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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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. 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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 critical for inducing FLP expression. A standard 37 &#176;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.

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

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 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64282/64282fig01.jpg" /> 
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 &#176;C for 1 h to activate FLP expression, the homozygous mutant cells with GFP-Atg8a expression can be generated in the y&#39; 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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64282/64282fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64282/64282fig02.jpg" /> 
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&#39; 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 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64282/64282fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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. ...

analyzing-starvation-induced-autophagy-drosophila-melanogaster-larval

Nanyang Technological University

Research

JoVE Journal

Biology

2.3K Views.  Zhejiang University. Dit protocol beschrijft hoe autofagie in het Drosophila melanogaster larvaal vetlichaam kan worden geïnduceerd via aminozuurdepletie en hoe de verschillen in autofagie tussen gemuteerde klonen kunnen worden geanalyseerd. Omdat autofagie zeer gevoelig is voor de beschikbaarheid van voeding in kweekomstandigheden, heeft klonale analyse, een methode die mutante cellen versus wilde cellen in hetzelfde weefsel analyseert, een voordeel bij het ontleden van autofagiedefecten. Begin met het introduc...

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

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in humans, but more tools and research efforts are needed to better elucidate the mechanisms involved. For over a century, the genetically highly tractable model of Drosophila has been instrumental in the discovery of key genes and molecular pathways that proved to be highly conserved across species. Many biological processes and disease mechanisms are functionally conserved in the fly, such as development (e.g., body plan, heart), cancer, and neurodegenerative disease. Recently, the study of obesity and secondary pathologies, such as heart disease in model organisms, has played a highly critical role in the identification of key regulators involved in metabolic syndrome in humans.
Here, we propose to use this model organism as an efficient tool to induce obesity, i.e., excessive fat accumulation, and develop an efficient protocol to monitor fat content in the form of TAGs accumulation. In addition to the highly conserved, but less complex genome, the fly also has a short lifespan for rapid experimentation, combined with cost-effectiveness. This paper provides a detailed protocol for High Fat Diet (HFD) feeding in Drosophila to induce obesity and a high throughput triacylglyceride (TAG) assay for measuring the associated increase in fat content, with the aim to be highly reproducible and efficient for large-scale genetic or chemical screening. These protocols offer new opportunities to efficiently investigate regulatory mechanisms involved in obesity, as well as provide a standardized platform for drug discovery research for rapid testing of the effect of drug candidates on the development or prevention of obesity, diabetes and related metabolic diseases.

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

Genetics

Single Drosophila Ommatidium Dissection and Imaging

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative diseases because of its powerful genetics1. The fly eye in particular has been the organ of choice for neurodegeneration research, being the most accessible and life-dispensable part of the Drosophila nervous system. However the major caveat of intact eyes is the difficulty, because of the intense autofluorescence of the pigment, in imaging intracellular events, such as autophagy dynamics2, which are paramount to understanding of neurodegeneration.
We have recently used the dissection and culture of single ommatidia3 that has been essential for our understanding of autophagic dysfunctions in a fly model of Dentatorubro-Pallidoluysian Atrophy (DRPLA)3, 4.
We now report a comprehensive description of this technique (Fig. 1), adapted from electrophysiological studies5, which is likely to expand dramatically the possibility of fly models for neurodegeneration. This method can be adapted to image live subcellular events and to monitor effective drug administration onto photoreceptor cells (Fig. 2). If used in combination with mosaic techniques6-8, the responses of genetically different cells can be assayed in parallel (Fig. 2).

Single Drosophila Ommatidium Dissection and Imaging

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.

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

Neuroscience

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, and issues commands to start or stop feeding. Deciphering the processes underlying the decision-making of timely and moderate feeding carries major implications in our understanding of physiological and psychological disorders related to feeding control. Simple, quantitative, and robust methods are required to measure the food ingestion of animals after experimental manipulation, such as forcibly increasing the activities of certain target neurons. Here, we introduced dye-labeling-based feeding assays to facilitate the neurogenetic study of feeding control in adult fruit flies. We review available feeding assays, and then describe our methods step-by-step from setup to analysis, which combine thermogenetic and optogenetic manipulation of neurons controlling feeding motivation with dye-labeled food intake assay. We also discuss the advantages and limitations of our methods, compared with other feeding assays, to help readers choose an appropriate assay.

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Quantitative food-intake assays with dyed food provide a robust and high-throughput means to evaluate feeding motivation. Combining the food consumption assay with thermogenetic and optogenetic screens is a powerful approach to investigate the neural circuits underlying appetite in adult Drosophila melanogaster.

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, ...

Behavior

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

Developmental Biology

Exploring the Regulation of Lipid Droplet Catabolism through Lipophagy

Macroautophagy, commonly referred to as autophagy, is a highly conserved cellular process responsible for the degradation of cellular components. This process is particularly prominent under conditions such as fasting, cellular stress, organelle damage, cellular damage, or aging of cellular components. During autophagy, a segment of the cytoplasm is enclosed within double-membrane vesicles known as autophagosomes, which then fuse with lysosomes. Following this fusion, the contents of autophagosomes undergo non-selective bulk degradation facilitated by lysosomes. However, autophagy also exhibits selective functionality, targeting specific organelles, including mitochondria, peroxisomes, lysosomes, nuclei, and lipid droplets (LDs). Lipid droplets are enclosed by a phospholipid monolayer that isolates neutral lipids from the cytoplasm, protecting cells from the harmful effects of excess sterols and free fatty acids (FFAs). Autophagy is implicated in various conditions, including neurodegenerative diseases, metabolic disorders, and cancer. Specifically, lipophagy -- the autophagy-dependent degradation of lipid droplets -- plays a crucial role in regulating intracellular FFA levels across different metabolic states. This regulation supports essential processes such as membrane synthesis, signaling molecule formation, and energy balance. Consequently, impaired lipophagy increases cellular vulnerability to death stimuli and contributes to the development of diseases such as cancer. Despite its significance, the precise mechanisms governing lipid droplet metabolism regulated by lipophagy in cancer cells remain poorly understood. This article aims to describe confocal imaging acquisition and quantitative imaging analysis protocols that enable the investigation of lipophagy associated with metabolic changes in cancer cells. The results obtained through these protocols may shed light on the intricate interplay between autophagy, lipid metabolism, and cancer progression. By elucidating these mechanisms, novel therapeutic targets may emerge for combating cancer and other metabolic-related diseases.

Lipophagy is a selective form of autophagy that involves the degradation of lipid droplets. Dysfunctions in this process are associated with cancer development. However, the precise mechanisms are not yet fully understood. This protocol describes quantitative imaging approaches to better understand the interplay between autophagy, lipid metabolism, and cancer progression.

Macroautophagy, commonly referred to as autophagy, is a highly conserved cellular process responsible for the degradation of cellular components. This ...

Cancer Research

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

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

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

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

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.

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

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

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating the neuronal regulation of these behaviors is essential for the understanding of the physiological and molecular mechanisms underlying nutritional homeostasis. The use of genetically tractable animal models such as worms, flies, and fish greatly facilitates these types of studies. In the last decade, the fruit fly Drosophila melanogaster has been used as a powerful animal model by neurobiologists investigating the neuronal control of feeding and foraging behaviors. While undoubtedly valuable, most studies examine adult flies. Here, we describe a protocol that takes advantage of the simpler larval nervous system to investigate neuronal substrates controlling feeding behaviors when larvae are exposed to diets differing in their protein and carbohydrates content. Our methods are based on a quantitative colorimetric no-choice feeding assay, performed in the context of a neuronal thermogenetic-activation screen. As a read-out, the amount of food eaten by larvae over a 1 h interval was used when exposed to one of the three dye-labeled diets that differ in their protein to carbohydrates (P:C) ratios. The efficacy of this protocol is demonstrated in the context of a neurogenetic screen in larval Drosophila, by identifying candidate neuronal populations regulating the amount of food eaten in diets of different macronutrient quality. We were also able to classify and group the genotypes tested into phenotypic classes. Besides a brief review of the currently available methods in the literature, the advantages and limitations of these methods are discussed and, also, some suggestions are provided about how this protocol might be adapted to other specific experiments.

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

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.

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating ...

A Buoyancy-based Method of Determining Fat Levels in Drosophila

Drosophila melanogaster is a key experimental system in the study of fat regulation. Numerous techniques currently exist to measure levels of stored fat in Drosophila, but most are expensive and/or laborious and have clear limitations. Here, we present a method to quickly and cheaply determine organismal fat levels in L3 Drosophila larvae. The technique relies on the differences in density between fat and lean tissues and allows for rapid detection of fat and lean phenotypes. We have verified the accuracy of this method by comparison to body fat percentage as determined by neutral lipid extraction and gas chromatography coupled with mass spectrometry (GCMS). We furthermore outline detailed protocols for the collection and synchronization of larvae as well as relevant experimental recipes. The technique presented below overcomes the major shortcomings in the most widely used lipid quantitation methods and provides a powerful way to quickly and sensitively screen L3 larvae for fat regulation phenotypes while maintaining the integrity of the larvae. This assay has wide applications for the study of metabolism and fat regulation using Drosophila.

A Buoyancy-based Method of Determining Fat Levels in Drosophila

Here we present a method to measure organismal fat levels in the third instar (L3) larval stage of Drosophila melanogaster. This method exploits the comparatively low density of fat tissue to differentiate between larvae with altered fat stores. Buoyancy-based analysis is a valuable tool for rapid, reproducible, and economical screening.

Drosophila melanogaster is a key experimental system in the study of fat regulation. Numerous techniques currently exist to measure levels of stored fat ...

Measuring Crop Motility and Food Passaging in Drosophila

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. Disordered GI motility and gastric emptying cause multiple diseases and symptoms. As a powerful genetic model organism, Drosophila can be used in GI motility research. The Drosophila crop is an organ that contracts and moves food into the midgut for further digestion, functionally similar to a mammalian stomach. Presented is a protocol to study Drosophila crop motility using simple measurement tools. A method for counting crop contractions to evaluate crop motility and a method for detecting the distribution of food dyed blue between the crop and gut using a spectrophotometer to investigate the effect of the crop on food passaging is described. The method was used to detect the difference in crop motility between control and nprl2 mutant flies. This protocol is both cost-efficient and highly sensitive to crop motility.

Measuring Crop Motility and Food Passaging in Drosophila

The goal of this protocol is to measure crop contraction and quantify food distribution in the Drosophila gut.

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. ...

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

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