Name: In Vivo Application of TurboID-based Proximity Labeling in Drosophila melanogaster (Video) | JoVE Journal
Uploaded: 2025-06-13T13:15:00.000+00:00
Duration: 9 min 59 s
Description: Learn TurboID-based proximity labeling to map zucchini protein interactions in Drosophila melanogaster germline cells. This method identifies novel interactors involved in protein folding and membrane organization.

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5 minutes

We thank the laboratory members for their discussions and help. This work was supported by the National Research Foundation (NRF) funded by the Korean government (MSIT) (RS-2024-00345327 to M.L.; RS-2023-00219563 to M.L.).

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<li>Nguyen, T. T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+profiling+of+the+zucchini+proximal+proteome+in+the+Drosophila+ovary%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">In vivo profiling of the zucchini proximal proteome in the Drosophila ovary.</a> Development. 150 (4), dev201220(2023).</li>
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</ol>

The authors declare no conflict of interest.

Here, we provide a comprehensive procedure for TurboID-based proximity labeling (PL) in the Drosophila ovary, developed through extensive troubleshooting to overcome various challenges. One key issue involved genetic constructs, as different fusion layouts significantly affected protein expression and biotinylation efficiency. For example, NES-V5-TurboID displayed weaker expression and biotinylation compared to other transgenes, occasionally complicating the interpretation of downstream assays. However, a revise...

Conventional protein-protein interaction (PPI) mapping methods, such as affinity purification-mass spectrometry (AP-MS) and yeast-2-hybrid (Y2H) systems, often fail to capture low-abundance, transiently expressed, or membrane-bound proteins. This limitation arises from their inability to replicate the native physiological conditions of a living cell1,2,3. To overcome these challenges, PL has emerged as a powerful technique for high-resolution proteome mapping at a sub-organelle level in vivo. PL leverages the fusion of a promiscuous enzyme with bait protein, enabling the enzyme to catalyze the formation of short-lived reactive molecules1. These reactive molecules, such as biotin, covalently label proteins within a few nanometers of the fusion protein4. Subsequently, biotinylated proteins are isolated and analyzed by mass spectrometry to facilitate large-scale protein identification5,6.
Over the past years, various promiscuous enzymes have been developed to advance PL techniques. Two widely used enzymes are Escherichia coli-derived R118G mutant biotin ligase (BioID) and pea-derived ascorbate peroxidase enzyme 2 (APEX2). BioID converts biotin and ATP into biotinyl-5&#39;-adenylate (bioAMP)1. This reactive molecule covalently binds to lysine residues of nearby proteins within &#177; 10 nm radius. A key advantage of BioID-based PL is the use of biotin. It is a naturally occurring and non-toxic biomolecule in living organisms, which allows for safe in vivo labeling6. However, BioID has limitations, including slow labeling kinetics (18-24 h) and the requirement for high temperature (37 &#176;C) for optimal catalytic activity4. These traits can hinder its application in studying dynamic biological processes. Meanwhile, APEX2 catalyzes the formation of biotin-phenoxyl radicals in the presence of biotin-phenol and hydrogen peroxide (H2O2)7. These radicals mainly react with the side chain of electron-rich amino acids like tyrosine and can also bind cysteine, histidine, and tryptophan8. The rapid labeling capability of the peroxidase enzyme (&#60; 1 min) is suitable for capturing dynamic and transient PPIs9. Moreover, it has a wider detection range (up to 20 nm) than biotin ligase. However, APEX2-based PL has its drawbacks, including the requirement for a toxic oxidizing agent, H2O2, and the low permeability of biotin-phenol, which limit the applicability in vivo10.
In 2018, Branon et al. developed a new biotin ligase called TurboID, a 35 kD mutant version of BirA (the biotin ligase found in E. coli)4. This enzyme incorporates a mutation at the R118 position (R118S) together with 15 other mutations relative to BirA. It exhibits two-fold higher catalytic activity than BioID. TurboID-induced biotinylation in 10 min produces proteomic data with a size and specificity comparable to 18 h of labeling by BioID. Furthermore, it demonstrates excellent labeling activity at 30 &#176;C4. Therefore, TurboID is more suitable for application in organisms like Drosophila or Caenorhabditis elegans, which are commonly reared at 25 &#176;C and 20 &#176;C, respectively.
In this study, we aim to utilize TurboID-based PL for interactome study in the Drosophila ovary. The protocols described here are employed to map the interactome of Zuc in germline cells. Zuc is an endonuclease localized on the outer mitochondrial membrane (OMM), which mediates the biogenesis of piRNAs, small non-coding RNAs critical for the maintenance of genome integrity11,12,13. By comparing biotinylated proteins identified from Zuc-TurboID analysis with those from two additional controls, NES-TurboID and Tom20-TurboID, we defined distinct Zuc-interacting candidates.

<table><tbody><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Goat anti-mouse IgG (H+L) secondary antibody, HRP</td><td>Invitrogen</td><td>31430</td><td>Analytical grade, light-sensitive</td></tr><tr><td>Goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, alexa fluor plus 488</td><td>Invitrogen</td><td>A32731</td><td>Analytical grade, light-sensitive</td></tr><tr><td>Goat anti-rabbit IgG (H+L) secondary antibody, HRP</td><td>Invitrogen</td><td>31460</td><td>Analytical grade, light-sensitive</td></tr><tr><td>HSP90 antibody (rabbit)</td><td>Cell Signaling Technology</td><td>4874</td><td>Polyclonal antibody</td></tr><tr><td>Pierce Streptavidin protein, HRP</td><td>Thermo Fisher Scientific</td><td>21126</td><td>Analytical grade</td></tr><tr><td>Streptavidin, Alexa Fluor 594 conjugate</td><td>Invitrogen</td><td>S11227</td><td>Light-sensitive, analytical grade</td></tr><tr><td>V5-tag antibody (mouse)</td><td>Invitrogen</td><td>R960-25</td><td>Monoclonal antibody</td></tr><tr><td>V5-tag antibody (rabbit) (D3H8Q)</td><td>Cell Signaling Technology</td><td>13202</td><td>Monoclonal antibody</td></tr><tr><td>&lt;strong&gt;Fly Lines&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>pUASp Tom20 V5-TurboID/TM3</td><td>Korea Drosophila Stock Center</td><td>2373-2</td><td>Fly stock</td></tr><tr><td>pUASp Zucchini V5-TurboID/Cyo</td><td>Korea Drosophila Stock Center</td><td>2188</td><td>Fly stock</td></tr><tr><td>pUASP_attB_V5-TurboID-NES/Cyo</td><td>Korea Drosophila Stock Center</td><td>2635-4</td><td>Fly stock</td></tr><tr><td>w[*]; P{w[+mC]=matalpha4-GAL-VP16}V2H</td><td>Bloomington Drosophila Stock Center</td><td>7062</td><td>Fly stock</td></tr><tr><td>&lt;strong&gt;Equipments and Consummables&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>Analytical Balance</td><td>Mettler Toledo</td><td>ME204</td><td>0.1 mg readibility</td></tr><tr><td>Amersham Protran 0.45 NC nitrocellulose western blotting membranes (300 mm &amp;times; 4 m)</td><td>Cytiva</td><td>10600002</td><td>-</td></tr><tr><td>Axygen 1.7 mL MaxyClear Snaplock Microcentrifuge Tube</td><td>Corning</td><td>MCT-175-C</td><td>Co-polymer</td></tr><tr><td>BRAND&amp;nbsp;Cover Glass (22 mm x 22 mm)</td><td>Sigma-Aldrich</td><td>BR470055</td><td>Borosilicate glass</td></tr><tr><td>Bulk Pipette Tips (10 &amp;micro;L)</td><td>Vertex</td><td>4110N00</td><td>Propylene</td></tr><tr><td>Bulk Pipette Tips (1000 &amp;micro;L)</td><td>Vertex</td><td>4330N00</td><td>Propylene</td></tr><tr><td>Bulk Pipette Tips (200 &amp;micro;L)</td><td>Vertex</td><td>4230N00</td><td>Propylene</td></tr><tr><td>Cell Culture Plate (96-well)</td><td>SPL</td><td>300096</td><td>Polystyrene, flat-bottom type</td></tr><tr><td>Centrifuge (with 1.5-2 mL tube-sized rotor)</td><td>Eppendorf</td><td>5424 R</td><td>Fixed-angle, refrigerated</td></tr><tr><td>Centrifuge (with 5 mL tube-sized rotor)</td><td>Eppendorf</td><td>5425 R</td><td>Fixed-angle, refrigerated</td></tr><tr><td>Chemical Spoon</td><td>-</td><td>-</td><td>Stainless steel</td></tr><tr><td>ChemiDoc Imaging system</td><td>Biorad</td><td>12003153</td><td>Western blot imaging system</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; fly pad</td><td>-</td><td>-</td><td>Anaesthetic board</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; tank &amp;amp; regulator</td><td>-</td><td>-</td><td>Caution of high pressure gas &amp;amp; dizziness</td></tr><tr><td>Compact incubator</td><td>JSR</td><td>JSGI-10T</td><td>Caution of high temperature</td></tr><tr><td>Confocal laser scanning microscope</td><td>Zeiss</td><td>LSM 710</td><td>Caution of intense laser beam</td></tr><tr><td>Conical tube (15 mL)</td><td>SPL</td><td>50015</td><td>Polypropylene /High density polyethylene&amp;nbsp;</td></tr><tr><td>Conical tube (50 mL)</td><td>SPL</td><td>50050</td><td>Polypropylene /High density polyethylene&amp;nbsp;</td></tr><tr><td>Digital balance</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Digital timer</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Dissection dish</td><td>-</td><td>-</td><td>Silicon</td></tr><tr><td>Dumont #5SF Forceps&amp;nbsp;</td><td>Fine Science Tools</td><td>11252-00</td><td>Super fine tips, caution of sharp object</td></tr><tr><td>DynaMag-2 magnet</td><td>Invitrogen</td><td>12321D</td><td>For 1.5-2 mL sized microcentrifuge tubes</td></tr><tr><td>Fisherbrand&amp;nbsp;Superfrost Plus microscope slides (25 mm x 75 mm)</td><td>Fisher Scientific</td><td>12-550-15</td><td>Caution of sharp object</td></tr><tr><td>Fly food bottles (with paper plug)</td><td>Hansol Tech</td><td>FFB-21010</td><td>-</td></tr><tr><td>Fly food vials (with cotton cap)</td><td>Hansol Tech</td><td>FFV-11010</td><td>-</td></tr><tr><td>Focused-ultrasonicator</td><td>Covaris</td><td>M220</td><td>Caution of ultrasonic wave</td></tr><tr><td>Gel knife</td><td>Invitrogen</td><td>EI9010</td><td>Stainless steel with plastic handle</td></tr><tr><td>Grade 3MM Chr blotting paper (46 cm &amp;times; 57 cm)</td><td>Cytiva</td><td>3030-917</td><td>Whatmann paper</td></tr><tr><td>Hand-held homogenizer and plastic pestle</td><td>Kimble</td><td>749540-0000</td><td>Cordless motor</td></tr><tr><td>Heat block</td><td>Fine PCR</td><td>ALB6400</td><td>Caution of high temperature</td></tr><tr><td>Lint-free tissue</td><td>Yuhan-Kimberly</td><td>41112</td><td>-</td></tr><tr><td>Liquid nitrogen</td><td>-</td><td>-</td><td>Caution of frostbite and burn</td></tr><tr><td>Microplate spectrophotometer</td><td>Agilent Technologies</td><td>Epoch</td><td>Monochromator-based optics</td></tr><tr><td>Microwave</td><td>-</td><td>-</td><td>Caution of high temperature</td></tr><tr><td>milliTUBE 1 mL AFA fiber</td><td>Covaris</td><td>PN 520135</td><td>Glass</td></tr><tr><td>Mini gel tank</td><td>Thermo Fisher Scientific</td><td>A25977</td><td>Caution of high temperature</td></tr><tr><td>NuPAGE Bis-tris mini protein gels, 4&amp;ndash;12%, 1.0&amp;ndash;1.5 mm, 10-well</td><td>Invitrogen</td><td>NP0323BOX</td><td>Contain polyacrylamide, caution of toxicity</td></tr><tr><td>NuPAGE Bis-tris mini protein gels, 4&amp;ndash;12%, 1.0&amp;ndash;1.5 mm, 15-well</td><td>Invitrogen</td><td>NP0321BOX</td><td>Contain polyacrylamide, caution of toxicity</td></tr><tr><td>Paint brush (&amp;Oslash; 1.8 mm)</td><td>Hwa hong</td><td>791240</td><td>-</td></tr><tr><td>Pipetman L (P1000L)</td><td>Gilson</td><td>FA10006M</td><td>Single channel micropipette with metal ejector</td></tr><tr><td>Pipetman L (P200L)</td><td>Gilson</td><td>FA10005M</td><td>Single channel micropipette with metal ejector</td></tr><tr><td>Pipetman L (P20L)</td><td>Gilson</td><td>FA10003M</td><td>Single channel micropipette with metal ejector</td></tr><tr><td>Pipetman L (P2L)</td><td>Gilson</td><td>FA10001M</td><td>Single channel micropipette with metal ejector</td></tr><tr><td>Power supply</td><td>Major science</td><td>MP-310</td><td>Caution of high voltage electricity</td></tr><tr><td>Programmable rotator</td><td>Biosan</td><td>RS-24</td><td>-</td></tr><tr><td>Protein LoBind&amp;nbsp;tubes</td><td>Eppendorf</td><td>30108302</td><td>-</td></tr><tr><td>Razor blade</td><td>Dorco</td><td>304020</td><td>Stainless steel, caution of sharp object</td></tr><tr><td>Roller</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Serological pipette (10 mL)</td><td>SPL</td><td>91010</td><td>Polystyrene</td></tr><tr><td>Serological pipette (25 mL)</td><td>SPL</td><td>91025</td><td>Polystyrene</td></tr><tr><td>Serological pipette (5 mL)</td><td>SPL</td><td>91005</td><td>Polystyrene</td></tr><tr><td>Serological pipette (50 mL)</td><td>SPL</td><td>91050</td><td>Polystyrene</td></tr><tr><td>Snap cap low retention microcentrifuge tubes (1.5 mL)</td><td>Thermo Scientific</td><td>3451PK</td><td>Polypropylene</td></tr><tr><td>Sponge pad</td><td>Invitrogen</td><td>EI9052</td><td>-</td></tr><tr><td>Stereo microscope</td><td>Zeiss</td><td>Stemi 508</td><td>-</td></tr><tr><td>Stereo microscope&amp;nbsp;</td><td>Leica</td><td></td><td></td></tr><tr><td>ThermoMixer&amp;nbsp;C</td><td>Eppendorf</td><td>5382000015</td><td>With 1.5 &amp;amp; 5 mL-sized blocks</td></tr><tr><td>Tweezer</td><td>Aven Tools</td><td>18526</td><td>Plastic, flat tips</td></tr><tr><td>Vortex mixer</td><td>Scientific Industries</td><td>SI-0236</td><td>-</td></tr><tr><td>XCell II Blot module</td><td>Invitrogen</td><td>EI9051</td><td>Caution of high temperature</td></tr><tr><td>XCell&amp;nbsp;SureLock mini-cell</td><td>Invitrogen</td><td>EI0001</td><td>Caution of high temperature</td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>Acetone</td><td>Sigma-Aldrich</td><td>650501</td><td>Analytical grade</td></tr><tr><td>Acetonitrile</td><td>Supelco</td><td>1.00029.1000</td><td>LC-MS grade, caution of inhalation toxicity and flammable</td></tr><tr><td>Albumin standard ampules (containing bovine serum albumin (BSA) at 2 mg/mL in 0.9% saline and 0.05% sodium azide)</td><td>Thermo Scientific</td><td>23209</td><td>Part of Pierce BCA Protein Assay Kits (Thermo Scientific, 23225)</td></tr><tr><td>Albumin, bovine serum</td><td>Bioshop</td><td>ALB001</td><td>Analytical grade</td></tr><tr><td>Ammonium bicarbonate</td><td>Sigma-Aldrich</td><td>09830</td><td>Analytical grade</td></tr><tr><td>Biotin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>B4639</td><td>Cell &amp;amp; tissue culture grade</td></tr><tr><td>Calcium chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>C5670</td><td>Cell-culture grade</td></tr><tr><td>Clarity western ECL substrate</td><td>Biorad</td><td>1705060</td><td>Analytical grade</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>MBD0015</td><td>Light-sensitive, analytical grade</td></tr><tr><td>Dithiothreitol (DTT)</td><td>Thermo Fisher Scientific</td><td>R0861</td><td>Analytical grade</td></tr><tr><td>Fetal bovine serum</td><td>Corning</td><td>35-015-CV</td><td>Cell culture grade</td></tr><tr><td>Formic acid</td><td>Sigma-Aldrich</td><td>F0507</td><td>Reagent grade, caution of inhalation toxicity</td></tr><tr><td>Grace&#039;s insect medium</td><td>Gibco</td><td>11595030</td><td>Cell culture grade</td></tr><tr><td>Instant dry yeast</td><td>No Brand</td><td>N1019</td><td>-</td></tr><tr><td>Iodoacetamide</td><td>Sigma-Aldrich</td><td>I1149</td><td>Light-sensitive, analytical grade</td></tr><tr><td>Methanol</td><td>Carlo Erba</td><td>41484</td><td>Analytical grade</td></tr><tr><td>Nail polish</td><td>-</td><td>-</td><td>Clear-type</td></tr><tr><td>Novex Tris-glycine SDS sample buffer (2X)</td><td>Invitrogen</td><td>LC2676</td><td>Analytical grade</td></tr><tr><td>NuPAGE MOPS SDS Running Buffer (20X)</td><td>Invitrogen</td><td>NP0001</td><td>Analytical grade</td></tr><tr><td>PageRuler Prestained protein ladder</td><td>Thermo Fisher Scientific</td><td>26616</td><td>Analytical grade</td></tr><tr><td>Phenylmethanesulfonyl fluoride&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>36978</td><td>Analytical grade</td></tr><tr><td>Phosphate buffer saline</td><td>Sigma-Aldrich</td><td>P4417</td><td>Analytical grade</td></tr><tr><td>Pierce 16% Formaldehyde (w/v)</td><td>Thermo Fisher Scientific</td><td>28908</td><td>Analytical grade, light-sensitive</td></tr><tr><td>Pierce BCA protein assay kits</td><td>Thermo Scientific</td><td>23225</td><td>Analytical grade</td></tr><tr><td>Pierce BCA reagent A (containing sodium carbonate, sodium bicarbonate, bicinchoninic acid and sodium tartrate in 0.1 M sodium hydroxide)</td><td>Thermo Scientific</td><td>23228</td><td>Part of Pierce BCA Protein Assay Kits (Thermo Scientific, 23225)</td></tr><tr><td>Pierce BCA reagent B (containing 4% cupric sulfate)</td><td>Thermo Scientific</td><td>1859078</td><td>Part of Pierce BCA Protein Assay Kits (Thermo Scientific, 23225)</td></tr><tr><td>Propionic acid</td><td>Sigma-Aldrich</td><td>402907</td><td>ACS grade</td></tr><tr><td>Protease inhibitor cocktail set III</td><td>Calbiochem</td><td>539134</td><td>Cell &amp;amp; tissue culture grade</td></tr><tr><td>SlowFade Gold Antifade Mountant</td><td>Invitrogen</td><td>S36936</td><td>Light-sensitive</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Bioneer</td><td>C-9025</td><td>Analytical grade</td></tr><tr><td>Sodium deoxycholate</td><td>Sigma-Aldrich</td><td>D6750</td><td>Analytical grade</td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma-Aldrich</td><td>71736</td><td>Molecular grade</td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Biosesang</td><td>SR2018-100-00</td><td>Analytical grade</td></tr><tr><td>Triple distilled water</td><td>-</td><td>-</td><td>Ultrapure (type 1)</td></tr><tr><td>Tris-buffered saline</td><td>Sigma-Aldrich</td><td>94158</td><td>Analytical grade</td></tr><tr><td>Tris-glycine transfer buffer</td><td>Koma Biotech</td><td>K0341001</td><td>Analytical grade</td></tr><tr><td>Tris-HCl, pH 8</td><td>Bioneer</td><td>C-9006</td><td>Analytical grade</td></tr><tr><td>Triton x-100</td><td>Sigma-Aldrich</td><td>93443</td><td>Molecular grade</td></tr><tr><td>Trypsin</td><td>Thermo Fisher Scientific</td><td>20233</td><td>Cell-culture grade</td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P9416</td><td>Molecular grade</td></tr><tr><td>Urea</td><td>Sigma-Aldrich</td><td>U5378</td><td>Molecular &amp;amp; cell-culture grade</td></tr></tbody></table>

in vivo application of turboid-based proximity labeling in drosophila melanogaster

In Vivo Application of TurboID-based Proximity Labeling in Drosophila melanogaster

Proximity labeling (PL) is a powerful method for mapping protein-protein interaction networks. Advances in labeling enzymes, especially TurboID, have broadened its use across biological systems. TurboID is particularly advantageous for in vivo applications due to its robustness, non-toxicity, and ease of use. This enzyme promiscuously biotinylates nearby biomolecules, including proteins and nucleic acids, within an approximately 10 nm range in the presence of biotin. We applied this method to investigate the interactome of Zucchini (Zuc), a key factor in piwi-interacting RNA (piRNA) biogenesis on the outer mitochondrial membrane (OMM) in germline cells of the Drosophila melanogaster ovary. By expressing Zuc-TurboID in germ cells and supplementing with biotin, we induced the biotinylation of Zuc-proximal proteins. Mass spectrometry analysis following the enrichment of biotinylated peptides identified proteins in close proximity to Zuc, including well-known interactors in piRNA biogenesis. Interestingly, we discovered novel interactors of Zuc involved in protein folding, membrane organization, and vesicle trafficking.

Example outputs are depicted in Figure 1, Figure 2, Figure 3, Figure 4 were previously published by Nguyen et al.20. Figure 1 illustrates the experimental procedure of TurboID-based PL in Drosophila ovary. First, a construct combining Zuc and TurboID was generated (Zuc-V5-TurboID). Well-known and distinct sub...

In this study, we establish a detailed protocol for TurboID-based proximity labeling (PL) in D. melanogaster ovary, covering steps from biotin supplementation and ovary dissection to transgene expression validation and enrichment of biotinylated peptides for mass spectrometry.

Proximity labeling (PL) is a powerful method for mapping protein-protein interaction networks. Advances in labeling enzymes, especially TurboID, have ...

in-vivo-application-turboid-based-proximity-labeling-drosophila

Research

JoVE Journal

Biology

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

Mesoscopic  fluorescence tomography operates beyond the penetration limits of  tissue-sectioning fluorescence microscopy. The technique is based on  multi-projection illumination and a photon transport description. We  demonstrate in-vivo whole-body 3D visualization of the morphogenesis of  GFP-expressing wing imaginal discs in Drosophila  melanogaster.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures  for up to a few days1.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

Imaging Through the Pupal Case of Drosophila melanogaster

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

Live-imaging of the Drosophila Pupal Eye

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

In the early Drosophila embryo, many organelles are motile. In principle, they can be imaged live via specific fluorescent probes, but the eggshell prevents direct application to the embryo. This protocol describes how to introduce such probes via microinjection, and then analyze bulk organelle motion via particle image velocimetry.

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

Learn TurboID-based proximity labeling to map zucchini protein interactions in Drosophila melanogaster germline cells. This method identifies novel interactors involved in protein folding and membrane organization.

In Vivo Application of TurboID-based Proximity Labeling in Drosophila melanogaster

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