Sep 16, 2022
Auxin-induced (AID) protein degradation in drosophila larvae
- Denis Jullien1,
- Adeline Payet1,
- Emmanuelle Guillou1,
- Henri-Marc Bourbon1,
- Sandra Bernat-Fabre1,
- muriel.boube-trey2
- 1Center for Integrative Biology, Molecular Cellular and Developmental (MCD) Biology Unit UMR 5077, University of Toulouse, Toulouse, France;
- 2RESTORE Research Center, Université de Toulouse, INSERM 1301, CNRS 5070, EFS, ENVT, Toulouse, France
Protocol Citation: Denis Jullien, Adeline Payet, Emmanuelle Guillou, Henri-Marc Bourbon, Sandra Bernat-Fabre, muriel.boube-trey 2022. Auxin-induced (AID) protein degradation in drosophila larvae . protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3949pg25/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 11, 2022
Last Modified: September 16, 2022
Protocol Integer ID: 69820
Keywords: AID, Auxin-induced degradation, NAA, targeted protein degradation, drosophila
Funders Acknowledgement:
Agence Nationale de Recherche
Grant ID: ANR-16 CE12-0021-01
Abstract
The present protocol describes how to operate the auxin degradation system in drosophila larvae.The auxin-induced degradation, also referred to as AID, is an efficient targeted protein degradation system widely used in many model organisms. Thanks to the fact that fast degradation is triggered once a small molecule, the auxin, is added to the cell environment, a tight temporal control of the loss of a protein of interest can be achieved. This unique control of when the proteolysis is triggered allows to study the precocious consequences of the loss of function. Importantly, the implementation of this protocol requires genetically modified flies that express the auxin-dependent F-box protein TIR1, usually under the control of the UAS/Gal4 system to achieve a spatial control of the degradation, and in which the AID degron has been inserted (typically using CRISPR) to the coding sequence of the gene of interest. The problem raised by the use of the AID system in drosophila larvae, as in all the metazoans that have thick tegument, is the penetration of the auxin to each indidual cell of the organism. Here we present a non-invasive strategy based on the ingestion by the larva of a nutritive medium that contains auxin. We detail how to prepare the auxin containing food, handle the larvae, and which food container to use. Our method allows a fast degradation in the imaginal discs, as early as 30 minutes after the larvae were transfered to the auxin containing medium, with little inter-individual difference.
Schematic view of the protocol used to operate the AID system in drosophila larvae. Here, a genetic configuration where the expression of TIR1 is driven by the Ubiquitin promoter is shown.
Attachments
Guidelines
The method described here to induce targeted protein degradation with the AID system in drosophila larva is based on feeding the animal with auxin containing nutritive medium. Once present in the digestive asparatus, auxin penetrates in the tissues of the larva.
The protocol workflow is:
Day 1: young well-fed adults are transfered to fresh fly tubes.
Allow the flies to lay eggs for 15 hours, then remove the adults from the tube. This allow to get larvae all at a similar stage, and also avoid overcrowding.
Day 3: prepare auxin containing petri dishes
30 mm plastic petri dishes containing a nutritive medium that contains auxin need to prepared.
Day 4 to day 5: collect larvae and trigger protein degradation, and proceed to phenotype analysis.
To trigger protein degradation, larvae are transfered using forceps to the auxin dishes.
Materials
-Bacteriological agarSigma AldrichCatalog #A5306
-SucroseSigma AldrichCatalog #S9378
- 1-Naphthaleneacetic acidSigma-aldrichCatalog #N0640
Equipment
Fine Forceps
NAME
Forceps
TYPE
Dumont
BRAND
11251-10
SKU
LINK
- 30 mm plastic petri dishes
- Drosophila malanogaster flies with a genotype similar to what shown in the following figure.
Genetic and molecular set-up used for the AID-mediated degradation of Med19 in drosophila. The expression of the auxin-dependent and AID-specific F-box protein TIR1 is controlled by the UAS/Gal4 system. The Gal4 driver is located in the second chromosome, when the UAS::TIR1 is positioned to the third chromosome where the Med19* gene lies. The F-box TIR1 incorporates the endogenous SCF (Skip1 Cul1 F-box) E3 ubiquitin complex c. Auxin (added to the fly food) triggers poly-ubiquitination of Med19* by the SCFTIR1 targeting the Mediator subunit to degradation by the proteasome.
Protocol materials
SucroseMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9378
Materials, Step 2.1
1-Naphthaleneacetic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #N0640
Materials, Step 2.1
Parafilm MMerck MilliporeSigma (Sigma-Aldrich)Catalog #P7793
Step 3.3
Bacteriological agarMerck MilliporeSigma (Sigma-Aldrich)Catalog #A5306
Materials, Step 2.1
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Auxin is toxic when used on cells above 50 mM.
Before start
Genetically modified flies need to be generated before the present protocol can be implemented.
These flies should contain:
1) a transgene allowing the expression of the auxin-dependent F-box protein TIR1, usually under the control of the UAS/Gal4 system (to achieve a spatial control of the degradation),
2) the AID degron inserted (typically using CRISPR) to the coding sequence of the gene of interest.
See materials for an illustrative figure.
.
Carry out AID protein degradation in drosophila larvae
Carry out AID protein degradation in drosophila larvae
5d 10h 28m
5d 10h 28m
Initiate larvae production
Note
In our hands, we found it is time saving to initially invest in generating genetic setups that are ready to use in term of induction of protein degradation by auxin (no crosses required to generate the larvae with the right genetic background allowing the AID system to work). As an example, in the case of degradation of Med19-AID (3rd chromosome), we recombined the UASt::TIR1 transgene (3rd Chromosome) with the Med19-AID allele, and use the 2nd chromosome to carry the Gal4 driver. For instance, for a degradation of Med19-AID directed in the posterior domain of the wing imaginal disc, the genotype for a ready to use system is enGal4; Med19-AID-GFP, UAStTIR1 (see materials for a figure).
Transfer in standard wheat cornmeal fly tubes about 50 healthy young adult flies (1/3 males, 2/3 females) per tube
Note
See the fly genotype required at this step in the previous note. It is essential to perform a no degradation control. Proper fashions to do so include using an identical genotype except that: possibility 1) it is devoid of the gal4 driver, or possibility 2) the gene of interest is devoid of the AID degron.
30m
Let the flies lay eggs Overnight at 25 °C
15h
Remove the flies from the tubes
30m
Incubate the emptied tubes for 96:00:00 at 25 °C until the wandering L3 Larvae appear
4d
Preparation of NAA containing medium
Note
NAA (1-Naphthaleneacetic acid) is a synthetic analog of Auxin offering better solubility and stability.
15h
Mix 1.2 g of agar Bacteriological agarSigma AldrichCatalog #A5306 , 8 g of sucroseSucroseSigma AldrichCatalog #S9378 , and 0.093 g of NAA 1-Naphthaleneacetic acidSigma AldrichCatalog #N0640 , with 200 mL of water.
Note
The composition of the degradation medium is 0.6% agar, 4% sucrose, 2.5mM 2.5*10^-3 Molarity (M) NAA, in water
If required, depending on the nature of the negative control, prepare the same mixture without NAA to produce the control medium. Note that in flies expressing TIR1, a leaking degradation of the AID-tagged protein occures in the absence of auxin.
30m
Microwave until complete agar melting
5m
Cool down to 70 °C
5m
Pour 30mm plastic petri dishes (5ml of melted agar solution per dish)
20m
Store in a plastic bag at 4°C protected from light
Note
The agar NAA medium is stable for two weeks when properly stored.
Triggering protein degradation in L3 larvae
30m
Equilibrate NAA 30 mm dishes, and control dishes, prepared as described in section 2, to room temperature, and add a pinch of yeast granulates to the dishes
30m
Using forceps, gently but quickly transfer up to 50 L3 larvae from tubes prepared as described in section 1 to a 30 mm dish containing NAA (same for control no NAA agar dish)
15m
Quickly seal the dishes using a piece of parafilmParafilm MSigma AldrichCatalog #P7793
to prevent the larvae from escaping
2m
Puncture tiny holes (small enough so that larvae cannot pass through) in the parafilm membrane using a needle in order to ensure oxygen and CO2 exchange
5m
Incubate the dishes at 25 °C the required amount of time
Note
Pronounced degradation is usually achieved within 30 minutes.
1h
Remove the parafilm seal
1m
Using forceps, gently collect the larvae from the agar dishes, and quickly proceed to desired operation, typically dissection of the imaginal discs.
5m