Rapid degradation ofC. 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, functioning through the activity of 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 method, 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 minutes 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 interacts with 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 provides a conceptual improvement to the AID system for dissecting gene function at the single-cell level during C. elegans development.

Auxin-Induced Degradation of Dream Proteins, Lin-9 and Lin-54, in Caenorhabditis Elegans

“…In these same cells it was observed that after removal from an auxin treatment GFP signal would begin to recover [10]. Since Nishimura's experiment, the system has successfully been used in fission yeast, Drosophila melanogaster, Caenorhabditis elegans, zebrafish, and mouse oocytes [2,3,4,11,12].…”

“…1). The SCF complex is highly conserved among eukaryotes, which allows the transplant of specific Fbox proteins from one organism to another to form a functional SCF complex that can direct the degradation of proteins tagged with the target degron [1,3,4]. In the C. elegans AID system, the exogenous F-box protein used is the Arabidopsis thaliana transport inhibitor response 1 (TIR1) protein [2].…”

“…In the C. elegans AID system, the exogenous F-box protein used is the Arabidopsis thaliana transport inhibitor response 1 (TIR1) protein [2]. TIR1 recognizes tagged substrates and mark them for degradation only in the presence of the plant phytohormone, auxin (indole-3-acetic acid, or IAA) [1,2,3,4]. Therefore, with an endogenous degron-tagged target protein and exogenous TIR1 expression, addition of auxin triggers rapid and efficient degradation of the targeted protein [2,3,4].…”

“…The AID system is not only highly effective in degrading target proteins, but the system has proven to be specific, quick, and reversible in a multitude of species [2,3,4,10,11,12]. The first successful use of the AID system in nonplant cells was completed by Nishimura et al using budding yeast and human cells.…”

“…However, these techniques are indirect, dependent on protein stability, and not easily reversible [1]. One new method to manipulate protein levels in cells and organisms, the auxin-inducible degron (AID) system, is effective in its depletion of proteins and is conditional, fast-acting, and reversible [1,2,3,4]. This system relies on both the presence of auxin and the expression of Arabidopsis thaliana F-box protein TIR1, which serves as the substrate recognition component for the SCF E3 ubiquitin ligase complex, resulting in the degradation of degron tagged proteins by a proteosome [1,2].…”