The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans

Abstract

Experimental manipulation of protein abundance in living cells or organisms is an essential strategy for investigation of biological regulatory mechanisms. Whereas powerful techniques for protein expression have been developed in Caenorhabditis elegans, existing tools for conditional disruption of protein function are far more limited. To address this, we have adapted the auxin-inducible degradation (AID) system discovered in plants to enable conditional protein depletion in C. elegans. We report that expression of a modified Arabidopsis TIR1 F-box protein mediates robust auxin-dependent depletion of degron-tagged targets. We document the effectiveness of this system for depletion of nuclear and cytoplasmic proteins in diverse somatic and germline tissues throughout development. Target proteins were depleted in as little as 20-30 min, and their expression could be re-established upon auxin removal. We have engineered strains expressing TIR1 under the control of various promoter and 3' UTR sequences to drive tissue-specific or temporally regulated expression. The degron tag can be efficiently introduced by CRISPR/Cas9-based genome editing. We have harnessed this system to explore the roles of dynamically expressed nuclear hormone receptors in molting, and to analyze meiosis-specific roles for proteins required for germ line proliferation. Together, our results demonstrate that the AID system provides a powerful new tool for spatiotemporal regulation and analysis of protein function in a metazoan model organism.

Keywords: Auxin; Auxin-inducible degradation; C. elegans; Degron; Genetic tool; Tissue-specific depletion.

Fig. 1.

The auxin-inducible degradation (AID) system enables degradation of cytoplasmic and nuclear proteins in larval and adult C. elegans. (A) Inducible degradation of the cytoplasmic dynein heavy chain, DHC-1, in the soma. Animals with a degron-GFP cassette inserted at the 3′ end of the endogenous dhc-1 gene in a P_eft-3::TIR1::mRuby::unc-54 3′ UTR genetic background were treated with (+) or without (−) 1 mM auxin for two hours. Worms were then immobilized and imaged. Wild-type (WT) worms treated with 1 mM auxin for two hours were included to measure background fluorescence. L2 larvae are shown for clarity because their germ line has not yet proliferated extensively, facilitating observation of somatic degradation; other developmental stages are shown in Fig. S2. (B) Quantification of DHC-1-degron-GFP degradation in A. Data are presented as the mean±s.d. from three independent experiments (n=18 worms). (C) Inducible degradation of nuclear SMU-2 protein in the soma. L2 larvae expressing degron-SMU-2-GFP from an extrachromosomal array and mRuby-tagged TIR1 from an integrated transgene were treated, immobilized and imaged as described in A. (D) Quantification of degron-SMU-2-GFP degradation in C. Data are presented as the mean±s.d. from three independent experiments (n=17 worms). (E) Inducible degradation in the adult soma. Young adult worms expressing degron-SMU-2-GFP and TIR1-mRuby were treated with (+) or without (−) 1 mM auxin for three hours and then immobilized and imaged as described in A. Scale bars: 50 μm.

Fig. 2.

AID-mediated degradation is rapid and reversible. (A) Young adult worms expressing degron-tagged GFP and TIR1-mRuby from the same somatic driver (P_eft-3; unc-54 3′ UTR) were treated with auxin in S basal buffer containing OP50. Worms were then lysed at various time points, and western blots were performed using antibodies against GFP and tubulin. (B) Degradation rates were determined using the blots shown in A. Data are presented as mean±s.d. from three independent experiments. (C) Low concentrations of auxin permit efficient degradation in larvae. L1 larvae expressing degron-GFP and TIR1-mRuby were treated with 25 μM or 1 mM auxin (+) or without (−) auxin for two hours. Worms were then immobilized and imaged as described in Fig. 1. (D) Conditional degradation is reversible following removal of auxin. L1 larvae treated with 25 μM auxin for two hours in C were transferred onto fresh NGM plates. Recovery of degron-tagged GFP was examined at the indicated time points. Worms without auxin treatment and those left on auxin plates were included as controls. (E) Quantification of the relative recovery rates in D (recovery from 25 μM auxin) and in Fig. S3 (recovery from 1 mM auxin). Data are presented as means±s.d. from three independent experiments. Scale bars: 50 μm.

Fig. 3.

The AID system enables functional analysis of nuclear hormone receptors during development. (A) Representative images of animals from the timed egg lay on control or 1 mM auxin plates following 60 h at 25°C. In the absence of auxin, no defects were seen in any genotype; however, in the presence of auxin, worms expressing degron-tagged NHR-25 and pan-somatic TIR1 showed molting defects (arrow indicated unshed cuticle) and gonadal defects such as tumorous germ lines (note lack of eggs and abnormal germ line). Animals expressing degron-tagged NHR-23 and pan-somatic TIR1 uniformly arrested as L1 larvae. Scale bar: 50 µm. (B) Temporal analysis of inducible protein degradation. Worms of the indicated genotypes were grown for six hours at 25°C following dauer release before 0.25% ethanol (control) or 1 mM auxin were added and samples harvested every 20 min. Lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Stain-free (Bio-Rad) analysis of total protein on each blot is provided as a loading control. Two isoforms of NHR-25 (a and b) are detected, as previously described (Ward, 2015).

Fig. 4.

The AID system permits tissue-specific degradation in C. elegans. (A) The ges-1 promoter was used to drive TIR1 expression in the intestine. L3 larvae carrying this transgene and degron-tagged SMU-2 from arrays were treated with (+) or without (−) 1 mM auxin for three hours. Worms were then dissected and intestines were extruded to monitor residual SMU-2-GFP in this tissue. DNA was stained with DAPI to indicate the nuclei. Insets show higher-magnification views of the outlined regions. (B) Quantification of degron-SMU-2-GFP degradation in the intestine. Data are presented as the mean±s.d. from three independent experiments (n=144 nuclei, 15 worms). (C) Tissue-specific degradation in adults. Young adult worms expressing degron-SMU-2-GFP from arrays and TIR1 in the intestine were treated with (+) or without (−) 1 mM auxin for three hours. Wild-type worms (WT) treated with auxin were included as background control. (D) Inducible degradation in the germ line. Young adults expressing TIR1 driven by the sun-1 promoter and 3′ UTR along with degron-tagged DHC-1 were treated with (+) or without (−) 1 mM auxin for two hours. Worms were then dissected, fixed, and imaged. (E) Inducible degradation in embryos. Eggs laid by hermaphrodites expressing dhc-1::degron::GFP and P_eft-3::TIR1::mRuby::unc-54 3′ UTR were treated with 1 mM or 4 mM auxin (+) or without (−) auxin in S basal buffer for indicated times. Scale bars: 50 µm in A,C,D; 5 μm in E.

Fig. 5.

Conditional depletion of DHC-1 in the germ line reveals its essential function in meiosis. (A) Rapid degradation of DHC-1-degron-GFP in the germ line. dhc-1::degron::GFP; P_sun-1::TIR1::mRuby young adult animals were treated with 1 mM auxin (+) or without (−) auxin for the indicated time. Worms were then dissected, fixed, stained, and imaged. Four enlarged images are included to indicate efficient degradation. (B) Low-magnification views of germ lines stained for SYP-1 (green) and HTP-3 (red) to monitor synapsis. SYP-1 is a synaptonemal complex protein, whereas HTP-3 is a component of the chromosome axes (MacQueen et al., 2002, 2005). Synapsis defect was indicated by mislocalization of SYP-1. dhc-1::degron::GFP; P_sun-1::TIR1::mRuby adults were treated with (+) or without (−) 1 mM auxin for the indicated times. (C) Higher magnification views from the corresponding regions in B. Scale bars: 50 μm in A; 5 μm in B,C.