Abstract
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.
Highlights
Antibody staining techniques provide a sensitive means to visualize cellular dynamics such as protein disposition, vesicular and membrane trafficking and organellar morphogenesis and function
The purpose of this study was to generate a panel of fluorescent protein (FP) markers for subcellular structures that could be used for multiplex imaging in C. elegans, one of the premier model organisms for studying cell biological and developmental processes in real-time [2, 3, 11]
The challenge was to select a FP that did not aggregate spontaneously in vivo, and displayed an Ex/Em spectra that was distinct from the popular FPs already adapted for use in C. elegans (e.g., GFP, YFP, CFP and mCherry) [1, 5, 7,8,9]
Summary
Antibody staining techniques provide a sensitive means to visualize cellular dynamics such as protein disposition, vesicular and membrane trafficking and organellar morphogenesis and function. The images are static and may be distorted by fixation and permeabilization. Subcellular imaging in C. elegans 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
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