Quantitative characterization of the auxin-inducible degron: a guide for dynamic protein depletion in single yeast cells

Abstract

Perturbations are essential for the interrogation of biological systems. The auxin-inducible degron harbors great potential for dynamic protein depletion in yeast. Here, we thoroughly and quantitatively characterize the auxin-inducible degron in single yeast cells. We show that an auxin concentration of 0.25 mM is necessary for fast and uniform protein depletion between single cells, and that in mother cells proteins are depleted faster than their daughters. Although, protein recovery starts immediately after removal of auxin, it takes multiple generations before equilibrium is reached between protein synthesis and dilution, which is when the original protein levels are restored. Further, we found that blue light, used for GFP excitation, together with auxin results in growth defects, caused by the photo-destruction of auxin to its toxic derivatives, which can be avoided if indole-free auxin substitutes are used. Our work provides guidelines for the successful combination of microscopy, microfluidics and the auxin-inducible degron, offering the yeast community an unprecedented tool for dynamic perturbations on the single cell level.

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.…”

“…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).…”

Auxin‐Inducible Degron System for Depletion of Proteins in Saccharomyces cerevisiae

“…We have found that both IAA and NAA can be reliably used to deplete proteins in liquid culture (Doris et al, 2018;Gopalakrishnan et al, 2019;Shetty et al, 2017), as well as on plates. However, certain conditions such as fluorescent light can affect the stability of the auxin and thereby necessitate the use of NAA over IAA (Papagiannakis, de Jonge, Zhang, & Heinemann, 2017). It is advisable to perform a depletion time course and growth on liquid and solid media using both forms of auxin to determine the best source for the particular protein of interest and experimental conditions used.…”

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).…”