than 60% loaded drug was release within 12 h with the calculation of ETE in FRET spectra and it was same to confocal laser scanning and flow cytometry results.

Interaction of promethazine and adiphenine to human hemoglobin: A comparative spectroscopic and computational analysis

The binding nature of amphiphilic drugs viz. promethazine hydrochloride (PMT) and adiphenine hydrochloride (ADP), with human hemoglobin (Hb) was unraveled by fluorescence, absorbance, time resolved fluorescence, fluorescence resonance energy transfer (FRET) and circular dichroism (CD) spectral techniques in combination with molecular docking and molecular dynamic simulation methods. The steady state fluorescence spectra indicated that both PMT and ADP quenches the fluorescence of Hb through static quenching mechanism which was further confirmed by time resolved fluorescence spectra. The UV–Vis spectroscopy suggested ground state complex formation. The activation energy (Ea) was observed more in the case of Hb-ADP than Hb-PMT interaction system. The FRET result indicates the high probability of energy transfer from β Trp37 residue of Hb to the PMT (r=2.02nm) and ADP (r=2.33nm). The thermodynamic data reveal that binding of PMT with Hb are exothermic in nature involving hydrogen bonding and van der Waal interaction whereas in the case of ADP hydrophobic forces play the major role and binding process is endothermic in nature. The CD results show that both PMT and ADP, induced secondary structural changes of Hb and unfold the protein by losing a large helical content while the effect is more pronounced with ADP. Additionally, we also utilized computational approaches for deep insight into the binding of these drugs with Hb and the results are well matched with our experimental results.

‘Forbidden’ States Boost Fluorescence Resonance Energy Transfer (FRET) Studies of Membrane Receptors in Live Cells

For probing biomolecular interactions or conformational changes in a live-cell setting the distance depending Fluorescence resonance energy transfer (FRET) is often the method of choice. The G-protein-coupled receptor (GPCR) superfamily mediates cellular responses and communication across cellular membranes, and is the largest known class of molecular targets with proven therapeutic value. On the one hand, real-time monitoring of membrane receptors and their interactions is vital to understand how they act in concert. On the other hand, the design of GPCR FRET probes is crucial to ensure unhampered functionality and binding kinetics of the molecular complex. Thus, such FRET probes usually require labeling compromises concerning donor-acceptor positions, distances and orientations, which limit the detectability of emitter signals in general, and the dynamic range of the FRET changes in particular. Here, we present an approach to optimize the energy transfer without changing the design of the FRET probe. We show that gold coated glass cover slips allow reinforcing the otherwise forbidden donor-acceptor energy transfer by virtual optimization of the dipole orientation. First, we confirm the resulting enhanced FRET on our nano-coatings for the inactive M1 muscarinic acetylcholine receptors (mAChRs) labeled with a CFP-FlAsH pair in classical bleaching experiments. Second, we demonstrate the advantage of this enhanced FRET technique for ligand binding studies in live cells by the increased dynamic FRET response between the inactive and active state of the M1 mAChR receptor. We believe that our techniques has particular potential for pharmaceutical drug screening to boost non-ideal FRET probes, and thus amplify signal responses without interfering with the well-characterized molecular assay.

In Situ Probing Intracellular Drug Release from Redox-Responsive Micelles by United FRET and AIE.

Redox-responsive micelles are versatile nanoplatforms for on-demand drug delivery, but the in situ evaluation of drug release is challenging. Fluorescence resonance energy transfer (FRET) technique shows potential for addressing this, while the aggregation-caused quenching effect limits the assay sensitivity. The aim of the current work is to combine aggregation-induced emission (AIE) probe with FRET to realize drug release assessment from micelles. Tetraphenylethene (TPE) is selected as AIE dye and curcumin (Cur) is chosen as the model drug as well as FRET receptor. The drug is covalently linked to a block copolymer via the disulfide bond linker and TPE is also chemically linked to the polymer via an amide bond; the obtained amphiphilic polymer conjugate self-assembles into micelles with a hydrodynamic size of ≈125 nm. Upon the supplement of glutathione or tris(2-carboxyethyl)phosphine) trigger (10 × 10-3 m), the drug release induces the fluorescence increase of both TPE and Cur. Accompanied with the FRET decay, absorption enhancement and particle size increase are observed. The same phenomenon is observed in MCF-7 cells. The FRET-AIE approach can be a useful addition to the spectrum of available methods for monitoring drug release from stimuli-responsive nanomedicine.

A partially-open inward-facing intermediate conformation of LeuT is associated with Na+ release and substrate transport

Neurotransmitter:sodium symporters (NSS), targets of antidepressants and psychostimulants, clear neurotransmitters from the synaptic cleft through sodium (Na+)-coupled transport. Substrate and Na+ are thought to be transported from the extracellular to intracellular space through an alternating access mechanism by coordinated conformational rearrangements in the symporter that alternately expose the binding sites to each side of the membrane. However, the mechanism by which the binding of ligands coordinates conformational changes occurring on opposite sides of the membrane is not well understood. Here, we report the use of single-molecule fluorescence resonance energy transfer (smFRET) techniques to image transitions between distinct conformational states on both the extracellular and intracellular sides of the prokaryotic NSS LeuT, including partially open intermediates associated with transport activity. The nature and functional context of these hitherto unidentified intermediate states shed new light on the allosteric mechanism that couples substrate and Na+ symport by the NSS family through conformational dynamics.

Spying on protein interactions in living cells with reconstituted scarlet light

The BiFC (bimolecular fluorescence complementation) assay and BiFC combined with FRET (fluorescence resonance energy transfer) technique have become important tools for molecular interaction studies in live cells. However, the real detection and cellular imaging performances of most existing red fluorescent protein-derived BiFC assays still suffer from relatively low ensemble brightness, high cytotoxicity, the red fluorescent proteins being prone-to-aggregation or severe residual dimerization, inefficient complementation and slow maturation at 37 °C physiological temperature in live mammalian cells. We developed a BiFC assay based on a recently evolved truly monomeric red fluorescent protein (FP) mScarlet-I with excellent cellular performances such as low cytotoxicity, fast and efficient chromophore maturation and the highest in-cell brightness among all previously reported monomeric red fluorescent proteins. In this work, a classic β-Fos/β-Jun constitutive heterodimerization model and a rapamycin-inducible FRB/FKBP interaction system were used to establish and test the performance of the mScarlet-I-based BiFC assay in live mammalian cells. Furthermore, simply by adopting the large-Stokes-shift fluorescent protein mAmetrine as the donor, β-Jun-β-Fos-NFAT1 ternary protein complex formation could be readily and efficiently detected and visualized with minimal spectral cross-talk in live HeLa cells by combining live-cell sensitized-emission FRET measurement with the mScarlet-I-based BiFC assay. The currently established BiFC assay in this work was also shown to be able to detect and visualize various protein-protein interactions (PPIs) at different subcellular compartments with high specificity and sensitivity at 37 °C physiological temperature in live mammalian cells.

Time-Resolved Fluorescence Spectroscopy Reveals Fine Structure and Dynamics of Poly(l-lysine) and Polyethylenimine Based DNA Polyplexes.

Structural dynamics of the polyethylenimine-DNA and poly(l-lysine)-DNA complexes (polyplexes) was studied by steady-state and time-resolved fluorescence spectroscopy using the fluorescence resonance energy transfer (FRET) technique. During the formation of the DNA polyplexes, the negative phosphate groups (P) of DNA are bound by the positive amine groups (N) of the polymer. At N/P ratio 2, nearly all of the DNA's P groups are bound by the polymer N groups: these complexes form the core of the polyplexes. The excess polymer, added to this system to increase the N/P ratio to the values giving efficient gene delivery, forms a positively charged shell around the core polyplex. We investigated whether the exchange between the core and shell regions of PEI and PLL polyplexes takes place. Our results demonstrated a clear difference between the two studied polymers. Shell PEI can replace PEIs previously attached to DNA in the polyplex core, while PLL cannot. Such a dynamic structure of PEI polyplexes compared to a more static one found for PLL polyplexes partially explains the observed difference in the DNA transfection efficiency of these polyplexes. Moreover, the time-resolved fluorescence spectroscopy revealed additional details on the structure of PLL polyplexes: in between the core and shell, there is an intermediate layer where both core and shell PLLs or their parts overlap.