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

Summary

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.

Abstract

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.

Introduction

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 memb....

Protocol

1. Drosophila crossing and egg laying

1. Introduce 3 male (genotype hsFLP ubiRFP FRT19A; cgGal4 UAS-GFP-Atg8a) and 15 female (genotype y' 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 °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....

Results

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, .......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
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