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. The malfunction of autophagy is associated with aging and multiple human diseases. The autophagy machinery is highly conserved-from yeast to humans. 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.
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.
1. Drosophila crossing and egg laying
2. Amino acid starvation induces autophagy
3. Third instar larvae dissection and sample tissue processing
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 number and/or size of GFP puncta is expected to be high.
In the protocol presented here, the FLP/FRT system induces mitotic recombination and generates tissues with both wild-type and mutant cells. While the wild-type cells express RFP, the mutant cells lack RFP expression. The expression of RFP in all fat body cells would indicate that FLP/FRT-mediated mitotic recombination was not successfully induced or that the mutation is cell-lethal. In the latter case (i.e., if the mutation is cell-lethal), the larval fat body is expected to have a few cells with higher level RFP signals than the surrounding cells.
In Figure 2, GFP-Atg8a (green) formed puncta in wild-type cells (red), implying that autophagy was successfully induced. In the mutant clones (RFP negative), the pattern of GFP-Atg8a puncta was different from that in the surrounding wild-type cells, suggesting autophagy defects. Very few GFP-ATG8a puncta were detected in Mu1 mutant clones (Figure 2B), indicating that autophagy was blocked at the initiation steps or accelerated autolysosome maturation. The numbers and size of GFP-Atg8a puncta were greatly increased in Mu2 mutant clones (Figure 2C), suggesting autophagosome-lysosome fusion or autolysosome acidification defects. Further experiments are required to distinguish these possibilities. Moreover, the sizes and number of puncta need to be quantified to determine whether the observed differences are statistically significant.
Figure 1: Schematic representation of the experimental procedures for monitoring the autophagy marker GFP-Atg8a in a mosaic larval fat body carrying mutant clones. The crossing scheme for generating flies that express GFP-Atg8a in the larval fat body tissues and carry mutant clones is shown (a strain with a lethal point mutation [Mu] on the X-chromosome serves as an example). After heat shock at 37 °C for 1 h to activate FLP expression, the homozygous mutant cells with GFP-Atg8a expression can be generated in the y' w* Mu FRT19A / hsFLP ubiRFP FRT19A; cgGal4 UAS-GFP-Atg8a / + larval fat body. Autophagy is induced in the fat body by incubating the larvae in 20% sucrose solution for 6 h. Please click here to view a larger version of this figure.
Figure 2: The patterns of GFP-Atg8a puncta in Mu1 and Mu2 clones differed from those in the control clones. Mu1 and Mu2 are two independent lethal mutants on the X chromosome. Isogenized y' w*, FRT19A flies serve as a control. GFP-Atg8a (green) patterns were analyzed in the control, Mu1, or Mu2 mosaic larval fat bodies. The mutant clones (or control clones) were negatively marked by RFP (red). (A) In the control clones (RFP negative), the patterns of GFP-Atg8a puncta were similar to those in the surrounding RFP positive cells. (B) In Mu1 mutant clones (RFP negative), the GFP-Atg8a puncta were greatly reduced. (C) In Mu2 mutant clones (RFP negative), the numbers and size of GFP-Atg8a puncta were increased. Scale bar: 10 µm. Please click here to view a larger version of this figure.
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 critical for inducing FLP expression. A standard 37 °C water bath for 1 h heat shock is recommended. Considering the increased possibility of embryonic/larval death during heat shock and starvation, multiple crossings must be set up for sufficient tissue sample generation for imaging.
In addition, some steps may need to be modified considering the actual culturing conditions. For example, the differences in environment and Drosophila culture media between laboratories may affect the growth rates of larvae. Therefore, the developmental time required for embryos to reach the early third instar stage and the duration of larval starvation (culturing in 20% sucrose to induce autophagy) may need to be standardized in each lab separately.
GFP-Atg8a is the most commonly used marker for determining autophagy. However, the changes in the GFP-Atg8a puncta pattern fail to pinpoint the exact autophagy defect or the affected step directly. Therefore, multiple markers must be analyzed to interpret such data accurately. The protocol presented here can be adapted for other autophagy-related proteins with their respective transgenic strains25. Multiple markers labeled with different fluorescent proteins (such as blue fluorescence protein [BFP]) can be analyzed simultaneously to elucidate the initiation or fusion processes. Controlled autophagy modification, using chemicals26 or RNA interference of critical genes27, can be combined with the present approach to elucidate autophagy mechanisms further. In addition, analyzing endogenous protein markers of autophagy (by using immunohistochemistry) in mosaic tissues is important to confirm the phenotypes28.
Although autophagy was originally recognized as a random, unselective process, increasing evidence indicates that it can be selective and selectively degrades cytoplasmic organelles such as mitochondria and the endoplasmic reticulum, among others29. Further, the procedure described here can be applied to explore selective autophagy. For example, mitophagy can be monitored in fly fat bodies by attaching fluorescent proteins (such as Keima or GFP-RFP tandem tags) to a mitochondrial targeting sequence30.
The authors have nothing to disclose.
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.
1.5 mL microcentrifuge tube | Axygen | MCT-150-C | |
#5 Forceps | Dumont | RS-5015 | |
9 Dressions Spot plate | PYREX | 7220-85 | |
Fluorescence Microscope | Nikon | SMZ1500 | |
Glycerol | Sangon Biotech | A100854-0100 | |
KCl | Sangon Biotech | A610440-0500 | Composition of 1x PBS solution |
KH2PO4 | Sangon Biotech | A600445-0500 | Composition of 1x PBS solution |
Laboratory spatula | Fisher | 14-375-10 | |
Long forceps | R' DEER | RST-14 | |
Microscope cover glass | CITOTEST | 80340-1130 | |
Microscope slides | CITOTEST | 80302-2104 | |
Na2HPO4 | Sangon Biotech | A501727-0500 | Composition of 1x PBS solution |
NaCl | Sangon Biotech | A610476-0005 | Composition of 1x PBS solution |
Paraformaldehyde | Sigma-Aldrich | 158127 | |
Petri dish | Corning | 430166 | |
Standard cornmeal/molasses/agar fly food | Tong Lab-made | ||
Stereo microscope | Nikon | SMZ745 | |
Sucrose | Sinopharm Chemical Reagent Co.,Ltd. | 10021418 | |
Vectashield antifade mounting medium with DAPI | Vectorlabratory | H-1200-10 | Recommended mounting medium |
Fly stocks | |||
y'w* Iso FRT19A | Tong Lab's fly stocks | ||
y'w* Mu1FRT19A/ FM7,Kr GFP | Tong Lab's fly stocks | ||
y'w* Mu2 FRT19A/ FM7,Kr GFP | Tong Lab's fly stocks | ||
hsFLP ubiRFP FRT19A; cgGal4 UAS-GFP-Atg8a | Tong Lab's fly stocks |