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Rapid degradation of C. elegans proteins at single-cell resolution with a synthetic auxin

View ORCID ProfileMichael A. Q. Martinez, View ORCID ProfileBrian A. Kinney, View ORCID ProfileTaylor N. Medwig-Kinney, View ORCID ProfileGuinevere Ashley, View ORCID ProfileJames M. Ragle, Londen Johnson, Joseph Aguilera, View ORCID ProfileChristopher M. Hammell, View ORCID ProfileJordan D. Ward, View ORCID ProfileDavid Q. Matus
doi: https://doi.org/10.1101/716837
Michael A. Q. Martinez
*Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794, USA.
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Brian A. Kinney
†Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
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Taylor N. Medwig-Kinney
*Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794, USA.
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Guinevere Ashley
‡Department of Molecular, Cell, and Developmental Biology, University of California-Santa Cruz, Santa Cruz, CA 95064, USA.
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James M. Ragle
‡Department of Molecular, Cell, and Developmental Biology, University of California-Santa Cruz, Santa Cruz, CA 95064, USA.
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Londen Johnson
‡Department of Molecular, Cell, and Developmental Biology, University of California-Santa Cruz, Santa Cruz, CA 95064, USA.
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Joseph Aguilera
‡Department of Molecular, Cell, and Developmental Biology, University of California-Santa Cruz, Santa Cruz, CA 95064, USA.
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Christopher M. Hammell
†Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
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Jordan D. Ward
‡Department of Molecular, Cell, and Developmental Biology, University of California-Santa Cruz, Santa Cruz, CA 95064, USA.
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David Q. Matus
*Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794, USA.
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  • For correspondence: david.matus@stonybrook.edu
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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.

Footnotes

  • 1. New data added on kinetics of AID::GFP degradation in the presence of K-NAA (see new Figure 3 and Figure S1) 2. Additional mechanistic insight into how the C. elegans SCF complex may interact with TIR1 (see new Figure 4 and Figures S2-S3) 3. Quantification of auxin-independent degradation (see new Figure S4).

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted September 28, 2019.
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Rapid degradation of C. elegans proteins at single-cell resolution with a synthetic auxin
Michael A. Q. Martinez, Brian A. Kinney, Taylor N. Medwig-Kinney, Guinevere Ashley, James M. Ragle, Londen Johnson, Joseph Aguilera, Christopher M. Hammell, Jordan D. Ward, David Q. Matus
bioRxiv 716837; doi: https://doi.org/10.1101/716837
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Rapid degradation of C. elegans proteins at single-cell resolution with a synthetic auxin
Michael A. Q. Martinez, Brian A. Kinney, Taylor N. Medwig-Kinney, Guinevere Ashley, James M. Ragle, Londen Johnson, Joseph Aguilera, Christopher M. Hammell, Jordan D. Ward, David Q. Matus
bioRxiv 716837; doi: https://doi.org/10.1101/716837

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