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  • Rapid Degradation of Caenorhabditis elegans Proteins at Single-Cell Resolution with a Synthetic Auxin.

Rapid Degradation of Caenorhabditis elegans Proteins at Single-Cell Resolution with a Synthetic Auxin.

G3 (Bethesda, Md.) (2019-11-16)
Michael A Q Martinez, Brian A Kinney, Taylor N Medwig-Kinney, Guinevere Ashley, James M Ragle, Londen Johnson, Joseph Aguilera, Christopher M Hammell, Jordan D Ward, David Q Matus
ABSTRACT

As developmental biologists in the age of genome editing, we now have access to an ever-increasing array of tools to manipulate endogenous gene expression. The auxin-inducible degradation system allows for spatial and temporal control of protein degradation via a hormone-inducible Arabidopsis F-box protein, transport inhibitor response 1 (TIR1). In the presence of auxin, TIR1 serves as a substrate-recognition component of the E3 ubiquitin ligase complex SKP1-CUL1-F-box (SCF), ubiquitinating auxin-inducible degron (AID)-tagged proteins for proteasomal degradation. Here, we optimize the Caenorhabditis elegans AID system by utilizing 1-naphthaleneacetic acid (NAA), an indole-free synthetic analog of the natural auxin indole-3-acetic acid (IAA). We take advantage of the photostability of NAA to demonstrate via quantitative high-resolution microscopy that rapid degradation of target proteins can be detected in single cells within 30 min of exposure. Additionally, we show that NAA works robustly in both standard growth media and physiological buffer. We also demonstrate that K-NAA, the water-soluble, potassium salt of NAA, can be combined with microfluidics for targeted protein degradation in C. elegans larvae. We provide insight into how the AID system functions in C. elegans by determining that TIR1 depends on C. elegans SKR-1/2, CUL-1, and RBX-1 to degrade target proteins. Finally, we present highly penetrant defects from NAA-mediated degradation of the FTZ-F1 nuclear hormone receptor, NHR-25, during C. elegans uterine-vulval development. Together, this work improves our use and understanding of the AID system for dissecting gene function at the single-cell level during C. elegans development.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
1-Naphthaleneacetic acid, technical grade
Sigma-Aldrich
1-Naphthylacetic acid, 1 mg/mL, BioReagent, suitable for plant cell culture