Rapid Degradation ofCaenorhabditis elegansProteins at Single-Cell Resolution with a Synthetic Auxin

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. elegansSKR-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.

Two isoforms of the essentialC. elegansArgonaute CSR-1 differentially regulate sperm and oocyte fertility

“…Instead, to assess a role for CSR-1b in sperm-based fertility, we would need to examine male fertility in a transgenerational mating assay similar to (Conine et al, 2013) or rescue the csr-1(tm892) null mutant with a csr-1a transgene expressed from the csr-1 promoter. Selective degradation of CSR-1b during spermatogenesis via a degron approach (Martinez et al, 2020) may also enable such an assessment. Related, any male specific roles for either CSR-1 isoform should also be explored in the future.…”

“…Our studies disentangle a significant amount of complexity surrounding the singly essential C. elegans AGO, CSR-1, and generate a myriad of additional questions surrounding the tissue-specific functions of CSR-1 and other AGOs. Newly developed means of expressing and degrading or inactivating proteins in a tissue specific manner will enable us to understand the roles of each CSR-1 isoform at a finer resolution, in specific cells and tissues (Martinez et al, 2020).…”

Auxin confers protection against ER stress in Caenorhabditis elegans

“…The AID system is increasingly used and constantly being improved to achieve rapid and reversible protein degradation in different systems ( Martinez et al, 2020 ; Sathyan et al, 2019 ). Our results suggest that caution should be applied when using this system to study stress related pathways in C. elegans , and perhaps in other species.…”

“…TIR1 is the Arabidopsis -specific substrate-recognition component of the conserved SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complex, and carries out its function only in the presence of auxin. Once bound to auxin, exogenous TIR1 targets AID-tagged proteins for ubiquitin-dependent proteasomal degradation, allowing for highly efficient, conditional protein depletion in many systems ( Camlin and Evans, 2019 ; Daniel et al, 2018 ; Holland et al, 2012 ; Lee and Lee, 2010 ; Natsume et al, 2016 ; Nishimura et al, 2009 ; Trost et al, 2016 ), including C. elegans ( Martinez et al, 2020 ; Zhang et al, 2015 ).…”

Combining Auxin-Induced Degradation and RNAi Screening Identifies Novel Genes Involved in Lipid Bilayer Stress Sensing in Caenorhabditis elegans

“…The auxin-inducible degradation (AID) system has been recently introduced to mediate fast and reversible degradation of targeted proteins in C. elegans (Zhang et al 2015). A protein of interest can be tagged with a short 68 amino acid sequence (degron), which is recognized by the E3 ubiquitin ligase TIR1, derived from Arabidopsis thaliana, in the presence of a small molecule called auxin (Zhang et al 2015;Martinez et al 2020). Ubiquitination targets the degron-tagged protein for fast degradation by the proteasome.…”