skip to main content

Explore Research Products in the NSF-PAR

Title: Methods for Rapid Protein Depletion in C. elegans using Auxin‐Inducible Degradation
Abstract

Numerous methods have been developed in model systems to deplete or inactivate proteins to elucidate their functional roles. InCaenorhabditis elegans, a common method for protein depletion is RNA interference (RNAi), in which mRNA is targeted for degradation.C. elegansis also a powerful genetic organism, amenable to large‐scale genetic screens and CRISPR‐mediated genome editing. However, these approaches largely lead to constitutive inhibition, which can make it difficult to study proteins essential for development or to dissect dynamic cellular processes. Thus, there have been recent efforts to develop methods to rapidly inactivate or deplete proteins to overcome these barriers. One such method that is proving to be exceptionally powerful is auxin‐inducible degradation. In order to apply this approach inC. elegans, a 44–amino acid degron tag is added to the protein of interest, and theArabidopsisubiquitin ligase TIR1 is expressed in target tissues. When the plant hormone auxin is added, it mediates an interaction between TIR1 and the degron‐tagged protein of interest, which triggers ubiquitination of the protein and its rapid degradation via the proteasome. Here, we have outlined multiple methods for inducing auxin‐mediated depletion of target proteins inC. elegans, highlighting the versatility and power of this method. © 2021 Wiley Periodicals LLC.

This article was corrected on 19 July 2022. See the end of the full text for details.

Basic Protocol 1: Long‐term auxin‐mediated depletion on plates

Support Protocol: Preparation of NGM and NGM‐auxin plates

Basic Protocol 2: Rapid auxin‐mediated depletion via soaking

Basic Protocol 3: Acute auxin‐mediated depletion in isolated embryos

Basic Protocol 4: Assessing auxin‐mediated depletion

  more » « less
NSF-PAR ID:
10237675
Author(s) / Creator(s):
 ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Current Protocols
Volume:
1
Issue:
2
ISSN:
2691-1299
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ; ; ; ; ; ; et al ( , Genetics)
    Greenstein, D (Ed.)
    Abstract The auxin-inducible degron (AID) system has emerged as a powerful tool to conditionally deplete proteins in a range of organisms and cell types. Here, we describe a toolkit to augment the use of the AID system in Caenorhabditis elegans. We have generated a set of single-copy, tissue-specific (germline, intestine, neuron, muscle, pharynx, hypodermis, seam cell, anchor cell) and pan-somatic TIR1-expressing strains carrying a co-expressed blue fluorescent reporter to enable use of both red and green channels in experiments. These transgenes are inserted into commonly used, well-characterized genetic loci. We confirmed that our TIR1-expressing strains produce the expected depletion phenotype for several nuclear and cytoplasmic AID-tagged endogenous substrates. We have also constructed a set of plasmids for constructing repair templates to generate fluorescent protein::AID fusions through CRISPR/Cas9-mediated genome editing. These plasmids are compatible with commonly used genome editing approaches in the C. elegans community (Gibson or SapTrap assembly of plasmid repair templates or PCR-derived linear repair templates). Together these reagents will complement existing TIR1 strains and facilitate rapid and high-throughput fluorescent protein::AID tagging of genes. This battery of new TIR1-expressing strains and modular, efficient cloning vectors serves as a platform for straightforward assembly of CRISPR/Cas9 repair templates for conditional protein depletion. 
    more » « less
  2. ; ; ; ; ; ; ; ; ( , GENETICS)
    Abstract

    The auxin-inducible degradation system has been widely adopted in the Caenorhabditis elegans research community for its ability to empirically control the spatiotemporal expression of target proteins. This system can efficiently degrade auxin-inducible degron (AID)-tagged proteins via the expression of a ligand-activatable AtTIR1 protein derived from A. thaliana that adapts target proteins to the endogenous C. elegans proteasome. While broad expression of AtTIR1 using strong, ubiquitous promoters can lead to rapid degradation of AID-tagged proteins, cell type-specific expression of AtTIR1 using spatially restricted promoters often results in less efficient target protein degradation. To circumvent this limitation, we have developed an FLP/FRT3-based system that functions to reanimate a dormant, high-powered promoter that can drive sufficient AtTIR1 expression in a cell type-specific manner. We benchmark the utility of this system by generating a number of tissue-specific FLP-ON::TIR1 drivers to reveal genetically separable cell type-specific phenotypes for several target proteins. We also demonstrate that the FLP-ON::TIR1 system is compatible with enhanced degron epitopes. Finally, we provide an expandable toolkit utilizing the basic FLP-ON::TIR1 system that can be adapted to drive optimized AtTIR1 expression in any tissue or cell type of interest.

     
    more » « less
  3. ; ; ; ; ( , Current Protocols)
    Abstract

    Visualization of gene products inCaenorhabditis eleganshas provided insights into the molecular and biological functions of many novel genes in their native contexts. Single‐molecule fluorescencein situhybridization (smFISH) and immunofluorescence (IF) enable the visualization of the abundance and localization of mRNAs and proteins, respectively, allowing researchers to ultimately elucidate the localization, dynamics, and functions of the corresponding genes. Whereas both smFISH and immunofluorescence have been foundational techniques in molecular biology, each protocol poses challenges for use in theC. elegansembryo. smFISH protocols suffer from high initial costs and can photobleach rapidly, and immunofluorescence requires technically challenging permeabilization steps and slide preparation. Most importantly, published smFISH and IF protocols have predominantly been mutually exclusive, preventing the exploration of relationships between an mRNA and a relevant protein in the same sample. Here, we describe protocols to perform immunofluorescence and smFISH inC. elegansembryos either in sequence or simultaneously. We also outline the steps to perform smFISH or immunofluorescence alone, including several improvements and optimizations to existing approaches. These protocols feature improved fixation and permeabilization steps to preserve cellular morphology while maintaining probe and antibody accessibility in the embryo, a streamlined, in‐tube approach for antibody staining that negates freeze‐cracking, a validated method to perform the cost‐reducing single molecule inexpensive FISH (smiFISH) adaptation, slide preparation using empirically determined optimal antifade products, and straightforward quantification and data analysis methods. Finally, we discuss tricks and tips to help the reader optimize and troubleshoot individual steps in each protocol. Together, these protocols simplify existing workflows for single‐molecule RNA and protein detection. Moreover, simultaneous, high‐resolution imaging of proteins and RNAs of interest will permit analysis, quantification, and comparison of protein and RNA distributions, furthering our understanding of the relationship between RNAs and their protein products or cellular markers in early development. © 2021 Wiley Periodicals LLC.

    Basic Protocol 1: Sequential immunofluorescence and single‐molecule fluorescencein situhybridization

    Alternate Protocol: Abbreviated protocol for simultaneous immunofluorescence and single‐molecule fluorescencein situhybridization

    Basic Protocol 2: Simplified immunofluorescence inC. elegansembryos

    Basic Protocol 3: Single‐molecule fluorescencein situhybridization or single‐molecule inexpensive fluorescencein situhybridization

     
    more » « less
  4. ; ; ; ; ( , Current Protocols in Microbiology)
    Abstract

    Facile bacterial genome sequencing has unlocked a veritable treasure trove of novel genes awaiting functional exploration. To make the most of this opportunity requires powerful genetic tools that can target all genes in diverse bacteria. CRISPR interference (CRISPRi) is a programmable gene‐knockdown tool that uses an RNA‐protein complex comprised of a single guide RNA (sgRNA) and a catalytically inactive Cas9 nuclease (dCas9) to sterically block transcription of target genes. We previously developed a suite of modular CRISPRi systems that transfer by conjugation and integrate into the genomes of diverse bacteria, which we call Mobile‐CRISPRi. Here, we provide detailed protocols for the modification and transfer of Mobile‐CRISPRi vectors for the purpose of knocking down target genes in bacteria of interest. We further discuss strategies for optimizing Mobile‐CRISPRi knockdown, transfer, and integration. We cover the following basic protocols: sgRNA design, cloning new sgRNA spacers into Mobile‐CRISPRi vectors, Tn7transfer of Mobile‐CRISPRi to Gram‐negative bacteria, and ICEBs1transfer of Mobile‐CRISPRi to Bacillales. © 2020 The Authors.

    Basic Protocol 1: sgRNA design

    Basic Protocol 2: Cloning of new sgRNA spacers into Mobile‐CRISPRi vectors

    Basic Protocol 3: Tn7transfer of Mobile‐CRISPRi to Gram‐negative bacteria

    Basic Protocol 4: ICEBs1transfer of Mobile‐CRISPRi to Bacillales

    Support Protocol 1: Quantification of CRISPRi repression using fluorescent reporters

    Support Protocol 2: Testing for gene essentiality using CRISPRi spot assays on plates

    Support Protocol 3: Transformation ofE. coliby electroporation

    Support Protocol 4: Transformation of CaCl2‐competentE. coli

     
    more » « less
  5. ; ; ; ; ; ; ; ; ; ; et al ( , Proceedings of the National Academy of Sciences)

    Auxin phytohormones control most aspects of plant development through a complex and interconnected signaling network. In the presence of auxin, AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) transcriptional repressors are targeted for degradation by the SKP1-CULLIN1-F-BOX (SCF) ubiquitin-protein ligases containing TRANSPORT INHIBITOR RESISTANT 1/AUXIN SIGNALING F-BOX (TIR1/AFB). CULLIN1-neddylation is required for SCFTIR1/AFBfunctionality, as exemplified by mutants deficient in the NEDD8-activating enzyme subunit AUXIN-RESISTANT 1 (AXR1). Here, we report a chemical biology screen that identifies small molecules requiring AXR1 to modulate plant development. We selected four molecules of interest, RubNeddin 1 to 4 (RN1 to -4), among which RN3 and RN4 trigger selective auxin responses at transcriptional, biochemical, and morphological levels. This selective activity is explained by their ability to consistently promote the interaction between TIR1 and a specific subset of AUX/IAA proteins, stimulating the degradation of particular AUX/IAA combinations. Finally, we performed a genetic screen using RN4, the RN with the greatest potential for dissecting auxin perception, which revealed that the chromatin remodeling ATPase BRAHMA is implicated in auxin-mediated apical hook development. These results demonstrate the power of selective auxin agonists to dissect auxin perception for plant developmental functions, as well as offering opportunities to discover new molecular players involved in auxin responses.

     
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email