Cargando…
CemOrange2 fusions facilitate multifluorophore subcellular imaging in C. elegans
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...
Autores principales: | , , , , , , , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2019
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6435234/ https://www.ncbi.nlm.nih.gov/pubmed/30913273 http://dx.doi.org/10.1371/journal.pone.0214257 |
_version_ | 1783406619844935680 |
---|---|
author | Thomas, Brian J. Wight, Ira E. Chou, Wendy Y. Y. Moreno, Marco Dawson, Zachary Homayouni, Arielle Huang, Huiyan Kim, Hyori Jia, Hanna Buland, Justin R. Wambach, Jennifer A. Cole, F. Sessions Pak, Stephen C. Silverman, Gary A. Luke, Cliff J. |
author_facet | Thomas, Brian J. Wight, Ira E. Chou, Wendy Y. Y. Moreno, Marco Dawson, Zachary Homayouni, Arielle Huang, Huiyan Kim, Hyori Jia, Hanna Buland, Justin R. Wambach, Jennifer A. Cole, F. Sessions Pak, Stephen C. Silverman, Gary A. Luke, Cliff J. |
author_sort | Thomas, Brian J. |
collection | PubMed |
description | 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 CemOrange2 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. |
format | Online Article Text |
id | pubmed-6435234 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-64352342019-04-08 CemOrange2 fusions facilitate multifluorophore subcellular imaging in C. elegans Thomas, Brian J. Wight, Ira E. Chou, Wendy Y. Y. Moreno, Marco Dawson, Zachary Homayouni, Arielle Huang, Huiyan Kim, Hyori Jia, Hanna Buland, Justin R. Wambach, Jennifer A. Cole, F. Sessions Pak, Stephen C. Silverman, Gary A. Luke, Cliff J. PLoS One Research Article 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 CemOrange2 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. Public Library of Science 2019-03-26 /pmc/articles/PMC6435234/ /pubmed/30913273 http://dx.doi.org/10.1371/journal.pone.0214257 Text en © 2019 Thomas et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Thomas, Brian J. Wight, Ira E. Chou, Wendy Y. Y. Moreno, Marco Dawson, Zachary Homayouni, Arielle Huang, Huiyan Kim, Hyori Jia, Hanna Buland, Justin R. Wambach, Jennifer A. Cole, F. Sessions Pak, Stephen C. Silverman, Gary A. Luke, Cliff J. CemOrange2 fusions facilitate multifluorophore subcellular imaging in C. elegans |
title | CemOrange2 fusions facilitate multifluorophore subcellular imaging in C. elegans |
title_full | CemOrange2 fusions facilitate multifluorophore subcellular imaging in C. elegans |
title_fullStr | CemOrange2 fusions facilitate multifluorophore subcellular imaging in C. elegans |
title_full_unstemmed | CemOrange2 fusions facilitate multifluorophore subcellular imaging in C. elegans |
title_short | CemOrange2 fusions facilitate multifluorophore subcellular imaging in C. elegans |
title_sort | cemorange2 fusions facilitate multifluorophore subcellular imaging in c. elegans |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6435234/ https://www.ncbi.nlm.nih.gov/pubmed/30913273 http://dx.doi.org/10.1371/journal.pone.0214257 |
work_keys_str_mv | AT thomasbrianj cemorange2fusionsfacilitatemultifluorophoresubcellularimagingincelegans AT wightirae cemorange2fusionsfacilitatemultifluorophoresubcellularimagingincelegans AT chouwendyyy cemorange2fusionsfacilitatemultifluorophoresubcellularimagingincelegans AT morenomarco cemorange2fusionsfacilitatemultifluorophoresubcellularimagingincelegans AT dawsonzachary cemorange2fusionsfacilitatemultifluorophoresubcellularimagingincelegans AT homayouniarielle cemorange2fusionsfacilitatemultifluorophoresubcellularimagingincelegans AT huanghuiyan cemorange2fusionsfacilitatemultifluorophoresubcellularimagingincelegans AT kimhyori cemorange2fusionsfacilitatemultifluorophoresubcellularimagingincelegans AT jiahanna cemorange2fusionsfacilitatemultifluorophoresubcellularimagingincelegans AT bulandjustinr cemorange2fusionsfacilitatemultifluorophoresubcellularimagingincelegans AT wambachjennifera cemorange2fusionsfacilitatemultifluorophoresubcellularimagingincelegans AT colefsessions cemorange2fusionsfacilitatemultifluorophoresubcellularimagingincelegans AT pakstephenc cemorange2fusionsfacilitatemultifluorophoresubcellularimagingincelegans AT silvermangarya cemorange2fusionsfacilitatemultifluorophoresubcellularimagingincelegans AT lukecliffj cemorange2fusionsfacilitatemultifluorophoresubcellularimagingincelegans |