Epitope-based labeling for improved live-imaging of endogenous proteins in C. elegans
Abstract
Visualizing protein expression dynamics with high temporal resolution is essential for understanding how cells acquire specific fates and functions during development, where key decisions can occur within minutes. Conventional direct fluorescent tagging often fails to capture these rapid changes in protein expression due to the relatively slow fluorophore maturation time. Indirect epitope-based labeling strategies offer a promising alternative, yet only a limited number of these systems have been developed and used in the context of multicellular organisms. Here, we evaluate and combine 4 epitope-based indirect labeling systems for live-imaging of proteins in Caenorhabditis elegans: the SunTag, Frankenbody, MoonTag, and AlfaTag systems. Each system uses a fluorescently labeled high-affinity single-chain antibody or nanobody to recognize short peptide epitopes fused to a protein of interest, enabling immediate labeling of newly synthesized proteins. We demonstrate that all 4 systems specifically label epitope-tagged endogenous proteins and show no detectable cross-reactivity when used in dual-color combinations, enabling simultaneous visualization of distinct proteins within the same embryo. In addition, we show that the SunTag system offers 3 major advantages over direct labeling: earlier detection of proteins and enhanced sensitivity through signal amplification (as illustrated by CAM-1) and less impact on the function (as demonstrated for ERM-1). Together, this work provides a resource for the C. elegans community by establishing 4 orthogonal epitope-based labeling systems for multiplex live-imaging of endogenous proteins, opening new opportunities to study rapid protein dynamics during development.
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BibTeX
@article{salm2026epitopebased,
title = {{Epitope-based labeling for improved live-imaging of endogenous proteins in C. elegans}},
author = {Elise van der Salm and Mette H Schroeder and Loes B Steller and Stephanie I Miller and Amelie Scheper and Gwen Nowee and Erik E Griffin and Suzan Ruijtenberg},
journal = {G3: Genes | Genomes | Genetics},
year = {2026},
month = aug,
doi = {10.1093/g3journal/jkag212},
url = {https://doi.org/10.1093/g3journal/jkag212},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Epitope-based labeling for improved live-imaging of endogenous proteins in C. elegans
AU - Elise van der Salm
AU - Mette H Schroeder
AU - Loes B Steller
AU - Stephanie I Miller
AU - Amelie Scheper
AU - Gwen Nowee
AU - Erik E Griffin
AU - Suzan Ruijtenberg
PY - 2026
DA - 2026/08/31
JO - G3: Genes | Genomes | Genetics
DO - 10.1093/g3journal/jkag212
UR - https://doi.org/10.1093/g3journal/jkag212
AB - Visualizing protein expression dynamics with high temporal resolution is essential for understanding how cells acquire specific fates and functions during development, where key decisions can occur within minutes. Conventional direct fluorescent tagging often fails to capture these rapid changes in protein expression due to the relatively slow fluorophore maturation time. Indirect epitope-based labeling strategies offer a promising alternative, yet only a limited number of these systems have been developed and used in the context of multicellular organisms. Here, we evaluate and combine 4 epitope-based indirect labeling systems for live-imaging of proteins in Caenorhabditis elegans: the SunTag, Frankenbody, MoonTag, and AlfaTag systems. Each system uses a fluorescently labeled high-affinity single-chain antibody or nanobody to recognize short peptide epitopes fused to a protein of interest, enabling immediate labeling of newly synthesized proteins. We demonstrate that all 4 systems specifically label epitope-tagged endogenous proteins and show no detectable cross-reactivity when used in dual-color combinations, enabling simultaneous visualization of distinct proteins within the same embryo. In addition, we show that the SunTag system offers 3 major advantages over direct labeling: earlier detection of proteins and enhanced sensitivity through signal amplification (as illustrated by CAM-1) and less impact on the function (as demonstrated for ERM-1). Together, this work provides a resource for the C. elegans community by establishing 4 orthogonal epitope-based labeling systems for multiplex live-imaging of endogenous proteins, opening new opportunities to study rapid protein dynamics during development.
SN - 2160-1836
ER -