Name: analyzing starvation-induced autophagy in the drosophila melanogaster larval fat body
Uploaded: 2022-08-04T15:00:00
Description: Watch this Scientific Journal Video about Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body at JoVE.com

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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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. ...

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