Auxin-inducible protein degradation as a novel approach for protein depletion and reverse genetic discoveries in mammalian oocytes†

Abstract

The disruption of protein expression is a major approach used for investigating protein function in mammalian oocytes. This is often achieved with RNAi/morpholino-mediated knockdown or gene knockout, leading to long-term loss of proteins of interest. However, these methods have noteworthy limitations, including (a) slow protein turnover can prohibit use of these approaches; (b) essential roles in early events precludes characterization of functions in subsequent events; (c) extended protein loss can allow time for compensatory mechanisms and other unanticipated events that confound interpretation of results. The work presented here examines the use of auxin-inducible degradation, a powerful new approach that overcomes these limitations through the depletion of one's protein of interest through controllable ubiquitin-mediated degradation. This method has been employed in yeast and mammalian cell lines, and here we demonstrate the utility of auxin-inducible degradation in mouse oocytes at multiple stages of meiosis, through degradation of exogenously expressed EGFP. We also evaluate important parameters for experimental design for use of this system in oocytes. This study thus expands the toolkit of researchers in oocyte biology, establishing the use of this unique and versatile approach for depleting proteins in oocytes, and providing researchers with valuable information to make use of this system.

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

“…A potentially more problematic limitation of IAA for live-cell imaging-based applications is cytotoxicity related to excitation with UV and blue light (Folkes and Wardman 2001;Srivastava 2002). Specifically, IAA has been shown in budding yeast (Papagiannakis et al 2017) and mouse oocytes (Camlin and Evans 2019) to cause cytotoxicity, likely due to acceleration of the oxidative decarboxylation of IAA to methylene-oxindole (Srivastava 2002). In budding yeast, IAA exposure during live-cell imaging suppressed cell proliferation (Papagiannakis et al 2017), while mammalian oocytes failed to complete meiotic maturation (Camlin and Evans 2019).…”

“…Specifically, IAA has been shown in budding yeast (Papagiannakis et al 2017) and mouse oocytes (Camlin and Evans 2019) to cause cytotoxicity, likely due to acceleration of the oxidative decarboxylation of IAA to methylene-oxindole (Srivastava 2002). In budding yeast, IAA exposure during live-cell imaging suppressed cell proliferation (Papagiannakis et al 2017), while mammalian oocytes failed to complete meiotic maturation (Camlin and Evans 2019). In both systems, the use of the synthetic auxin, 1-naphthaleneacetic acid (NAA), rescued these cytotoxic responses.…”

“…in C. elegans (Zhang et al 2015) as well as in other commonly used model systems including budding yeast (Nishimura et al 2009), Drosophila (Trost et al 2016;Chen et al 2018), zebrafish (Daniel et al 2018), cultured mammalian cells (Nishimura et al 2009;Holland et al 2012;Natsume et al 2016), and mouse oocytes (Camlin and Evans 2019). This protein degradation system relies on the expression of an Arabidopsis F-box protein called transport inhibitor response 1 (TIR1).…”

“…We also use the water-soluble, potassium salt of NAA (K-NAA) to demonstrate rapid degradation kinetics of an AID-tagged transgenic protein in a C. elegans-based microfluidic device for the first time (Keil et al 2017). Unlike the natural auxin indole-3-acetic acid (IAA), these synthetic auxins are photostable (Yamakawa et al 1979;Papagiannakis et al 2017;Camlin and Evans 2019). Their use in microscopy-based experiments can prevent the production of toxic indole-derivatives during GFP excitation with blue light (Folkes and Wardman 2001;Srivastava 2002) and overcome unwanted phenotypes associated with IAA exposure (Papagiannakis et al 2017;Camlin and Evans 2019).…”

Rapid degradation ofC. elegansproteins at single-cell resolution with a synthetic auxin

“…A potentially more problematic limitation of IAA for live-cell imaging-based applications is 282 cytotoxicity related to excitation with UV and blue light. Specifically, IAA has been shown 283 in yeast (Papagiannakis et al 2017) and mouse oocytes (Camlin and Evans 2019) to 284 cause cytotoxicity, likely due to acceleration of the oxidative decarboxylation of IAA to 285 methylene-oxindole (Srivastava 2002). In yeast, IAA exposure during live-cell imaging 286 suppressed cell proliferation (Papagiannakis et al 2017) and mammalian oocytes failed 287 to complete meiotic maturation (Camlin and Evans 2019).…”