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 w...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy:+Molecular+machinery+for+self-eating.">Autophagy: Molecular machinery for self-eating.</a> Cell Death &amp; Differentiation. 12 (2), 1542-1552 (2005).</li><li>Jin, S., White, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+autophagy+in+cancer:+Management+of+metabolic+stress.">Role of autophagy in cancer: Management of metabolic stress.</a> Autophagy. 3 (1), 28-31 (2007).</li><li>Mizushima, N., Levine, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+human+diseases.">Autophagy in human diseases.</a> New England Journal of Medicine. 383 (16), 1564-1576 (2020).</li><li>Mizushima, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+fights+disease+through+cellular+self-digestion.">Autophagy fights disease through cellular self-digestion.</a> Nature. 451 (7182), 1069-1075 (2008).</li><li>Yang, Z., Klionsky, D. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atg8+controls+phagophore+expansion+during+autophagosome+formation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cargo+proteins+facilitate+the+formation+of+transport+vesicles+in+the+cytoplasm+to+vacuole+targeting+pathway.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+regulation+by+nutrient+signaling.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+and+why+to+study+autophagy+in+Drosophila:+It's+more+than+just+a+garbage+chute.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+FLP+recombinase+of+yeast+catalyzes+site-specific+recombination+in+the+Drosophila+genome.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directionality+in+FLP+protein-promoted+site-specific+recombination+is+mediated+by+DNA-DNA+pairing.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+analysis+in+Drosophila.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E2F/Dp+inactivation+in+fat+body+cells+triggers+systemic+metabolic+changes.">E2F/Dp inactivation in fat body cells triggers systemic metabolic changes.</a> eLife. 10, 67753 (2021).</li><li>Demerec, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology of Drosophila. , (1965).</li><li>Rizki, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+larval+fat+body+surfaces.">Drosophila larval fat body surfaces.</a> Wilhelm Roux's Archives of Developmental Biology. 192 (1), 1-7 (1983).</li><li>Szeto, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ALIS+are+stress-induced+protein+storage+compartments+for+substrates+of+the+proteasome+and+autophagy.">ALIS are stress-induced protein storage compartments for substrates of the proteasome and autophagy.</a> Autophagy. 2 (3), 189-199 (2006).</li><li>Renna, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+inducers+of+autophagy+that+enhance+the+clearance+of+mutant+proteins+in+neurodegenerative+diseases.">Chemical inducers of autophagy that enhance the clearance of mutant proteins in neurodegenerative diseases.</a> Journal of Biological Chemistry. 285 (15), 11061-11067 (2010).</li><li>Guo, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large-scale+RNA+interference+screen+identifies+genes+that+regulate+autophagy+at+different+stages.">A large-scale RNA interference screen identifies genes that regulate autophagy at different stages.</a> Scientific Reports. 8, 1-15 (2018).</li><li>Tian, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+antibody+for+analysis+of+autophagy+induction.">An antibody for analysis of autophagy induction.</a> Nature Methods. 17 (2), 232-239 (2020).</li><li>Nezis, I. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>KH2PO4</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&#39; 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>Na2HPO4</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>Fly stocks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>y&#39;w* Iso FRT19A</td>
			<td></td>
			<td></td>
			<td>Tong Lab&#39;s fly stocks</td>
		</tr>
		<tr>
			<td>y&#39;w* Mu1FRT19A/ FM7,Kr GFP</td>
			<td></td>
			<td></td>
			<td>Tong Lab&#39;s fly stocks</td>
		</tr>
		<tr>
			<td>y&#39;w* Mu2 FRT19A/ FM7,Kr GFP</td>
			<td></td>
			<td></td>
			<td>Tong Lab&#39;s fly stocks</td>
		</tr>
		<tr>
			<td>hsFLP ubiRFP FRT19A; cgGal4 UAS-GFP-Atg8a</td>
			<td></td>
			<td></td>
			<td>Tong Lab&#39;s fly stocks</td>
		</tr>
	</tbody>
</table>

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, ...

Watch this Scientific Journal Video about Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body at JoVE.com

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

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.

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. ...

This protocol describes how to induce autophagy in the Drosophila melanogaster larval fat body via amino acid depletion and how to analyze the differences in autophagy among mutant clones. As autophagy is highly sensitive to nutrition availability in culture conditions, clonal analysis, a method that analyzes mutant cells versus wild type cells in the same tissue, has an advantage in dissecting autophagy defects. Begin by introducing three males and 15 female adult flies into a culture vial with standard cornmeal or molasses or agar Drosophila media at 25 degrees Celsius for mating.At 48 hours post-induction, tap the mating vial until the flies are stunned and drop down onto the media at the base of the vial. Unplug the mating vial and cover its mouth with an unplugged inverted culture vial with fresh media. Then flip and tap the vials to transfer the flies to the fresh culture vial.Plug the fresh culture vial and discard the old vial. Place the fresh culture vial in a 25 degree Celsius incubator for egg laying on the fresh media. Remove the flies after six hours of egg laying and discard the flies by dumping them into a flask containing 75%ethanol.Incubate the vial with embryos in a 37 degree Celsius water bath for one hour to induce flippase expression. Subsequently, place them in a 25 degree Celsius incubator and allow the embryos to continue to develop. Using a laboratory spatula, scoop out the media containing the developing larvae into a Petri dish 75 hours post egg laying.Add 1X PBS to the dish and gently separate out the culture media and the larvae using long forceps. Then select 10 to 15 early third instar larvae. Fill the wells of a nine-well glass depression spot plate with 1X PBS and place the separated third instar larvae in the wells using long forceps.Wash the larvae thoroughly to remove all the media residues. Take five milliliters of 20%sucrose solution in an empty vial and place the clean third instar larvae in this solution using long forceps. Incubate this vial in a 25 degree Celsius incubator for six hours before harvesting them for dissection.Add 1X PBS into each well of the nine-well glass depression spot plate and transfer the larvae into the wells with long forceps. Place one larvae in a well with the dorsal side facing up. 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.Transfer the larval carcass into a 1.5 milliliter microcentrifuge tube containing 500 microliters of 4%PFA and incubate for 30 minutes at 25 degrees Celsius without shaking the tubes. After 30 minutes, pipette out the PFA solution. Add 500 microliters of 1X PBS into the tube.Gently shake the tube on a flat rotator for 10 minutes and then discard the 1X PBS solution. Repeat this three times. Using long forceps, transfer the fixed and washed larval carcass to a well in the nine depression spot plate filled with 1X PBS.Remove all the non-fat body tissues with 5 forceps. Finally, mount the pieces of the fat body on a microscope slide using 80%glycerol as the mounting medium and lay a coverslip on top. Mutant one and mutant two are two independent lethal mutants on the X chromosome and isogenized yellow white FRT 19A flies serve as a control here.GFP-Atg8a patterns were analyzed in the control, mutant one, or mutant two mosaic larval fat bodies, and the mutant clones or control clones were negatively marked by RFP. In the control clones, the patterns of GFP-Atg8a Puncta were similar to those in the surrounding RFP positive cells. In mutant one mutant clones, the GFP-Atg8a Puncta were greatly reduced.And in mutant two mutant clones, the numbers and size of GFP-Atg8a Puncta were increased. The larvae developmental stage is critical here. The time for development and the duration of starvation need to be modified according to each lab's culture medium recipes.This procedure can be applied to explore selective autophagy. For example, mitophagy can be monitored in fly fat bodies by attaching a fluorescent protein to a mitochondrial targeting sequence.

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JoVE Journal

Biology

2.0K 조회수.  Zhejiang University. 이 프로토콜은 아미노산 고갈을 통해 초파리 멜라노가스터 유충 지방체에서 자가포식을 유도하는 방법과 돌연변이 클론 간의 자가포식 차이를 분석하는 방법을 설명합니다. 자가포식은 배양 조건에서 영양 가용성에 매우 민감하기 때문에 동일한 조직에서 돌연변이 세포와 야생형 세포를 분석하는 방법인 클론 분석은 자가포식 결함을 해부하는 데 이점이 있습니다. 짝짓기?...