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

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. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Long‐term auxin‐mediated depletion on plates Support Protocol: Preparation of NGM and NGM‐auxin plates Basic Protocol 2: Rapid auxin‐mediated depletion via soaking Basic Protocol 3: Acute auxin‐mediated depletion in isolated embryos Basic Protocol 4: Assessing auxin‐mediated depletion

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“…We used the auxin inducible degradation (AID) method, in which a protein of interest is tagged at the endogenous locus with a short 44 amino acid degron tag, and the Arabidopsis ubiquitin ligase TIR1 is expressed in the tissue where the protein of interest will be degraded. Addition of the plant hormone auxin mediates an interaction between TIR1 and the degron tag, and subsequently leads to the ubiquitination and degradation of the degron-tagged protein via the proteasome [ 25 , 26 ]. We inserted a degron and GFP coding sequence into the endogenous air-2 gene ( degron ∷ GFP ∷ air-2 ) in a strain expressing TIR1 in the germ line using the sun-1 promoter [ 25 ]; this enabled auxin-inducible degradation of endogenous AIR-2 specifically in the germ cells of the worm.…”

“…In the absence of auxin, the localization of tagged endogenous AIR-2 recapitulated previously-described AIR-2 enrichment at the central region of each bivalent (the “midbivalent”) in each of these strains, indicating that expression of the WT and KD AIR-2 transgenes does not affect the localization of the tagged endogenous protein ( Fig 1C ). We then transferred late larval stage (L4) worms onto auxin plates and incubated them until early adulthood to deplete the endogenously-expressed degron∷GFP∷AIR-2 from the germ line (via overnight incubation on auxin-containing plates) [ 26 ]. Upon auxin treatment, we observed that the anti-degron immunofluorescence was reduced to background levels in all three strains, demonstrating that degron-tagged endogenous AIR-2 was efficiently depleted from the oocytes, either substantially reducing AIR-2 in the germ line ( Fig 1C , “No AIR-2”) or leaving one of the transgenic AIR-2 versions in place of the endogenous ( Fig 1C ; “AIR-2 WT” and “AIR-2 KD”).…”

“…Auxin treatments were performed as detailed in Divekar et al [ 26 ] using the protocol “Long term auxin mediated deletion on plates”; that publication also includes information about applying this technique more broadly. Briefly, NGM plates were poured with 1mM Auxin and spotted using OP50 bacteria.…”

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“…Slides were moved quickly to the Spinning Disk, and acquisition was started as soon as an embryo could be found in order to begin imaging prior to full protein depletion. More detailed auxin treatment protocols are described in (Divekar et al, 2021b).…”

“…Fifteen adult worms, grown on either control RNAi or experimental RNAi, were picked into a 10μL drop of Meiosis Media in the center of a custom-made apparatus for live imaging (Laband et al, 2018;Divekar et al, 2021b). All worms were quickly dissected, and an eyelash pick was used to push remaining worm bodies to the outside of the drop, leaving only the embryos in the center to avoid disruption from worm movement during the imaging process.…”