TY - JOUR
T1 - CemOrange2 fusions facilitate multifluorophore subcellular imaging in C. Elegans
AU - Thomas, Brian J.
AU - Wight, Ira E.
AU - Chou, Wendy Y.Y.
AU - Moreno, Marco
AU - Dawson, Zachary
AU - Homayouni, Arielle
AU - Huang, Huiyan
AU - Kim, Hyori
AU - Jia, Hanna
AU - Buland, Justin R.
AU - Wambach, Jennifer A.
AU - Cole, F. Sessions
AU - Pak, Stephen C.
AU - Silverman, Gary A.
AU - Luke, Cliff J.
N1 - Funding Information:
This work was supported by National Institute of Health grants R01DK104946 (https:// www.niddk.nih.gov/, GAS), R01DK114047 (https:// www.niddk.nih.gov/, CJL, GAS), UDN U01HG010215 (https://www.genome.gov/, FSC, JAW), the Children’s Discovery Institute of St. Louis Children’s Hospital Foundation (http:// www.childrensdiscovery.org, CJL, SCP). Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (https://orip.nih.gov/, P40 OD010440). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work was supported by National Institute of Health grants R01DK104946 (https://www. niddk.nih.gov/, GAS), R01DK114047 (https://www.niddk.nih.gov/, CJL, GAS), UDN U01HG010215 (https://www.genome.gov/, FSC, JAW), the Children’s Discovery Institute of St. Louis Children’s Hospital Foundation (http://www.childrensdiscovery.org, CJL, SCP). Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (https://orip.nih.gov/, P40 OD010440). The plasmid constructs described in this manuscript (including CemOrange2, CemCardinal2 and CemNeptune2) were deposited with Addgene (https://www.addgene.org/). C. elegans strains with subcellular structures labeled by CemOrange2 fusion proteins were deposited in the Caenorhabditis Genetics Center (https://cgc.umn.edu/).
Publisher Copyright:
© 2019 Thomas et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2019/3
Y1 - 2019/3
N2 - Due to its ease of genetic manipulation and transparency, Caenorhabditis elegans (C. elegans) has become a preferred model system to study gene function by microscopy. The use of Aequorea victoria green fluorescent protein (GFP) fused to proteins or targeting sequences of interest, further expanded upon the utility of C. elegans by labeling subcellular structures, which enables following their disposition during development or in the presence of genetic mutations. Fluorescent proteins with excitation and emission spectra different from that of GFP accelerated the use of multifluorophore imaging in real time. We have expanded the repertoire of fluorescent proteins for use in C. elegans by developing a codon-optimized version of Orange2 (CemOrange2). Proteins or targeting motifs fused to CemOr-ange2 were distinguishable from the more common fluorophores used in the nematode; such as GFP, YFP, and mKate2. We generated a panel of CemOrange2 fusion constructs, and confirmed they were targeted to their correct subcellular addresses by colocalization with independent markers. To demonstrate the potential usefulness of this new panel of fluorescent protein markers, we showed that CemOrange2 fusion proteins could be used to: 1) monitor biological pathways, 2) multiplex with other fluorescent proteins to determine colocalization and 3) gain phenotypic knowledge of a human ABCA3 orthologue, ABT-4, trafficking variant in the C. elegans model organism.
AB - Due to its ease of genetic manipulation and transparency, Caenorhabditis elegans (C. elegans) has become a preferred model system to study gene function by microscopy. The use of Aequorea victoria green fluorescent protein (GFP) fused to proteins or targeting sequences of interest, further expanded upon the utility of C. elegans by labeling subcellular structures, which enables following their disposition during development or in the presence of genetic mutations. Fluorescent proteins with excitation and emission spectra different from that of GFP accelerated the use of multifluorophore imaging in real time. We have expanded the repertoire of fluorescent proteins for use in C. elegans by developing a codon-optimized version of Orange2 (CemOrange2). Proteins or targeting motifs fused to CemOr-ange2 were distinguishable from the more common fluorophores used in the nematode; such as GFP, YFP, and mKate2. We generated a panel of CemOrange2 fusion constructs, and confirmed they were targeted to their correct subcellular addresses by colocalization with independent markers. To demonstrate the potential usefulness of this new panel of fluorescent protein markers, we showed that CemOrange2 fusion proteins could be used to: 1) monitor biological pathways, 2) multiplex with other fluorescent proteins to determine colocalization and 3) gain phenotypic knowledge of a human ABCA3 orthologue, ABT-4, trafficking variant in the C. elegans model organism.
UR - http://www.scopus.com/inward/record.url?scp=85063478829&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0214257
DO - 10.1371/journal.pone.0214257
M3 - Article
C2 - 30913273
AN - SCOPUS:85063478829
SN - 1932-6203
VL - 14
JO - PloS one
JF - PloS one
IS - 3
M1 - e0214257
ER -