Name: Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body (Video) | JoVE Journal
Uploaded: 2022-08-04T15:00:00.000+00:00
Duration: 6 min 2 s
Description: Learn to induce starvation-induced autophagy in Drosophila melanogaster larval fat body using amino acid depletion. This protocol enables clonal analysis of mutant autophagy patterns via GFP-Atg8a imaging.

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5 minutes

We are grateful to THFC and BDSC for providing the fly strains. Dr. Tong Chao is supported by the National Natural Science Foundation of China (32030027, 91754103, 92157201) and Fundamental research funds for the central universities. We thank the core facility in the Life Sciences Institute (LSI) for providing services.

1. Drosophila crossing and egg laying
<ol>
	<li>Introduce 3 male (genotype hsFLP ubiRFP FRT19A; cgGal4 UAS-GFP-Atg8a) and 15 female (genotype y&#39; w* Mu FRT19A/ FM7, Kr GFP) adult flies (see Table of Materials) into a culture vial (with standard cornmeal/molasses/agar Drosophila media at 25 &#176;C) for mating. 
	NOTE: Multiple culture vials of the same cross must be set up to ensure enough larvae for further experiments. The male fly strain with genotype hsFLP ubiRFP FRT19A; cgGal4 UAS-GFP-Atg8a carries an FRT site at the X chromosome near the centromere (FRT19A). It also expresses FLP upon heat shock at 37 &#176;C. A transgene with ubiquitous RFP expression is inserted on the X chromosome. GFP-Atg8a is expressed under the control of cgGal4 in the larval fat body22. The female fly strain with genotype y&#39; w* Mu FRT19A/ FM7, Kr GFP carries a lethal mutation (Mu) and FRT19A on the X chromosome. The detailed crossing scheme is shown in Figure 1.</li>
	<li>For egg laying, transfer the flies to a new culture vial. At 48 h post introduction, tap the &#34;mating&#34; vial until the flies are stunned and drop down on the media at the base of the vial.
	<ol>
		<li>Unplug the &#34;mating&#34; vial and cover its mouth with an unplugged inverted vial with fresh media (fresh culture vial). Then, transfer the flies to the fresh culture vial by flipping and tapping the vials.</li>
		<li>Plug the fresh culture vial (with the transferred flies) and discard the old culture vial. Place this fresh culture vial in a 25 &#176;C incubator for egg laying on the fresh media. 
		NOTE: Prewarming the fresh culture vials to 25 &#176;C for 15 min before the fly transfer process will help the flies adapt to the new environment quickly and hasten egg laying.</li>
	</ol>
	</li>
	<li>Remove the flies after 6 h of egg laying. If the experiments need to be repeated, transfer these flies into another culture vial (as described in step 1.2). Otherwise, discard the flies by dumping them into a flask containing 75% ethanol).
	<ol>
		<li>Incubate the vial with embryos in a 37 &#176;C water bath for 1 h to induce FLP expression. Subsequently, place them in a 25 &#176;C incubator and allow the embryos to continue to develop. 
		&#8203;NOTE: The successful formation of mutant clones can be confirmed subsequently through imaging. The absence of RFP signals marks the mutant clones.</li>
	</ol>
	</li>
</ol>
2. Amino acid starvation induces autophagy
<ol>
	<li>Using a laboratory spatula, scoop out the media containing the developing larvae into a Petri dish 75 h post egg-laying. Add 3 mL of 1x PBS to the dish and gently separate out the culture media and the larvae using long forceps. Select 10 to 15 early third instar larvae. 
	NOTE: Early third instar larvae need to be chosen carefully. The larvae&#39;s developmental stage is critical for this protocol&#39;s success. Due to the restricted egg laying duration, most larvae in the culture vial are expected to be in the early third instar stage by 75 h of incubation. However, different culture media recipes may lead to variations in the developmental timing of the larvae. The criteria for distinguishing early third instar larvae are the body length, the presence of anterior and posterior spiracles, as well as the mandibular hooks of the mouth apparatus23.</li>
	<li>Fill the wells of a 9-well glass depression spot plate (see Table of Materials) with 1x PBS. Place the separated third instar larvae in the wells using long forceps and wash the larvae thoroughly to remove all the media residues.</li>
	<li>Take 5 mL of 20% sucrose (in 1x PBS) solution in an empty vial and place the clean third instar larvae in this solution using long forceps. Incubate this vial in a 25 &#176;C incubator for 6 h before harvesting them for dissection. 
	&#8203;NOTE: The 20% sucrose (in 1x PBS) solution serves as the amino acid-deficient starvation medium.</li>
</ol>
3. Third instar larvae dissection and sample tissue processing
<ol>
	<li>Sharpen two pairs of #5 forceps (see Table of Materials) evenly on both sides with a sharpening stone.</li>
	<li>Add 400 &#181;L of 1x PBS into each well of the 9-well glass depression spot plate and transfer the larvae into the wells with long forceps. Place one larva in a well with the dorsal side (the side with the trachea) facing up.
	<ol>
		<li>Grip the cuticle of the larva with two #5 forceps in the middle of the larval trunk and gently tear open the cuticles. The exposed fat bodies, along with other larval internal tissues, will still be attached to the carcass of the larva. Pull enough to expose the internal organs as much as possible. Repeat this step for all the larvae. 
		NOTE: Each larva has two large pieces of fat body along the body trunk. Fat body tissue is a white, opaque, and flat monolayer that can be easily distinguished under the dissection microscope24.</li>
	</ol>
	</li>
	<li>Transfer the larval carcass into a 1.5 mL microcentrifuge tube containing 500 &#181;L of 4% paraformaldehyde (PFA). Incubate for 30 min at 25 &#176;C without shaking the tubes. 
	CAUTION: 4% PFA is toxic. Wear gloves and masks for protection. 
	NOTE: Preparing 4% PFA in 1x PBS buffer is recommended. PFA powder does not dissolve in 1x PBS instantly. Hence, the mixture must be incubated at 65 &#176;C in an incubator overnight or shaken intermittently for 45 min at 25 &#176;C.</li>
	<li>After 30 min incubation of the carcass with 4% PFA, pipette out the PFA solution, add 500 &#181;L of 1x PBS into the tube, and gently shake the tube on a flat rotator for 10 min before discarding the 1x PBS solution (repeat 3x).</li>
	<li>Using long forceps, transfer the fixed and washed larval carcass to a well in the 9-depression spot plate filled with 1x PBS. Using #5 forceps, remove all the non-fat body tissues.</li>
	<li>Use #5 forceps to mount the pieces of the fat body on a microscope slide using 80% glycerol as the mounting medium and lay a coverslip on top. 
	NOTE: Before mounting, check the pH of the 80% glycerol (using pH papers, pH = 7 is optimal) to protect the GFP signal. Mounting media (see Table of Materials) with DAPI in 80% glycerol will help in imaging the nuclei of fat body cells. These slides are stable for 1 week when stored in the dark at 4 &#176;C.</li>
</ol>

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<li>Xie, Z., Nair, U., Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Atg8+controls+phagophore+expansion+during+autophagosome+formation%5BTitle%5D)+AND+%22Molecular+Biology+of+the+Cell%22%5BJournal%5D)">Atg8 controls phagophore expansion during autophagosome formation.</a> Molecular Biology of the Cell. 19 (8), 3290-3298 (2008).</li>
<li>Shintani, T., Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cargo+proteins+facilitate+the+formation+of+transport+vesicles+in+the+cytoplasm+to+vacuole+targeting+pathway%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Cargo proteins facilitate the formation of transport vesicles in the cytoplasm to vacuole targeting pathway.</a> Journal of Biological Chemistry. 279 (29), 29889-29894 (2004).</li>
<li>Russell, R. C., Yuan, H. X., Guan, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autophagy+regulation+by+nutrient+signaling%5BTitle%5D)+AND+%22Cell+Research%22%5BJournal%5D)">Autophagy regulation by nutrient signaling.</a> Cell Research. 24 (1), 42-57 (2014).</li>
<li>Nagy, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+and+why+to+study+autophagy+in+Drosophila%3A+It%27s+more+than+just+a+garbage+chute%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">How and why to study autophagy in Drosophila: It's more than just a garbage chute.</a> Methods. 75, 151-161 (2015).</li>
<li>Golic, K. G., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+FLP+recombinase+of+yeast+catalyzes+site-specific+recombination+in+the+Drosophila+genome%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome.</a> Cell. 59 (3), 499-509 (1989).</li>
<li>Senecoff, J. F., Cox, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Directionality+in+FLP+protein-promoted+site-specific+recombination+is+mediated+by+DNA-DNA+pairing%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Directionality in FLP protein-promoted site-specific recombination is mediated by DNA-DNA pairing.</a> Journal of Biological Chemistry. 261 (16), 7380-7386 (1986).</li>
<li>Germani, F., Bergantinos, C., Johnston, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mosaic+analysis+in+Drosophila%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Mosaic analysis in Drosophila.</a> Genetics. 208 (2), 473-490 (2018).</li>
<li>Zappia, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((E2F%2FDp+inactivation+in+fat+body+cells+triggers+systemic+metabolic+changes%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">E2F/Dp inactivation in fat body cells triggers systemic metabolic changes.</a> eLife. 10, 67753(2021).</li>
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<li>Rizki, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drosophila+larval+fat+body+surfaces%5BTitle%5D)+AND+%22Wilhelm+Roux%27s+Archives+of+Developmental+Biology%22%5BJournal%5D)">Drosophila larval fat body surfaces.</a> Wilhelm Roux's Archives of Developmental Biology. 192 (1), 1-7 (1983).</li>
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</ol>

The authors declare no conflicts of interest.

The present protocol describes the methods to (1) generate flies carrying mutant clones in the larval fat bodies, (2) induce autophagy through amino acid starvation, and (3) dissect the larval fat bodies. In order to generate clones successfully in the larval fat bodies, the following critical steps need to be carried out diligently. (1) Timing the heat shock accurately is crucial because mitotic recombination only happens when the tissue is undergoing mitosis, and (2) both heat shock temperature and duration are critica...

Autophagy is a "self-eating" process induced by various stresses, including amino acid starvation1. Macroautophagy (hereafter referred to as autophagy) is the most well-studied type of autophagy and plays an irreplaceable role in maintaining cellular homeostasis2. The malfunction of autophagy is associated with several human diseases3. In addition, some autophagy-related genes are potential targets for treating various diseases4. 
Autophagy is regulated in a highly sophisticated manner5. Upon starvation, the isolation membranes sequester cytoplasmic materials to form double-membraned autophagosomes6. Autophagosomes then fuse with endosomes and lysosomes to form amphisomes and autolysosomes. With the help of lysosomal hydrolytic enzymes, the engulfed cytoplasmic contents are degraded, and the nutrients are recycled7. 
Autophagy is an evolutionarily conserved process8. Drosophila melanogaster is a great model for studying the autophagy process in vivo. Amino acid starvation easily induces autophagy in fly fat body tissue, an analog of human liver and adipose tissue9. Defects in autophagy disrupt the distinct puncta patterns of several autophagy-related proteins, such as Atg8, Atg9, Atg18, Syx17, Rab7, LAMP1, and p62, among others10. Therefore, analyzing the patterns of these autophagy markers will help discern the occurrence of autophagy defects and the defective autophagy step. For example, the ubiquitin-like protein Atg8 is the most commonly used autophagy marker11. In Drosophila melanogaster, transgenic strains with a green fluorescent protein (GFP)-tagged Atg8a have been successfully developed12. GFP-Atg8a is diffused in the cytosol and nuclei in the fed cells. Upon starvation, GFP-Atg8a is processed and modified by phosphatidylethanolamine (PE) and forms puncta, which label the isolation membranes and fully developed autophagosomes13,14. Through direct fluorescence microscopy, the autophagy induction can be easily observed as an increase in GFP-Atg8 puncta formation15. Atg8a puncta would not form in response to starvation in the presence of an autophagy initiation defect. As GFP-Atg8a can be quenched and digested by the low pH in autolysosomes, GFP-Atg8a puncta may increase in numbers if autophagy is blocked at late stages16.
As autophagy is highly sensitive to nutrition availability17, slight differences in culture conditions often lead to variations in phenotypes. Therefore, clonal analysis, a method that analyzes mutant cells versus wild-type control cells in the same tissue, has a major advantage in dissecting autophagy defects18. Taking advantage of flippase/flippase recognition target (FLP/FRT)-mediated site-specific recombination between homologous chromosomes, flies carrying mosaic tissues are readily made19,20. The wild-type cells surrounding the mutant cells form a perfect internal control to avoid individual differences21.
The present study describes how to induce autophagy by amino acid starvation and generate GFP-Atg8a-expressing mosaic fat body tissues. These protocols can be used for analyzing differences in autophagy among mutant clones.

<table><tbody><tr><td>1.5 mL microcentrifuge tube</td><td>Axygen</td><td>MCT-150-C</td><td></td></tr><tr><td>#5 Forceps</td><td>Dumont</td><td>RS-5015</td><td></td></tr><tr><td>9 Dressions Spot plate</td><td>PYREX</td><td>7220-85</td><td></td></tr><tr><td>Fluorescence Microscope</td><td>Nikon</td><td>SMZ1500</td><td></td></tr><tr><td>Glycerol</td><td>Sangon Biotech</td><td>A100854-0100</td><td></td></tr><tr><td>KCl</td><td>Sangon Biotech</td><td>A610440-0500</td><td>Composition of 1x PBS solution</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sangon Biotech</td><td>A600445-0500</td><td>Composition of 1x PBS solution</td></tr><tr><td>Laboratory spatula</td><td>Fisher</td><td>14-375-10</td><td></td></tr><tr><td>Long forceps</td><td>R&#039; DEER</td><td>RST-14</td><td></td></tr><tr><td>Microscope cover glass</td><td>CITOTEST</td><td>80340-1130</td><td></td></tr><tr><td>Microscope slides</td><td>CITOTEST</td><td>80302-2104</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sangon Biotech</td><td>A501727-0500</td><td>Composition of 1x PBS solution</td></tr><tr><td>NaCl</td><td>Sangon Biotech</td><td>A610476-0005</td><td>Composition of 1x PBS solution</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Petri dish</td><td>Corning</td><td>430166</td><td></td></tr><tr><td>Standard cornmeal/molasses/agar fly food</td><td></td><td></td><td>Tong Lab-made</td></tr><tr><td>Stereo microscope</td><td>Nikon</td><td>SMZ745</td><td></td></tr><tr><td>Sucrose</td><td>Sinopharm Chemical Reagent Co.,Ltd.</td><td>10021418</td><td></td></tr><tr><td>Vectashield antifade mounting medium with DAPI</td><td>Vectorlabratory</td><td>H-1200-10</td><td>Recommended mounting medium</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Fly stocks&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;y&#039;w* Iso FRT19A&lt;/em&gt;</td><td></td><td></td><td>Tong Lab&#039;s fly stocks</td></tr><tr><td>&lt;em&gt;y&#039;w* Mu1FRT19A/ FM7,Kr GFP&lt;/em&gt;</td><td></td><td></td><td>Tong Lab&#039;s fly stocks</td></tr><tr><td>&lt;em&gt;y&#039;w* Mu2 FRT19A/ FM7,Kr GFP&lt;/em&gt;</td><td></td><td></td><td>Tong Lab&#039;s fly stocks</td></tr><tr><td>&lt;em&gt;hsFLP ubiRFP FRT19A; cgGal4 UAS-GFP-Atg8a&lt;/em&gt;</td><td></td><td></td><td>Tong Lab&#039;s fly stocks</td></tr></tbody></table>

analyzing starvation-induced autophagy in the drosophila melanogaster larval fat body

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. The malfunction of autophagy is associated with aging and multiple human diseases. The autophagy machinery is highly conserved-from yeast to humans.&#160;The larval fat body of Drosophila melanogaster, an analog for vertebrate liver and adipose tissue, provides a unique model for monitoring autophagy in vivo. Autophagy can be easily induced by nutrient starvation in the larval fat body. Most autophagy-related genes are conserved in Drosophila. Many transgenic fly strains expressing tagged autophagy markers have been developed, which facilitates the monitoring of different steps in the autophagy process. The clonal analysis enables a close comparison of autophagy markers in cells with different genotypes in the same piece of tissue. The current protocol details procedures for (1) generating somatic clones in the larval fat body, (2) inducing autophagy via amino acid starvation, and (3) dissecting the larval fat body, aiming to create a model for analyzing differences in autophagy using an autophagosome marker (GFP-Atg8a) and clonal analysis.

Under fed conditions, the GFP-tagged ubiquitin-like protein, GFP-Atg8a, is diffused inside the cells. Upon starvation, it forms green puncta and labels autophagosomes. Once autophagosomes fuse with lysosomes, GFP is quenched in the acidic autolysosomes, and the green puncta disappear. If autophagy is not induced or the autophagosome maturation is accelerated, the number of GFP puncta is expected to be low. However, if the fusion between autophagosomes and lysosomes is blocked or the pH of the autolysosome becomes basic, ...

The present protocol describes the induction of autophagy in the Drosophila melanogaster larval fat body via nutrient depletion and analyzes changes in autophagy using transgenic fly strains.

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. ...

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JoVE Journal

Biology

The limiting factor in the use of the adult Drosophila eye to study neurodegeneration and cell biology is the difficult imaging of intracellular processes. We describe the dissection of single ommatidia to generate a bona-fide primary neuronal cell culture, which can be subject to drug treatment and advanced imaging.

Single Drosophila Ommatidium Dissection and Imaging

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

Functional Analysis of the Larval Feeding Circuit in Drosophila

We herein describe a phagocytosis assay using the dispersed embryonic cells of Drosophila. It enables us to easily and precisely quantify in vivo phagocytosis levels, and to identify new molecules required for the phagocytosis of apoptotic cells.

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and...

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

Here, we provide protocols to perform three simple injury-induced axon degeneration (axon death) assays in Drosophila melanogaster to evaluate the morphological and functional preservation of severed axons and their synapses.

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Described here is a protocol that enables the colorimetric quantification of the amount of food eaten within a defined interval of time by Drosophila melanogaster larvae exposed to diets of different macronutrient quality. These assays are conducted in the context of a neuronal thermogenetic screen.

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Here we present a high-resolution respirometry protocol to analyze bioenergetics in PINK1B9-null mutant fruit flies. The method uses the Substrate-Uncoupler-Inhibitor-Titration (SUIT) protocol.

Analyzing Mitochondrial Function in a Drosophila melanogaster PINK1B9-Null Mutant Using High-resolution Respirometry

The protocol presents a high-throughput, cost-effective method for assessing neurotoxicity in the fruit fly through the quantification of locomotor dysfunction as an alternative to traditional mammalian models. It aims to evaluate the neurotoxic effects of pharmaceutical compounds, environmental agents, or genetic modifications using a sensitive, reproducible, and ethically favorable model.

Behavioral Analysis of Locomotor Dysfunction in Drosophila melanogaster as a Readout for Neurotoxicity

Learn to induce starvation-induced autophagy in Drosophila melanogaster larval fat body using amino acid depletion. This protocol enables clonal analysis of mutant autophagy patterns via GFP-Atg8a imaging.

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

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