Dataset Information

Direct Visualization of Live Zebrafish Glycans via Single-Step Metabolic Labeling with Fluorophore-Tagged Nucleotide Sugars.

ABSTRACT: Dynamic turnover of cell-surface glycans is involved in a myriad of biological events, making this process an attractive target for in?vivo molecular imaging. Metabolic glycan labeling coupled with bioorthogonal chemistry has paved the way for visualizing glycans in living organisms. However, a two-step labeling sequence is required, which suffers from the tissue-penetration difficulties of the imaging probes. Here, by exploring the substrate promiscuity of endogenous glycosyltransferases, we developed a single-step fluorescent glycan labeling strategy by using fluorophore-tagged analogues of the nucleotide sugars. Injecting fluorophore-tagged sialic acid and fucose into the yolk of zebrafish embryos at the one-cell stage enables systematic imaging of sialylation and fucosylation in live zebrafish embryos at distinct developmental stages. From these studies, we obtained insights into the role of sialylated and fucosylated glycans in zebrafish hematopoiesis.

SUBMITTER: Hong S

PROVIDER: S-EPMC6820142 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Publications

Direct Visualization of Live Zebrafish Glycans via Single-Step Metabolic Labeling with Fluorophore-Tagged Nucleotide Sugars.

Hong Senlian S Sahai-Hernandez Pankaj P Chapla Digantkumar Gopaldas DG Moremen Kelley W KW Traver David D Wu Peng P

Angewandte Chemie (International ed. in English) 20190828 40

Dynamic turnover of cell-surface glycans is involved in a myriad of biological events, making this process an attractive target for in vivo molecular imaging. Metabolic glycan labeling coupled with bioorthogonal chemistry has paved the way for visualizing glycans in living organisms. However, a two-step labeling sequence is required, which suffers from the tissue-penetration difficulties of the imaging probes. Here, by exploring the substrate promiscuity of endogenous glycosyltransferases, we de ...[more]

PMID: 31295389

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Project description:Within infected host cells, mammalian orthoreovirus (MRV) forms viral factories (VFs), which are sites of viral transcription, translation, assembly, and replication. The MRV nonstructural protein μNS comprises the structural matrix of VFs and is involved in recruiting other viral proteins to VF structures. Previous attempts have been made to visualize VF dynamics in live cells, but due to current limitations in recovery of replicating reoviruses carrying large fluorescent protein tags, researchers have been unable to directly assess VF dynamics from virus-produced μNS. We set out to develop a method to overcome this obstacle by utilizing the 6-amino-acid (CCPGCC) tetracysteine (TC) tag and FlAsH-EDT2 reagent. The TC tag was introduced into eight sites throughout μNS, and the capacity of the TC-μNS fusion proteins to form virus factory-like (VFL) structures and colocalize with virus proteins was characterized. Insertion of the TC tag interfered with recombinant virus rescue in six of the eight mutants, likely as a result of loss of VF formation or important virus protein interactions. However, two recombinant (r)TC-μNS viruses were rescued and VF formation, colocalization with associating virus proteins, and characterization of virus replication were subsequently examined. Furthermore, the rTC-μNS viruses were utilized to infect cells and examine VF dynamics using live-cell microscopy. These experiments demonstrate active VF movement with fusion events as well as transient interactions between individual VFs and demonstrate the importance of microtubule stability for VF fusion during MRV infection. This work provides important groundwork for future in-depth studies of VF dynamics and host cell interactions.IMPORTANCE MRV has historically been used as a model to study the double-stranded RNA (dsRNA) Reoviridae family, the members of which infect and cause disease in humans, animals, and plants. During infection, MRV forms VFs that play a critical role in virus infection but remain to be fully characterized. To study VFs, researchers have focused on visualizing the nonstructural protein μNS, which forms the VF matrix. This work provides the first evidence of recovery of replicating reoviruses in which VFs can be labeled in live cells via introduction of a TC tag into the μNS open reading frame. Characterization of each recombinant reovirus sheds light on μNS interactions with viral proteins. Moreover, utilizing the TC-labeling FlAsH-EDT2 biarsenical reagent to visualize VFs, evidence is provided of dynamic VF movement and interactions at least partially dependent on intact microtubules.

Dataset Information

Direct Visualization of Live Zebrafish Glycans via Single-Step Metabolic Labeling with Fluorophore-Tagged Nucleotide Sugars.

Publications

Direct Visualization of Live Zebrafish Glycans via Single-Step Metabolic Labeling with Fluorophore-Tagged Nucleotide Sugars.

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