Organelle membrane-specific chemical labeling and dynamic imaging in living cells.

Lipids play crucial roles as structural elements, signaling molecules and material transporters in cells. However, the functions and dynamics of lipids within cells remain unclear because of a lack of methods to selectively label lipids in specific organelles and trace their movement by live-cell imaging. We describe here a technology for the selective labeling and fluorescence imaging (microscopic or nanoscopic) of phosphatidylcholine in target organelles. This approach involves the metabolic incorporation of azido-choline, followed by a spatially limited bioorthogonal reaction that enables the visualization and quantitative analysis of interorganelle lipid transport in live cells. More importantly, with live-cell imaging, we obtained direct evidence that the autophagosomal membrane originates from the endoplasmic reticulum. This method is simple and robust and is thus powerful for real-time tracing of interorganelle lipid trafficking.

An in vitro single-molecule assay for eukaryotic cap-dependent translation initiation kinetics.

Eukaryotic mRNAs are predominantly translated via the cap-dependent pathway. Initiation is a rate-limiting step in cap-dependent translation and is the main target of translational control mechanisms. There is a lack of high-resolution techniques for characterizing the cap-dependent initiation kinetics. Here, we report an in vitro single-molecule assay that allows characterization of both initiation and peptide chain elongation kinetics for cap-dependent translation. Surprisingly, the histogram of the first-round initiation time is highly asymmetrical and spans a large time range that is several-fold greater than the average peptide synthesis time in translation reactions with a firefly luciferase-encoding mRNA. Both the histogram and single-molecule trajectories reveal an unexpected high-degree of asynchrony in translation activity between mRNA molecules. Furthermore, by inserting a small stem-loop (ΔG = -4.8 kcal/mol) in the middle of the mRNA 5' untranslated region (UTR), our assay robustly detects small changes in budding yeast initiation kinetics, which could not be resolved by bulk luminescence kinetics. Lastly, we demonstrate the general applicability of this assay to distinct cell-free translation systems by using extracts prepared from budding yeast, wheat germ, and rabbit reticulocyte lysates. This assay should facilitate mechanistic studies of eukaryotic cap-dependent translation initiation and translational control.

Mechanism of Cyanine5 to Cyanine3 Photoconversion and Its Application for High-Density Single-Particle Tracking in a Living Cell.

Cyanine (Cy) dyes are among the most useful organic fluorophores that have found a wide range of applications in single-molecule and super-resolution imaging as well as in other biophysical studies. However, recent observations that blueshifted derivatives of Cy dyes are formed via photoconversion have raised concerns as to the potential artifacts in multicolor imaging. Here, we report the mechanism for the photoconversion of Cy5 to Cy3 that occurs upon photoexcitation during fluorescent imaging. Our studies show that the formal C2H2 excision from Cy5 occurs mainly through an intermolecular pathway involving a combination of bond cleavage and reconstitution while unambiguously confirming the identity of the fluorescent photoproduct of Cy5 to be Cy3 using various spectroscopic tools. The carbonyl products generated from singlet oxygen-mediated photooxidation of Cy5 undergo a sequence of carbon-carbon bond-breaking and -forming events to bring about the novel dye-to-dye transformation. We also show that the deletion of a two-methine unit from the polymethine chain, which results in the formation of blueshifted products, commonly occurs in other cyanine dyes, such as Alexa Fluor 647 (AF647) and Cyanine5.5. The formation of a blueshifted congener dye can obscure the multicolor fluorescence imaging, leading to misinterpretation of the data. We demonstrate that the potentially deleterious photoconversion, however, can be exploited to develop a new photoactivation method for high-density single-particle tracking in a living cell without using UV illumination and cell-toxic additives.

Unsymmetrical cyanine dye via in vivo hitchhiking endogenous albumin affords high-performance NIR-II/photoacoustic imaging and photothermal therapy.

Herein, an unprecedented synergistic strategy for the development of high-performance NIR-II fluorophore is proposed and validated. Based on an unsymmetrical cyanine dye design strategy, the NIR-II emissive dye NIC was successfully developed by replacing only one of the indoline donors of symmetrical cyanine dye ICG with a fully conjugated benz[c,d]indole donor. This minor structural change maximally maintains the high extinction coefficient advantage of cyanine dyes. NIC-ER with endogenous albumin-hitchhiking capability was constructed to further enhance its in vivo fluorescence brightness. In the presence of HSA (Human serum albumin), NIC-ER spontaneously resides in the albumin pocket, and a brilliant ~89-fold increase in fluorescence was observed. Due to its high molar absorptivity and moderate quantum yield, NIC-ER in HSA exhibits bright NIR-II emission with high photostability and significant Stokes shift (>110 nm). Moreover, NIC-ER was successfully employed for tumor-targeted NIR-II/PA imaging and efficient photothermal tumor elimination. Overall, our strategy may open up a new avenue for designing and constructing high-performance NIR-II fluorophores.

Minimizing ATP depletion by oxygen scavengers for single-molecule fluorescence imaging in live cells.

The stability of organic dyes against photobleaching is critical in single-molecule tracking and localization microscopy. Since oxygen accelerates photobleaching of most organic dyes, glucose oxidase is commonly used to slow dye photobleaching by depleting oxygen. As demonstrated here, pyranose-2-oxidase slows bleaching of Alexa647 dye by ∼20-fold. However, oxygen deprivation may pose severe problems for live cells by reducing mitochondrial oxidative phosphorylation and ATP production. We formulate a method to sustain intracellular ATP levels in the presence of oxygen scavengers. Supplementation with metabolic intermediates including glyceraldehyde, glutamine, and α-ketoisocaproate maintained the intracellular ATP level for at least 10 min by balancing between FADH2 and NADH despite reduced oxygen levels. Furthermore, those metabolites supported ATP-dependent synthesis of phosphatidylinositol 4,5-bisphosphate and internalization of PAR2 receptors. Our method is potentially relevant to other circumstances that involve acute drops of oxygen levels, such as ischemic damage in the brain or heart or tissues for transplantation.

Comparative Antimicrobial Activity of Hp404 Peptide and Its Analogs against Acinetobacter baumannii.

An amphipathic α-helical peptide, Hp1404, was isolated from the venomous gland of the scorpion Heterometrus petersii. Hp1404 exhibits antimicrobial activity against methicillin-resistant Staphylococcus aureus but is cytotoxic. In this study, we designed antimicrobial peptides by substituting amino acids at the 14 C-terminal residues of Hp1404 to reduce toxicity and improve antibacterial activity. The analog peptides, which had an amphipathic α-helical structure, were active against gram-positive and gram-negative bacteria, particularly multidrug-resistant Acinetobacter baumannii, and showed lower cytotoxicity than Hp1404. N-phenyl-1-naphthylamine uptake and DisC3-5 assays demonstrated that the peptides kill bacteria by effectively permeating the outer and cytoplasmic membranes. Additionally, the analog peptides inhibited biofilm formation largely than Hp1404 at low concentrations. These results suggest that the analog peptides of Hp1404 can be used as therapeutic agents against A. baumannii infection.

Assessing G4-Binding Ligands In Vitro and in Cellulo Using Dimeric Carbocyanine Dye Displacement Assay.

G-quadruplexes (G4) are the most actively studied non-canonical secondary structures formed by contiguous repeats of guanines in DNA or RNA strands. Small molecule mediated targeting of G-quadruplexes has emerged as an attractive tool for visualization and stabilization of these structures inside the cell. Limited number of DNA and RNA G4-selective assays have been reported for primary ligand screening. A combination of fluorescence spectroscopy, AFM, CD, PAGE, and confocal microscopy have been used to assess a dimeric carbocyanine dye B6,5 for screening G4-binding ligands in vitro and in cellulo. The dye B6,5 interacts with physiologically relevant DNA and RNA G4 structures, resulting in fluorescence enhancement of the molecule as an in vitro readout for G4 selectivity. Interaction of the dye with G4 is accompanied by quadruplex stabilization that extends its use in primary screening of G4 specific ligands. The molecule is cell permeable and enables visualization of quadruplex dominated cellular regions of nucleoli using confocal microscopy. The dye is displaced by quarfloxin in live cells. The dye B6,5 shows remarkable duplex to quadruplex selectivity in vitro along with ligand-like stabilization of DNA G4 structures. Cell permeability and response to RNA G4 structures project the dye with interesting theranostic potential. Our results validate that B6,5 can serve the dual purpose of visualization of DNA and RNA G4 structures and screening of G4 specific ligands, and adds to the limited number of probes with such potential.

Membrane potential is vital for rapid permeabilization of plasma membranes and lipid bilayers by the antimicrobial peptide lactoferricin B.

Lactoferricin B (LfcinB) is a cationic antimicrobial peptide, and its capacity to damage the bacterial plasma membrane is suggested to be a main factor in LfcinB's antimicrobial activity. However, the specific processes and mechanisms in LfcinB-induced membrane damage are unclear. In this report, using confocal laser-scanning microscopy, we examined the interaction of LfcinB with single Escherichia coli cells and spheroplasts containing the water-soluble fluorescent probe calcein in the cytoplasm. LfcinB induced rapid calcein leakage from single E. coli cells and from single spheroplasts, indicating that LfcinB interacts directly with the plasma membrane and induces its rapid permeabilization. The proton ionophore carbonyl cyanide m-chlorophenylhydrazone suppressed this leakage. Next, we used the single giant unilamellar vesicle (GUV) method to examine LfcinB's interaction with GUVs comprising polar lipid extracts of E. coli containing a water-soluble fluorescent probe, Alexa Fluor 647 hydrazide (AF647). We observed that LfcinB stochastically induces local rupture in single GUVs, causing rapid AF647 leakage; however, higher LfcinB concentrations were required for AF647 leakage from GUVs than from E. coli cells and spheroplasts. To identify the reason for this difference, we examined the effect of membrane potential on LfcinB-induced pore formation, finding that the rate of LfcinB-induced local rupture in GUVs increases greatly with increasing negative membrane potential. These results indicate that membrane potential plays an important role in LfcinB-induced local rupture of lipid bilayers and rapid permeabilization of E. coli plasma membranes. On the basis of these results, we discuss the mode of action of LfcinB's antimicrobial activity.

Series of Mitochondria/Lysosomes Self-Targetable Near-Infrared Hemicyanine Dyes for Viscosity Detection.

Six mitochondria/lysosomes self-targetable and viscosity-sensitive dyes (1a-1f) were developed via simple structure modification on cyanine-derived dyes. They all showed remarkable OFF-ON fluorescent response to viscosity in the near-infrared region (652-690 nm) and exhibited good linear relationship with solution viscosity. The transient absorption spectra were used to evaluate the excited-state lifetime of dye 1a in different viscosity environments. Furthermore, cellular imaging assays indicated that different derivatives (1a-1f) with the same chromophore core exhibited different organelle-targeting abilities. Among them, dyes 1a-1c could sense lysosomal viscosity fluctuations while dyes 1d-1f could be applied in mitochondrial viscosity detections.

Sulfenic Acid-Mediated on-Site-Specific Immobilization of Mitochondrial-Targeted NIR Fluorescent Probe for Prolonged Tumor Imaging.

Mitochondria plays pivotal roles in energy production and apoptotic pathways. Mitochondria-targeting strategy has been recognized as a promising way for cancer theranostics. Thus, spatiotemporally manipulating the prolonged retention of theranostic agents within mitochondria is considerably significant in cancer diagnosis and therapy. Herein, as a proof-of concept, we for the first time report a sulfenic acid-responsive platform on controlled immobilization of probes within mitochondria for prolonged tumor imaging. A novel near-infrared (NIR) probe DATC constructed with a NIR dye (Cy5) as signal unit, a cationic triphenylphosphonium (TPP) for mitochondria targeting, and a sulfenic acid-reactive group (1,3-cyclohexanedione) for mitochondrial fixation was rationally designed and synthesized. This probe displayed good target ability to mitochondria and could act as a promising fluorescent probe for specific visualization of endogenous protein sulfenic acids expressed in the mitochondria. Moreover, the probe could be spontaneously fixed on site through the specific reaction and covalent binding to the sulfenic acids of oxidized proteins under oxidative stress, resulting in enhanced intracellular uptake and prolonged retention. We thus believe that this mitochondria-targeted and locational immobilization strategy may offer a new insight for long-term tumor imaging and effective therapy.

Role of Albumin in Accumulation and Persistence of Tumor-Seeking Cyanine Dyes.

Some heptamethine cyanine dyes accumulate in solid tumors in vivo and persist there for several days. The reasons why they accumulate and persist in tumors were incompletely defined, but explanations based on uptake into cancer cells via organic anion transporting polypeptides (OATPs) have been widely discussed. All cyanine-based "tumor-seeking dyes" have a chloride centrally placed on the heptamethine bridge (a "meso-chloride"). We were intrigued and perplexed by the correlation between this particular functional group and tumor uptake, so the following study was designed. It features four dyes (1-Cl, 1-Ph, 5-Cl, and 5-Ph) with complementary properties. Dye 1-Cl is otherwise known as MHI-148, and 1-Ph is a close analog wherein the meso-chloride has been replaced by a phenyl group. Data presented here shows that both 1-Cl and 1-Ph form noncovalent adducts with albumin, but only 1-Cl can form a covalent one. Both dyes 5-Cl and 5-Ph have a methylene (CH2) unit replaced by a dimethylammonium functionality (N+Me2). Data presented here shows that both these dyes 5 do not form tight noncovalent adducts with albumin, and only 5-Cl can form a covalent one (though much more slowly than 1-Cl). In tissue culture experiments, uptake of dyes 1 is more impacted by the albumin in the media than by the pan-OATP uptake inhibitor (BSP) that has been used to connect uptake of tumor-seeking dyes in vivo with the OATPs. Uptake of 1-Cl in media containing fluorescein-labeled albumin gave a high degree of colocalization of intracellular fluorescence. No evidence was found for the involvement of OATPs in uptake of the dyes into cells in media containing albumin. In an in vivo tumor model, only the two dyes that can form albumin adducts (1-Cl and 5-Cl) gave intratumor fluorescence that persisted long enough to be clearly discerned over the background (∼4 h); this fluorescence was still observed at 48 h. Tumors could be imaged with a higher contrast if 5-Cl is used instead of 1-Cl, because 5-Cl is cleared more rapidly from healthy tissues. Overall, the evidence is consistent with in vitro and in vivo results and indicates that the two dyes in the test series that accumulate in tumors and persist there (1-Cl and 5-Cl, true tumor-seeking dyes) do so as covalent albumin adducts trapped in tumor tissue via uptake by some cancer cells and via the enhanced permeability and retention (EPR) effect.

Fluorescent probes for super-resolution imaging in living cells.

In 1873, Ernst Abbe discovered that features closer than approximately 200 nm cannot be resolved by lens-based light microscopy. In recent years, however, several new far-field super-resolution imaging techniques have broken this diffraction limit, producing, for example, video-rate movies of synaptic vesicles in living neurons with 62 nm spatial resolution. Current research is focused on further improving spatial resolution in an effort to reach the goal of video-rate imaging of live cells with molecular (1-5 nm) resolution. Here, we describe the contributions of fluorescent probes to far-field super-resolution imaging, focusing on fluorescent proteins and organic small-molecule fluorophores. We describe the features of existing super-resolution fluorophores and highlight areas of importance for future research and development.

Florescence Imaging Lung Cancer with a Small Molecule MHI-148.

MHI-148 is a type of heptamethine cyanine dye that can cross the cytoplasmic membrane of lung cancer cells. Here we tested the cytotoxic, in vivo imaging of MHI-148 in lung-cancer nude mice model. Ex vivo imaging was also been measured by testing the major tissue fluorescence intensity. And, the small molecular compound MHI-148 had low cytotoxicity which could be visualized at 1 h post-injection in tumor. From ex vivo fluorescence imaging, the tumor showed the highest uptake of MHI-148 among all the selected organs expect for the time point of 2 h. MHI-148 could be used for effective imaging in lung cancer tissue with good stability and specificity, which suggested that MHI-148 could be an effective tumor clinical imaging agent.

A Complex Comprising a Cyanine Dye Rotaxane and a Porphyrin Nanoring as a Model Light-Harvesting System.

A nanoring-rotaxane supramolecular assembly with a Cy7 cyanine dye (hexamethylindotricarbocyanine) threaded along the axis of the nanoring was synthesized as a model for the energy transfer between the light-harvesting complex LH1 and the reaction center in purple bacteria photosynthesis. The complex displays efficient energy transfer from the central cyanine dye to the surrounding zinc porphyrin nanoring. We present a theoretical model that reproduces the absorption spectrum of the nanoring and quantifies the excitonic coupling between the nanoring and the central dye, thereby explaining the efficient energy transfer and demonstrating similarity with structurally related natural light-harvesting systems.

Integrin αvß3-Specific Hydrocyanine for Cooperative Targeting of Glioblastoma with High Sensitivity and Specificity.

Glioblastoma is a highly malignant brain tumor with poor prognosis and survival rate because of a lack of effective diagnostic methods. Hydrocyanines are a type of reactive oxygen species (ROS)-responsive fluorescent probes, allowing for distinguishing tumor cells from normal cells based on their different intracellular levels of ROS. However, their diagnostic applications for glioblastoma have been limited because of the inability to discriminate between tumor cells and other tissues with high ROS production, leading to high false-positive diagnosis. Therefore, tumor-responsive and -specific hydrocyanines with cooperative targeting ability have great potential for improving the diagnosis and treatment of glioblastoma. Integrin αvß3 plays a critical role in the progression and angiogenesis of glioblastoma and has become a promising target for diagnosing glioblastoma. Herein, we identify a specific peptide ligand for integrin αvß3, Arg-Trp-(d-Arg)-Asn-Arg (RWrNR), which shows high binding affinity to human glioblastoma U87MG cells. Importantly, hydro-Cy5-RWrNR conjugation allowed for distinguishing U87MG cells from normal cells in response to intracellular ROS. Particularly, hydro-Cy5-RWrNR could not only selectively accumulate in orthotopic U87MG tumor with minimal background fluorescence but also effectively discriminate between glioblastoma and inflammatory tissues for the first time, leading to detection of glioblastoma in vivo with high target-to-background ratios and minimal background fluorescence. Therefore, hydro-Cy5-RWrNR is the first integrin αvß3-specific hydrocyanine probe and has great potential in precise tumor diagnosis because of its cooperative targeting of integrin αvß3 and ROS.

Strong Binding and Off-On Signaling Functions of Deep-Red Fluorescent TO-PRO-3 for Influenza†A Virus RNA Promoter Region.

The RNA promoter region of the influenza A virus has recently attracted much attention as an RNA target for the development of anti-influenza drugs. However, there are very few reports on small RNA-binding ligands targeting this region. In this work, it is reported that TO-PRO-3, a thiazole orange analogue with a trimethine bridge, exhibits strong and selective binding to the internal loop structure of the influenza A virus RNA promoter. This binding accompanies the remarkable light-up response of TO-PRO-3 in the deep-red spectral region. By virtue of these binding and fluorescence signaling functions, TO-PRO-3 can act as a useful indicator for the assessment of the binding capabilities of various test compounds for this RNA region, with a view toward the development of anti-influenza drug candidates.

Comparison of JC-1 and MitoTracker probes for mitochondrial viability assessment in stored canine platelet concentrates: A flow cytometry study.

Mitochondria perform crucial roles in many biochemical processes, and mitochondrial depolarization is an early sign of platelet apoptosis. The mitochondrial membrane potential is usually evaluated through JC-1 probe, but it can also be assessed with MitoTracker probes. Our aim was to evaluate mitochondrial viability in stored canine platelet concentrates (PCs) with the fluorescent probes JC-1 and MitoTracker. Platelets from 22 canine PCs were stained with JC-1 and MitoTracker probes on days 1, 3, and 5 of storage. Data on metabolic parameters were also collected for correlation studies. Results of JC-1 and MitoTracker revealed a decrease in mitochondrial membrane potential in day 5 of storage compared to days 1 and 3, providing evidence of mitochondrial depolarization, a finding that was confirmed by the data on metabolic parameters. MitoTracker probes also added information regarding platelet swelling. In conclusion, MitoTracker probes offered a more complete mitochondrial analysis in the evaluation of stored canine PCs. © 2018 International Society for Advancement of Cytometry.

Involvement of multiple influx and efflux transporters in the accumulation of cationic fluorescent dyes by Escherichia coli.

BACKGROUND: It is widely believed that most xenobiotics cross biomembranes by diffusing through the phospholipid bilayer, and that the use of protein transporters is an occasional adjunct. According to an alternative view, phospholipid bilayer transport is negligible, and several different transporters may be involved in the uptake of an individual molecular type. We recognise here that the availability of gene knockout collections allows one to assess the contributions of all potential transporters, and flow cytometry based on fluorescence provides a convenient high-throughput assay for xenobiotic uptake in individual cells. RESULTS: We used high-throughput flow cytometry to assess the ability of individual gene knockout strains of E coli to take up two membrane-permeable, cationic fluorescent dyes, namely the carbocyanine diS-C3(5) and the DNA dye SYBR Green. Individual strains showed a large range of distributions of uptake. The range of modal steady-state uptakes for the carbocyanine between the different strains was 36-fold. Knockouts of the ATP synthase α- and ß-subunits greatly inhibited uptake, implying that most uptake was ATP-driven rather than being driven by a membrane potential. Dozens of transporters changed the steady-state uptake of the dye by more than 50% with respect to that of the wild type, in either direction (increased or decreased); knockouts of known influx and efflux transporters behaved as expected, giving credence to the general strategy. Many of the knockouts with the most reduced uptake were transporter genes of unknown function ('y-genes'). Similarly, several overexpression variants in the 'ASKA' collection had the anticipated, opposite effects. Similar results were obtained with SYBR Green (the range being approximately 69-fold). Although it too contains a benzothiazole motif there was negligible correlation between its uptake and that of the carbocyanine when compared across the various strains (although the membrane potential is presumably the same in each case). CONCLUSIONS: Overall, we conclude that the uptake of these dyes may be catalysed by a great many transporters of putatively broad and presently unknown specificity, and that the very large range between the 'lowest' and the 'highest' levels of uptake, even in knockouts of just single genes, implies strongly that phospholipid bilayer transport is indeed negligible. This work also casts serious doubt upon the use of such dyes as quantitative stains for representing either bioenergetic parameters or the amount of cellular DNA in unfixed cells (in vivo). By contrast, it opens up their potential use as transporter assay substrates in high-throughput screening.

Photoactivated biscarbocyanine dye with two conjugated chromophores: complexes with albumin, photochemical and phototoxic properties.

Complexes of photosensitizers with blood proteins play an essential role in their delivery to the cell, as well as in the efficacy of photodynamic therapy. Biscarbocyanine dye non-covalently binds human serum albumin (HSA), the dissociation constant of the dye with albumin being Kd = (1.7 ± 0.1) × 10-5 M. According to time correlated single photon counting (TCSPC) fluorescence lifetime spectroscopy data, two types of complexes with lifetimes of 1.0 ns and 2.5 ns are formed between the dye and HSA. Confocal fluorescence microscopy has unambiguously shown the penetration of biscarbocyanine into endoplasmic reticulum, lysosomes, mitochondria and nuclei of the cells. The dye demonstrates photocytotoxicity towards the colon carcinoma HCT116 cells with IC50 = 0.3 µM. Hydrophobicity of the polymethine chain and the presence of two positive charges on the dye molecule contribute to the effective binding of the dye with HSA and the penetration into cells. These facts allow considering the biscarbocyanine dye as a promising agent for the photodynamic therapy of cancer.