Limits...
In vivo imaging of Caenorhabditis elegans glycans.

Laughlin ST, Bertozzi CR - ACS Chem. Biol. (2009)

Bottom Line: We treated worms with azidosugar variants of N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), and N-acetylmannosamine (ManNAc), resulting in the metabolic labeling of their cell-surface glycans with azides.Subsequently, the worms were reacted via copper-free click reaction with fluorophore-conjugated difluorinated cyclooctyne (DIFO) reagents.We identified prominent localization of mucins in the pharynx of all four larval stages, in the adult hermaphrodite pharynx, vulva and anus, and in the tail of the adult male.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.

ABSTRACT
The nematode Caenorhabditis elegans is an excellent model organism for studies of glycan dynamics, a goal that requires tools for imaging glycans in vivo. Here we applied the bioorthogonal chemical reporter technique for the molecular imaging of mucin-type O-glycans in live C. elegans. We treated worms with azidosugar variants of N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), and N-acetylmannosamine (ManNAc), resulting in the metabolic labeling of their cell-surface glycans with azides. Subsequently, the worms were reacted via copper-free click reaction with fluorophore-conjugated difluorinated cyclooctyne (DIFO) reagents. We identified prominent localization of mucins in the pharynx of all four larval stages, in the adult hermaphrodite pharynx, vulva and anus, and in the tail of the adult male. Using a multicolor, time-resolved imaging strategy, we found that the distribution and dynamics of the glycans varied anatomically and with respect to developmental stage.

Show MeSH

Related in: MedlinePlus

In vivo imaging of glycan distribution in larval and adult C. elegans. a) C. elegans adult hermaphrodites that had been incubated with Ac4GalNAz (+) or no sugar (–) were reacted with DIFO-488 and imaged alive. Fluorescence was observed in the pharynx (b), vulva (c), and anus (d) of Ac4GalNAz-treated (+) but not in the no sugar control (−) worms. b–d) Higher magnification images of the pharynx (b), vulva (c), and anus (d) regions. e) C. elegans hermaphrodites at larval stage L1−L4 that had been incubated with Ac4GalNAz (+) or no sugar (−) were reacted with DIFO-488. Fluorescence was observed in the pharynx region of Ac4GalNAz-treated worms. f) C. elegans strain CA151 adult males metabolically labeled with Ac4GalNAz (+) or no sugar (−) were reacted with DIFO-488. Fluorescence was observed in the tail region of Ac4GalNAz-treated worms. DIC denotes differential interference contrast. 488 denotes DIFO-488 fluorescence. Scale bars: (a) 100 μm; (b–f) 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2807738&req=5

fig2: In vivo imaging of glycan distribution in larval and adult C. elegans. a) C. elegans adult hermaphrodites that had been incubated with Ac4GalNAz (+) or no sugar (–) were reacted with DIFO-488 and imaged alive. Fluorescence was observed in the pharynx (b), vulva (c), and anus (d) of Ac4GalNAz-treated (+) but not in the no sugar control (−) worms. b–d) Higher magnification images of the pharynx (b), vulva (c), and anus (d) regions. e) C. elegans hermaphrodites at larval stage L1−L4 that had been incubated with Ac4GalNAz (+) or no sugar (−) were reacted with DIFO-488. Fluorescence was observed in the pharynx region of Ac4GalNAz-treated worms. f) C. elegans strain CA151 adult males metabolically labeled with Ac4GalNAz (+) or no sugar (−) were reacted with DIFO-488. Fluorescence was observed in the tail region of Ac4GalNAz-treated worms. DIC denotes differential interference contrast. 488 denotes DIFO-488 fluorescence. Scale bars: (a) 100 μm; (b–f) 25 μm.

Mentions: Mixed-stage populations of Ac4GalNAz-treated or untreated (“no sugar”) C. elegans were reacted with DIFO-488 for 1 h, washed briefly, and then anesthetized prior to imaging (2). In the adult hermaphrodite, we observed GalNAz-specific labeling in the pharynx, vulva, and anus (2, panels a–d). The labeled glycans were concentrated in the pharynx of all larval and adult worms (2, panels a, b, e), though higher exposure times did reveal some staining of the worms’ outer coat, the cuticle (2, panel d). Like the adult hermaphrodites, the adult males stained intensely in the pharynx region and minimally in the anus and cuticle. However, the male tail also showed striking labeling of the fan, spicule, and rays (2, panel f). Since the C. elegans cuticle is notoriously impenetrable by small molecules (23), we suspect that Ac4GalNAz enters the worm by ingestion. The intense fluorescence observed in the pharynx is consistent with this presumption. Control worms that were not incubated with azidosugar showed minimal background fluorescence, and neither azidosugar metabolism nor DIFO-488 labeling produced observable toxicity. Importantly, the observed fluorescence is not due to Escherichia coli reacting with DIFO-conjugates and adhering to the worms. E. coli were visible in high magnification images next to worms and were not labeled with DIFO-fluorophore conjugates (Supplementary Figure 3).


In vivo imaging of Caenorhabditis elegans glycans.

Laughlin ST, Bertozzi CR - ACS Chem. Biol. (2009)

In vivo imaging of glycan distribution in larval and adult C. elegans. a) C. elegans adult hermaphrodites that had been incubated with Ac4GalNAz (+) or no sugar (–) were reacted with DIFO-488 and imaged alive. Fluorescence was observed in the pharynx (b), vulva (c), and anus (d) of Ac4GalNAz-treated (+) but not in the no sugar control (−) worms. b–d) Higher magnification images of the pharynx (b), vulva (c), and anus (d) regions. e) C. elegans hermaphrodites at larval stage L1−L4 that had been incubated with Ac4GalNAz (+) or no sugar (−) were reacted with DIFO-488. Fluorescence was observed in the pharynx region of Ac4GalNAz-treated worms. f) C. elegans strain CA151 adult males metabolically labeled with Ac4GalNAz (+) or no sugar (−) were reacted with DIFO-488. Fluorescence was observed in the tail region of Ac4GalNAz-treated worms. DIC denotes differential interference contrast. 488 denotes DIFO-488 fluorescence. Scale bars: (a) 100 μm; (b–f) 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2807738&req=5

fig2: In vivo imaging of glycan distribution in larval and adult C. elegans. a) C. elegans adult hermaphrodites that had been incubated with Ac4GalNAz (+) or no sugar (–) were reacted with DIFO-488 and imaged alive. Fluorescence was observed in the pharynx (b), vulva (c), and anus (d) of Ac4GalNAz-treated (+) but not in the no sugar control (−) worms. b–d) Higher magnification images of the pharynx (b), vulva (c), and anus (d) regions. e) C. elegans hermaphrodites at larval stage L1−L4 that had been incubated with Ac4GalNAz (+) or no sugar (−) were reacted with DIFO-488. Fluorescence was observed in the pharynx region of Ac4GalNAz-treated worms. f) C. elegans strain CA151 adult males metabolically labeled with Ac4GalNAz (+) or no sugar (−) were reacted with DIFO-488. Fluorescence was observed in the tail region of Ac4GalNAz-treated worms. DIC denotes differential interference contrast. 488 denotes DIFO-488 fluorescence. Scale bars: (a) 100 μm; (b–f) 25 μm.
Mentions: Mixed-stage populations of Ac4GalNAz-treated or untreated (“no sugar”) C. elegans were reacted with DIFO-488 for 1 h, washed briefly, and then anesthetized prior to imaging (2). In the adult hermaphrodite, we observed GalNAz-specific labeling in the pharynx, vulva, and anus (2, panels a–d). The labeled glycans were concentrated in the pharynx of all larval and adult worms (2, panels a, b, e), though higher exposure times did reveal some staining of the worms’ outer coat, the cuticle (2, panel d). Like the adult hermaphrodites, the adult males stained intensely in the pharynx region and minimally in the anus and cuticle. However, the male tail also showed striking labeling of the fan, spicule, and rays (2, panel f). Since the C. elegans cuticle is notoriously impenetrable by small molecules (23), we suspect that Ac4GalNAz enters the worm by ingestion. The intense fluorescence observed in the pharynx is consistent with this presumption. Control worms that were not incubated with azidosugar showed minimal background fluorescence, and neither azidosugar metabolism nor DIFO-488 labeling produced observable toxicity. Importantly, the observed fluorescence is not due to Escherichia coli reacting with DIFO-conjugates and adhering to the worms. E. coli were visible in high magnification images next to worms and were not labeled with DIFO-fluorophore conjugates (Supplementary Figure 3).

Bottom Line: We treated worms with azidosugar variants of N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), and N-acetylmannosamine (ManNAc), resulting in the metabolic labeling of their cell-surface glycans with azides.Subsequently, the worms were reacted via copper-free click reaction with fluorophore-conjugated difluorinated cyclooctyne (DIFO) reagents.We identified prominent localization of mucins in the pharynx of all four larval stages, in the adult hermaphrodite pharynx, vulva and anus, and in the tail of the adult male.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.

ABSTRACT
The nematode Caenorhabditis elegans is an excellent model organism for studies of glycan dynamics, a goal that requires tools for imaging glycans in vivo. Here we applied the bioorthogonal chemical reporter technique for the molecular imaging of mucin-type O-glycans in live C. elegans. We treated worms with azidosugar variants of N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), and N-acetylmannosamine (ManNAc), resulting in the metabolic labeling of their cell-surface glycans with azides. Subsequently, the worms were reacted via copper-free click reaction with fluorophore-conjugated difluorinated cyclooctyne (DIFO) reagents. We identified prominent localization of mucins in the pharynx of all four larval stages, in the adult hermaphrodite pharynx, vulva and anus, and in the tail of the adult male. Using a multicolor, time-resolved imaging strategy, we found that the distribution and dynamics of the glycans varied anatomically and with respect to developmental stage.

Show MeSH
Related in: MedlinePlus