Methods for Rapid Protein Depletion in C. elegans using Auxin-Inducible Degradation

Nikita S. Divekar; Hannah E. Horton; Sarah M. Wignall

doi:10.1002/cpz1.16

Methods for Rapid Protein Depletion in C. elegans using Auxin-Inducible Degradation

Nikita S. Divekar, Hannah E. Horton, Sarah M. Wignall^*

^*Corresponding author for this work

Molecular Biosciences

Research output: Contribution to journal › Article › peer-review

7 Scopus citations

Abstract

Numerous methods have been developed in model systems to deplete or inactivate proteins to elucidate their functional roles. In Caenorhabditis elegans, a common method for protein depletion is RNA interference (RNAi), in which mRNA is targeted for degradation. C. elegans is also a powerful genetic organism, amenable to large-scale genetic screens and CRISPR-mediated genome editing. However, these approaches largely lead to constitutive inhibition, which can make it difficult to study proteins essential for development or to dissect dynamic cellular processes. Thus, there have been recent efforts to develop methods to rapidly inactivate or deplete proteins to overcome these barriers. One such method that is proving to be exceptionally powerful is auxin-inducible degradation. In order to apply this approach in C. elegans, a 44–amino acid degron tag is added to the protein of interest, and the Arabidopsis ubiquitin ligase TIR1 is expressed in target tissues. When the plant hormone auxin is added, it mediates an interaction between TIR1 and the degron-tagged protein of interest, which triggers ubiquitination of the protein and its rapid degradation via the proteasome. Here, we have outlined multiple methods for inducing auxin-mediated depletion of target proteins in C. elegans, highlighting the versatility and power of this method.

Original language	English (US)
Article number	e16
Journal	Current Protocols
Volume	1
Issue number	2
DOIs	https://doi.org/10.1002/cpz1.16
State	Published - Feb 2021

Funding

We would like to thank Drs. Liangyu Zhang and Abby Dernburg, who pioneered the AID system in , for their invaluable help and advice as we began working with the degron system in our lab. We also thank Wignall lab members past and present for their contributions in optimizing these protocols, especially Gabriel Cavin‐Meza and Dr. Tim Mullen. This work was supported by Cellular and Molecular Basis of Disease (CMBD) Training Grant NIH T32 GM00806 (to HEH) and by NIH R01GM124354 (to SMW). Microscopy was performed at the Biological Imaging Facility at Northwestern University, supported by the Chemistry for Life Processes Institute, the Northwestern University Office for Research, and the Department of Molecular Biosciences. C. elegans

Keywords

AID
C. elegans
auxin
degron
protein degradation

ASJC Scopus subject areas

General Neuroscience
General Biochemistry, Genetics and Molecular Biology
General Immunology and Microbiology
Pharmacology, Toxicology and Pharmaceutics(all)
Health Informatics
Medical Laboratory Technology

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10.1002/cpz1.16

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abstract = "Numerous methods have been developed in model systems to deplete or inactivate proteins to elucidate their functional roles. In Caenorhabditis elegans, a common method for protein depletion is RNA interference (RNAi), in which mRNA is targeted for degradation. C. elegans is also a powerful genetic organism, amenable to large-scale genetic screens and CRISPR-mediated genome editing. However, these approaches largely lead to constitutive inhibition, which can make it difficult to study proteins essential for development or to dissect dynamic cellular processes. Thus, there have been recent efforts to develop methods to rapidly inactivate or deplete proteins to overcome these barriers. One such method that is proving to be exceptionally powerful is auxin-inducible degradation. In order to apply this approach in C. elegans, a 44–amino acid degron tag is added to the protein of interest, and the Arabidopsis ubiquitin ligase TIR1 is expressed in target tissues. When the plant hormone auxin is added, it mediates an interaction between TIR1 and the degron-tagged protein of interest, which triggers ubiquitination of the protein and its rapid degradation via the proteasome. Here, we have outlined multiple methods for inducing auxin-mediated depletion of target proteins in C. elegans, highlighting the versatility and power of this method.",

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Methods for Rapid Protein Depletion in C. elegans using Auxin-Inducible Degradation

Abstract

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Keywords

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