Expanded palette of Nano-lanterns for real-time multicolor luminescence imaging.

Fluorescence live imaging has become an essential methodology in modern cell biology. However, fluorescence requires excitation light, which can sometimes cause potential problems, such as autofluorescence, phototoxicity, and photobleaching. Furthermore, combined with recent optogenetic tools, the light illumination can trigger their unintended activation. Because luminescence imaging does not require excitation light, it is a good candidate as an alternative imaging modality to circumvent these problems. The application of luminescence imaging, however, has been limited by the two drawbacks of existing luminescent protein probes, such as luciferases: namely, low brightness and poor color variants. Here, we report the development of bright cyan and orange luminescent proteins by extending our previous development of the bright yellowish-green luminescent protein Nano-lantern. The color change and the enhancement of brightness were both achieved by bioluminescence resonance energy transfer (BRET) from enhanced Renilla luciferase to a fluorescent protein. The brightness of these cyan and orange Nano-lanterns was ∼20 times brighter than wild-type Renilla luciferase, which allowed us to perform multicolor live imaging of intracellular submicron structures. The rapid dynamics of endosomes and peroxisomes were visualized at around 1-s temporal resolution, and the slow dynamics of focal adhesions were continuously imaged for longer than a few hours without photobleaching or photodamage. In addition, we extended the application of these multicolor Nano-lanterns to simultaneous monitoring of multiple gene expression or Ca(2+) dynamics in different cellular compartments in a single cell.

Compact halo-ligand-conjugated quantum dots for multicolored single-molecule imaging of overcrowding GPCR proteins on cell membranes.

To detect single molecules within the optical diffraction limit (< ca. 200 nm), a multicolored imaging technique is developed using Halo-ligand conjugated quantum dots (Halo-QDs; <6 nm in diameter). Using three types of Halo-QDs, multicolored single-molecule fluorescence imaging of GPCR proteins in Dictyostelium cells is achieved.

Molecular shuttle between extracellular and cytoplasmic space allows for monitoring of GAG biosynthesis in human articular chondrocytes.

BACKGROUND: Cell surface proteoglycans play vital functional roles in various biological processes such as cell proliferation, differentiation, adhesion, inflammation, immune response, sustentation of cartilage tissue and intensity of tissues. We show here that serglycin-like synthetic glycopeptides function efficiently as a molecular shuttle to hijack glycosaminoglycan (GAG) biosynthetic pathway within cells across the plasma membrane. METHODS: Fluorescence (FITC)-labeled tetrapeptide (H-Ser(1)-Gly(2)-Ser(3)-Gly(4)-OH) carrying Galß(1➝4)Xylß1➝ defined as proteoglycan initiator (PGI) monomer and its tandem repeating PGI polymer was employed for direct imaging of cellular uptake and intracellular traffic by confocal laser-scanning microscopy. Novel method for enrichment analysis of GAG-primed PGIs by combined use of anti-FITC antibody and LC/mass spectrometry was established. RESULTS: PGI monomer was incorporated promptly into human articular chondrocytes and distributed in whole cytoplasm including ER/Golgi while PGI polymer localized specifically in nucleus. It was demonstrated that PGIs become good substrates for GAG biosynthesis within the cells and high molecular weight GAGs primed by PGIs is chondroitin sulfate involving N-acetyl-d-galactosamine residues substituted by 4-O-sulfate or 6-O-sulfate group as major components. PGIs activated chondrocytes proliferation and induced up-regulation of the expression level of type II collagen, suggesting that PGIs can function as new class cytokine-like molecules to stimulate cell growth. CONCLUSION: Synthetic serglycin-type PGIs allow for live cell imaging during proteoglycan biosynthesis and structural characterization of GAG-primed PGIs by an antibody-based enrichment protocol. GENERAL SIGNIFICANCE: Novel glycomics designated for investigating proteoglycan biosynthesis, namely real-time GAGomics using synthetic glycopeptides as PGIs, should facilitate greatly dynamic profiling of GAGs in the living cells. This article is part of a Special Issue entitled Glycoproteomics.

Importance of sialic acid residues illuminated by live animal imaging using phosphorylcholine self-assembled monolayer-coated quantum dots.

Glycans are expected to be one of the potential signal molecules for controlling drug targeting/delivery or long-term circulation of biopharmaceuticals. However, the effect of the carbohydrates of artificially glycosylated derivatives on in vivo dynamic distribution profiles after intravenous injection of model animals remains unclear due to the lack of standardized methodology and a suitable platform. We report herein an efficient and versatile method for the preparation of multifunctional quantum dots (QDs) displaying common synthetic glycosides with excellent solubility and long-term stability in aqueous solution without loss of quantum yields. Combined use of an aminooxy-terminated thiol derivative, 11,11'-dithio bis[undec-11-yl 12-(aminooxyacetyl)amino hexa(ethyleneglycol)], and a phosphorylcholine derivative, 11-mercaptoundecylphosphorylcholine, provided QDs with novel functions for the chemical ligation of ketone-functionalized compounds and the prevention of nonspecific protein adsorption concurrently. In vivo near-infrared (NIR) fluorescence imaging of phosphorylcholine self-assembled monolayer (SAM)-coated QDs displaying various simple sugars (glyco-PC-QDs) after administration into the tail vein of the mouse revealed that distinct long-term delocalization over 2 h can be achieved in cases of QDs modified with α-sialic acid residue (Neu5Ac-PC-QDs) and control PC-QDs, while QDs bearing other common sugars, such as α-glucose (Glc-PC-QDs), α-mannose (Man-PC-QDs), α-fucose (Fuc-PC-QDs), lactose (Lac-PC-QDs), ß-glucuronic acid (GlcA-PC-QDs), N-acetyl-ß-D-glucosamine (GlcNAc-PC-QDs), and N-acetyl-ß-D-galactosamine (GalNAc-PC-QDs) residues, accumulated rapidly (5-10 min) in the liver. Sequential enzymatic modifications of GlcNAc-PC-QDs gave Galß1,4GlcNAc-PC-QDs (LacNAc-PC-QDs), Galß1,4(Fucα1,3)GlcNAc-PC-QDs (Le(x)-PC-QDs), Neu5Acα2,3Galß1,4GlcNAc-PC-QDs (sialyl LacNAc-PC-QDs), and Neu5Acα2,3Galß1,4(Fucα1,3)GlcNAc-PC-QDs (sialyl Le(x)-PC-QDs) in quantitative yield as monitored by direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry analyses. Live animal imaging uncovered for the first time that Le(x)-PC-QDs also distributed rapidly in the liver after intravenous injection and almost quenched over 1 h in similar profiles to those of LacNAc-PC-QDs and Lac-PC-QDs. On the other hand, sialyl LacNAc-PC-QDs and sialyl Le(x)-PC-QDs were still retained stably in the whole body after 2 h, while they showed significantly different in vivo dynamics in the tissue distribution, suggesting that structure/sequence of the neighboring sugar residues in the individual sialyl oligosaccharides might influence the final organ-specific distribution. The present results clearly visualize the evidence of an essential role of the terminal sialic acid residue(s) for achieving prolonged in vivo lifetime and biodistribution of various glyco-PC-QDs as a novel class of functional platforms for nanomaterial-based drug targeting/delivery. A standardized protocol using multifunctional PC-QDs should facilitate live animal imaging of ligand-displayed QDs using versatile NIR fluorescence photometry without influence of size-dependent accumulation/excretion pathway for nanoparticles (e.g., viruses) >10 nm in hydrodynamic diameter by the liver.

Compact and stable SNAP ligand-conjugated quantum dots as a fluorescent probe for single-molecule imaging of dynein motor protein.

Compact SNAP ligand-conjugated quantum dots (<10 nm) with high colloidal stability over a wide range of pH (5-9) have been synthesized as fluorescent probe for the single-molecule imaging of dynein motor protein.

Bioluminescence resonance energy transfer coupled near-infrared quantum dots using GST-tagged luciferase for in vivo imaging.

Bioluminescence resonance energy transfer coupled near-infrared quantum dots using glutathione-s-transferase (GST) tagged luciferase were synthesized as luminescent probes for in vivo imaging.

Novel thiosialosides tethered to metal nanoparticles as potent influenza A virus haemagglutinin blockers.

BACKGROUND: The purpose of this study was to develop a new class of influenza A virus haemagglutinin (HA) blockers by tethering thiosialoside molecules to metal nanoparticles and producing glycoclusters that enhance the affinity of HA binding by N-acetylneuraminic acid. METHODS: Oxygen of the glycoside bond of sialoside was replaced with sulfur to prevent hydrolytic digestion of the N-acetylneuraminic acid residue by viral neuraminidase. Two novel thiosialosides, α-2-S-[p-(N-levulinyl)aminophenyl]-5-N-acetylneuraminic acid (Neu5Ac-S-Lev) and α-2-S-[m-(N-levulinyl)aminobenzyl]-5-N-acetylneuraminic acid (Neu5Ac-S-CH2-Lev), were tethered onto the surface of metal nanoparticles via an aminooxy functionalized thiol linker in a glycoblotting reaction. Gold (Au) and silver (Ag) nanoparticles were coated simultaneously with 11-mercaptoundecyl phosphorylcholine to reduce non-specific adsorption of proteins. Phosphorylcholine self-assembled monolayer-coated metals displaying clustered Neu5Ac (Neu5Ac-PCSAM-Au and Neu5Ac-PCSAM-Ag) were subjected to haemagglutination inhibition (HI) assays using the influenza A virus strain A/PR/8/1934 (H1N1). RESULTS: Glyconanoparticles with thiosialosides had potent HI activities. In particular, Neu5Ac-PCSAM-Au with a diameter of 20 nm corresponding to 9.8 µM monosaccharide Neu5Ac was the most potent HA inhibitor. The versatility of this strategy was demonstrated by similar submicromolar HI activities of Neu5Ac-PCSAM-Ag with diameters of 50 nm and 150 nm. CONCLUSIONS: Glycosylated metal nanoparticles were designed and synthesized as potent influenza A virus HA blockers. This study may contribute to the acceleration of the discovery of a new class of nanoparticle anti-influenza drugs.

Fluorescence polarization-based assay using N-glycan-conjugated quantum dots for screening in hemagglutinin blockers for influenza A viruses.

Attachment of influenza virus to susceptible cells is mediated by viral protein hemagglutinin (HA), which recognizes cell surface glycoconjugates that terminate in α-sialosides. To develop anti-influenza drugs based on inhibition of HA-mediated infection, novel fluorescent nanoparticles displaying multiple biantennary N-glycan chains with α-sialosides (A2-PC-QDs) that have high affinity for the HA were designed and constructed. The A2-PC-QDs enabled an easy and efficient fluorescence polarization (FP) assay for detection of interaction with the HA and competitive inhibition even by small molecule compounds against A2-PC-QDs-HA binding. The quantum dot (QD)-based FP assay established in the present study is a useful tool for high-throughput screening and to accelerate the development of novel and more effective blockers of the viral attachment of influenza virus.