In Vivo Imaging of Membrane-Associated Glycans in Developing Zebrafish

Abstract

Glycans are attractive targets for molecular imaging but have been inaccessible because of their incompatibility with genetically encoded reporters. We demonstrated the noninvasive imaging of glycans in live developing zebrafish, using a chemical reporter strategy. Zebrafish embryos were treated with an unnatural sugar to metabolically label their cell-surface glycans with azides. Subsequently, the embryos were reacted with fluorophore conjugates by means of copper-free click chemistry, enabling the visualization of glycans in vivo at subcellular resolution during development. At 60 hours after fertilization, we observed an increase in de novo glycan biosynthesis in the jaw region, pectoral fins, and olfactory organs. Using a multicolor detection strategy, we performed a spatiotemporal analysis of glycan expression and trafficking and identified patterns that would be undetectable with conventional molecular imaging approaches.The cell-surface glycome is a rich source of information that reports on the cell's physiological state. For example, changes in glycan structures serve as markers of altered gene expression during development (1) and disease progression (2). The dynamics of glycans at the plasma membrane reflect the activity of the cell's secretory machinery (3), and their relative abundances report on flux in metabolic pathways inside the cell (4). Glycans are therefore attractive targets for in vivo imaging but have been inaccessible because of their incompatibility with genetically encoded reporters (5).To image glycans in vivo, we employed a strategy in which an azide is introduced into target biomolecules, priming them for selective covalent reaction with fluorescent probes (5). The azide is small, stable in biological systems, and selectively reactive with phosphines or activated alkynes. Previously, the Staudinger ligation (6,7) or copper-catalyzed click chemistry (8,9) have been used to detect azide-labeled biomolecules on cells ex vivo. However, in vivo †To whom correspondence should be addressed. E-mail: E-mail: crb@berkeley.edu. * These authors contributed equally to this work. imaging of dynamic biological processes using these chemistries could be complicated by slow reaction kinetics or reagent toxicity. The copper-free click reaction of azides with difluorinated cyclooctyne (DIFO) reagents (10) overcomes these limitations, suggesting its potential application to in vivo imaging.We chose zebrafish as a model organism because of their well-defined developmental program (11), emerging disease models (12), and amenability to optical imaging. The metabolic substrate peracetylated N-azidoacetylgalactosamine (Ac 4 GalNAz) was selected on the basis of its known incorporation into mucin-type O-linked glycoproteins in mammalian cells and mice via the N-acetylgalactosamine (GalNAc) salvage pathway (13,14) ( fig. S1). We envisioned an imaging experiment ( Before performing imaging experiments, we confirmed that the zebrafish glycan biosynthetic enzymes are permissive of the unnatural sugar. The...

Molecular protein adaptor with genetically encoded interaction sites guiding the hierarchical assembly of plasmonically active nanoparticle architectures

“…Nevertheless, the yield could not be increased. To finally overcome constraints associated with copper and sodium ascorbate 46 , a newly synthesized rhodamine dye (COMBOrhod) 47 was attached by copper-free ring strain-promoted cycloaddition 48 between the azide-bearing unnatural amino acid and the cyclooctyne group of the dye. This reaction allows for fast site-specific conjugation without the need for copper.…”

Selective chemical protein modification

“…To improve the rate, both difluorinated cyclooctynes 92 (DIFO) and dibenzocylooctynes 93 were independently reported allowing the visualisation of dynamic processes. In a particularly striking example, Laughlin et al 94 utilized DIFOs to visualize the development of glycans during zebrafish embryo growth, demonstrating a high degree of specificity and 'bio-orthogonality' at slightly faster rates than previously reported using the Staudinger ligation. Further enhancements in rates have been reported through the generation of biarylazacyclooctynones 95 and cyclopropyl-fused bicyclononynes 96 (Fig.…”

Tissue-based metabolic labeling of polysialic acids in living primary hippocampal neurons

“…On the other hand, Bertozzi and coworkers (19)(20)(21)(22) metabolically introduced monosaccharidebased chemical reporters-N-acyl derivatives of ManNAc, N-acetyl-D-glucosamine, or N-acetyl-D-galactosamine containing a ketone or azide group-onto cellular surface glycans followed by bioorthogonal, chemoselective coupling with a fluorescent dye or an affinity tag bearing hydrazide/aminooxy (for ketones) or phosphine/alkyne (for azides) group. This elegant methodology, combining metabolic engineering and bioorthogonal reactions, enabled in situ imaging or proteomic enrichment of one glycan type (19)(20)(21)(22) and has been applied to many different cell lines (e.g., Jurkat, HeLa, CHO, and neuron-like blastoma cells) and organisms (e.g., zebrafish, mice, and microbes) for the selective labeling of sialic acid (23)(24)(25), N-acetyl-D-galactosamine residue (in mucin-type O-linked glycan) (26)(27)(28), fucose residue (29)(30)(31)(32), and LPSs/O-antigen (33,34). Although there are innumerable previous reports, the metabolic labeling and the imaging of glycan structures in primary neurons have yet to be achieved, although the surface glycans, especially PSA-NCAMs, play important roles in neuronal development.…”