We thank Jordan Davis, Yanyan Lin, Eric Schadler and Jimiao Zheng for their technical help with this study. This work was supported by NSF CAREER grant MCB-1054962 to A.L.A.

1. Preparing Large Apple Juice-agar Plates
<ol>
	<li>To make 4 plates, add 375 ml H2O, 11.25 g fly agar and a stir bar to a 1,000&#160;ml flask. This is Mix A. Autoclave Mix A with the flask lid loosely capped on one 30 min-sterilization cycle for liquid goods.</li>
	<li>Add 125 ml apple juice, 12.5 g table sugar and a stir bar to a 500&#160;ml beaker. This is Mix B. Heat Mix B on a heated platform while stirring and maintain the temperature at approximately 70 &#176;C until the autoclaving of Mix A is finished.</li>
	<li>Upon the completion of Mix A autoclaving, transfer Mix B to Mix A. Keep stirring the combined mixture gently and let it cool for 15 min. Avoid over-cooling and the resulted agar solidification before the addition of preservative.</li>
	<li>Make 10% (weight/volume) methyl 4-hydroxybenzoate solution in ethanol. This will act as a preservative. Add 3.75 ml of the solution to the above apple juice-agar mixture. Keep stirring for another 5 min.</li>
	<li>Pour 125 ml apple juice-agar mixture with preservative into each 300 cm2 plastic or styrofoam plate. Let it cool to RT and solidify. Avoid the forming of air bubbles while pouring. 
	Note: The apple juice-agar plates are ready to use immediately. Cover unused plates with clear wraps and store at 4 &#176;C for up to a week.</li>
</ol>
2. Drosophila&#160;Embryo Collection and Pre-crosslinking Treatment
<ol>
	<li>Load a population cage with approximately 40,000 to 50,000 flies from which embryos will be collected.</li>
	<li>Make two plates each containing 125 ml of standard cornmeal-molasses medium12 and sprinkle approximately 4 g of dried baker&#39;s yeast on each plate. Place these plates into the population cage in a 25 &#176;C incubator for 48 hr&#160;to fatten the flies.</li>
	<li>Warm 2 apple juice-agar plates to 25 &#176;C. Sprinkle approximately 1 g of dried baker&#39;s yeast on each plate. Place the 2 plates in the population cage to start collecting 0-1 hr-old embryos.</li>
	<li>Take out the plates at the end of the 1 hr collection. Replace with 2 new plates if another round of collection is needed.</li>
	<li>Add enough water to cover the plates and resuspend the embryos gently with a fine paint brush. Pour the resuspended embryos through a two layer sieve made out of stainless steel wire mesh. 
	Note: The top layer is made of medium pore size (850 &#956;m) mesh which collects large fly parts while letting through embryos. The bottom layer is made of fine mesh (75 &#956;m) which collects embryos while letting through yeast and water.</li>
	<li>Use the fine paint brush to transfer the collected embryos into a collection basket made with a 50&#160;ml tube and fine nylon mesh. Rinse the embryos briefly with water then blot the mesh on paper towels to dry.</li>
	<li>Immerse the embryos in 50% bleach with gentle swirling for 3 min. Rinse thoroughly with water to remove any remaining bleach. Blot the mesh on paper towels to dry.</li>
	<li>Immerse the dechorionated embryos in isopropanol with gentle agitation for 15 sec or until the breakdown of multi-embryo clumps (this should not take longer than 30 sec). Blot the mesh on paper towels to dry thoroughly.</li>
	<li>Immerse the embryos in heptane for 5 min while using a pipette to stream heptane over the embryos to keep them resuspended. Blot the mesh on paper towels to dry.</li>
	<li>Rinse the embryos with streams of Phosphate Buffered Saline, pH 7.4 (PBS) containing 0.1% Triton X-100 (PBST) for 3 min.</li>
	<li>Disassemble the collection basket and transfer the embryos along with the mesh to a 15&#160;ml tube containing 10 ml PBST. Invert the tube gently to wash the embryos off the mesh.</li>
	<li>Remove the mesh from the tube. Put the tube in a vertical position and let the embryos sink to the bottom of the tube by gravity. 
	Note: Approximately 100 - 300 &#956;l of embryos is expected for a 1 hr collection from one cage.</li>
	<li>Remove the PBST and continue to perform crosslinking immediately.</li>
</ol>
3. In vivo&#160;Crosslinking of Collected Embryos
<ol>
	<li>Prepare 0.2% formaldehyde in PBST immediately before use. Add 10 ml of the formaldehyde fixative to the embryos and incubate at 25 &#176;C with gentle agitation on a rotary shaker for 10 min. Discard unused fixative by the end of the day.</li>
	<li>Quench the crosslinking reaction
	<ol>
		<li>At the end of the crosslinking, let the embryos sink to the bottom of the tube.</li>
		<li>Remove the formaldehyde solution. Immediately add 10 ml of 0.25 M glycine in PBST to quench the crosslinking reaction. Incubate at 25 &#176;C with gentle agitation on a rotary shaker for 5 min.</li>
		<li>Let the embryos sink to the bottom of the tube. Remove the quench solution. Wash the embryos with 10 ml of PBST three times.</li>
		<li>Completely remove the PBST wash solution using a pipette. Store embryos at -80 &#176;C until use.</li>
	</ol>
	</li>
</ol>
4. Protein Sample Preparation and&#160;Immunoprecipitation
<ol>
	<li>Homogenize 500 &#956;l crosslinked embryos using a Dounce homogenizer in 2 ml lysis buffer (0.5 M urea, 0.01% SDS, 2% Triton-X 100, 2 mM phenylmethanesulfonyl fluoride [PMSF] and protease inhibitor cocktail in PBS). 
	Note: Perform this and the following steps at RT unless otherwise specified.</li>
	<li>Transfer the lysate to 1.5&#160;ml centrifuge tubes and gently rock on a rotating platform for 15 min to ensure thorough lysis. Centrifuge the lysate at 16,000 x g for 5 min to pellet cellular debris. Save the supernatant in a clean 15&#160;ml tube.</li>
	<li>Add 40 &#956;l anti-HA agarose beads slurry to the supernatant and incubate on a rotating platform for 2.5 hr.</li>
	<li>Pellet the beads by centrifugation at 2,500 x g for 5 min. Remove the supernatant but leave approximately 250 &#956;l in the tube. Resuspend the beads in the remaining supernatant and transfer to a 1.5&#160;ml spin column with 10-&#956;m pore filter.</li>
	<li>Centrifuge the spin column at 12,000 x g for 10 sec and discard the flow through.</li>
	<li>Wash the beads by adding 200 &#956;l wash buffer (0.1 M glycine, 1 M NaCl, 1% IGEPAL CA-630, 0.1% Tween-20, in PBS). Centrifuge the column at 12,000 x g for 10 sec and discard the wash. Repeat the wash two more times.</li>
	<li>Add 40 &#956;l elution buffer (2 mg/ml HA-peptide in PBS) to the beads and incubate at 37 &#176;C for 15 min with gentle agitation every 5 min. Centrifuge the column at 12,000 x g for 10 sec and collect the eluate.</li>
	<li>Confirm elution of the complex by western-blot analysis using anti-HA antibody13.</li>
</ol>

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The authors have nothing to disclose.

Formaldehyde has been commonly used as a crosslinking reagent for identifying protein-protein and protein-nucleic acid interactions. Its good solubility and cell membrane permeability, together with the compatibility with downstream mass spectrometry procedures, make formaldehyde an ideal candidate agent for intracellular crosslinking applications3,15-17. In particular, it was successfully used to identify mRNAs associated with Vasa, a critical germ cell RNA helicase in Drosophila11. In add...

Isolation of multisubunit protein assemblies and DNA- or RNA-protein complexes is performed to identify protein complexes, genomic loci recognized by DNA-binding regulatory proteins or RNA targets of RNA binding proteins. Different methods allow genome-wide identification of DNA sites recognized by transcription factors or chromatin proteins (ChIP-seq)1 and RNA targets associated with a given RNA-binding protein (CLIP-seq)2. The libraries of RNA-derived cDNAs or DNA targets are then deeply sequenced. These methods use chemical or UV-induced cross-linking to stabilize the complexes followed by immunoprecipitation (IP) with an antibody against a protein component of the studied complex.
During development of an organism, many protein complexes form transiently. Therefore, it is crucial to analyze the composition and function of these complexes in vivo to understand the molecular mechanisms that control development. Such an in vivo analysis would be superior to in vitro approach since it is virtually impossible to reproduce native concentrations of the interacting components and cellular biochemical environment in vitro. Here we demonstrate an in vivo approach that we successfully use to isolate large protein complexes from Drosophila embryos. In this method, protein complexes in the living embryos are crosslinked with a low concentration of formaldehyde and subsequently protein complexes of interest are isolated by IP with an antibody against a known component of the complexes followed by gel purification of the complexes and mass spectrometry analysis to identify unknown complex components. Since formaldehyde is able to permeate the cell membrane and has a crosslinking range of 2.3-2.7 &Aring;3, protein complexes can be crosslinked in vivo and the complex components are likely to be close to each other. In this article we describe this method using the isolation of Tudor (Tud) protein complex as an example. Tud is a germline protein which is essential for germline development4-7. This protein contains 11 Tud domains known to interact with methylated arginines or lysines of other polypeptides8-10.
Previously, we have generated a transgenic Drosophila line which expresses HA-tagged functional Tud5 and therefore, specific anti-HA antibody is used to pull down Tud complex after crosslinking.
In addition to protein-protein crosslinks, formaldehyde can generate nucleic acid-protein crosslinks and is used in ChIP-seq experiments. Furthermore, in Drosophila, in vivo crosslinking with formaldehyde has allowed the identification of an RNA target of Vasa RNA helicase protein11.
While in this article we describe a method for in vivo crosslinking and purification of protein complexes from Drosophila embryos, this method can be adapted for other organisms and tissues.

<table><tbody><tr><td>&lt;em&gt;Drosophila&lt;/em&gt; agar</td><td>Lab Scientific (http://www.labscientific.com/)</td><td>FLY-8020-1</td><td>Do not autoclave the water and agar mix for prolonged period of time as it will cause the apple juice plates to become fragile</td></tr><tr><td>Methyl 4-hydroxybenzoate</td><td>Sigma (http://www.sigmaaldrich.com/united-states.html)</td><td>H5501</td><td>Other name: TEGOSEPT</td></tr><tr><td>Population cage</td><td>Flystuff (http://www.flystuff.com/)</td><td>59-104</td><td></td></tr><tr><td>Fine nylon mesh</td><td>Flystuff (http://www.flystuff.com/)</td><td>57-102</td><td>When making the collection basket, cut the mesh slightly larger than the opening of the falcon tube cap to ensure a tight seal</td></tr><tr><td>Dounce homogenizer</td><td>Sigma (http://www.sigmaaldrich.com/united-states.html)</td><td>D8938-1SET</td><td>Chill the homogenizer on ice and prerinse with cold lysis buffer before use to prevent protein degradation</td></tr><tr><td>Protease inhibitor cocktail&amp;nbsp;</td><td>Roche (http://www.rocheusa.com/portal/usa)</td><td>4693132001</td><td>PBS could be used to prepare concentrated protease inhibitor stock solution</td></tr><tr><td>Anti-HA agarose beads</td><td>MBL international (http://www.mblintl.com/)</td><td>561-8</td><td>The kit also includes HA-peptide and spin columns</td></tr><tr><td>HA-peptide</td><td>MBL international (http://www.mblintl.com/)</td><td>561-8</td><td>Prepare to 2 mg/ml with PBS&amp;nbsp;</td></tr><tr><td>Spin Column</td><td>MBL international (http://www.mblintl.com/)</td><td>561-8</td><td>Spin columns are included as part of the kit</td></tr><tr><td>Isopropanol</td><td>Fisher Scientific (www.fishersci.com/&amp;lrm;)</td><td>BP26324</td><td></td></tr><tr><td>Triton X-100</td><td>Fisher Scientific (www.fishersci.com/&amp;lrm;)</td><td>BP151-500</td><td></td></tr><tr><td>Heptane</td><td>Fisher Scientific (www.fishersci.com/&amp;lrm;)</td><td>H350-4</td><td></td></tr><tr><td>PBS</td><td>Invitrogen (https://www.lifetechnologies.com/us/en/home.html)</td><td>AM9625</td><td>Dilute from 10X to 1X with nanopure water before use</td></tr><tr><td>Formaldehyde</td><td>Fisher Scientific (www.fishersci.com/&amp;lrm;)</td><td>BP531-500</td><td></td></tr><tr><td>Glycine</td><td>BioRad (www.bio-rad.com/&amp;lrm;)</td><td>161-0724</td><td></td></tr><tr><td>SDS</td><td>BioRad (www.bio-rad.com/&amp;lrm;)</td><td>161-0301</td><td></td></tr><tr><td>Urea</td><td>BioRad (www.bio-rad.com/&amp;lrm;)</td><td>161-0730</td><td></td></tr><tr><td>Phenylmethanesulfonyl fluoride</td><td>Sigma (http://www.sigmaaldrich.com/united-states.html)</td><td>P7626-1G</td><td>Prepare 200 mM stock solution in isopropanol then dilute to working concentration of 2 mM in lysis buffer</td></tr><tr><td>IGEPAL CA-630</td><td>Sigma (http://www.sigmaaldrich.com/united-states.html)</td><td>I8896-50ML</td><td></td></tr><tr><td>Tween 20</td><td>Fisher Scientific (www.fishersci.com/&amp;lrm;)</td><td>BP337-100</td><td></td></tr><tr><td>15-ml tubes</td><td>USA Scientific (http://www.usascientific.com/)</td><td>1475-0511</td><td></td></tr><tr><td>Bleach</td><td>Clorox brand</td><td></td><td>Dilute with equal volume of nanopure water to make 50% bleach</td></tr><tr><td>50-ml tubes</td><td>BD Biosciences (http://www.bdbiosciences.com/home.jsp)</td><td>352098</td><td></td></tr><tr><td>Top layer sieve</td><td>Fisher Scientific (www.fishersci.com/&amp;lrm;)</td><td>04-884-1AK</td><td></td></tr><tr><td>Bottom layer sieve</td><td>Fisher Scientific (www.fishersci.com/&amp;lrm;)</td><td>04-884-1BA</td><td></td></tr></tbody></table>

an in vivo crosslinking approach to isolate protein complexes from drosophila embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

The effectiveness of the crosslinking and the successful purification of the crosslinked Tud protein complex were analyzed by SDS-PAGE on a 3% - 7% step gel (illustrated in Figure 1) followed by western blot (Figure 2).
The purpose of using the 3% - 7% step gel is based on the effective separation of crosslinked Tud protein complex from the remaining uncrosslinked Tud protein and concentration of the complex. Under our in vivo crosslinking conditions ...

Watch this Scientific Journal Video about An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos at JoVE.com

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

an-vivo-crosslinking-approach-to-isolate-protein-complexes-from

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Biology

16.8K Views.  Murray State University. Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis. 

Video: An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, organism development, and disease. It is often desirable to obtain PPI information using in vivo material, to gain the most natural and unbiased view of the interaction networks. The fruit fly Drosophila melanogaster is an excellent platform to study PPIs in vivo, and lends itself to straightforward approaches to isolating material for biochemical experiments. In particular, fruit fly embryos represent a convenient type of tissue to study PPIs, due to the ease of collecting animals at this developmental stage and the fact that the majority of proteins are expressed in embryogenesis, thus providing a relevant environment to reveal most PPIs. Here we present a protocol for collection of Drosophila embryos at medium scale (0.5-1 g), which is an ideal amount for a wide range of proteomic applications, including analysis of PPIs by affinity purification-mass spectrometry (AP-MS). We describe our designs for 1 L and 5 L cages for embryo collections that can be easily and inexpensively set up in any laboratory. We also provide a general protocol for embryo collection and protein extraction to generate lysates that can be directly used in downstream applications such as AP-MS. Our goal is to provide an accessible means for all researchers to carry out the analyses of PPIs in vivo.

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

The goal of this protocol is to provide a straightforward and inexpensive approach to collecting Drosophila embryos at medium scale (0.5-1 g) and preparing protein extracts that can be used in downstream proteomic applications, such as affinity purification-mass spectrometry (AP-MS).

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, ...

Developmental Biology

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

Neuroscience

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

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of embryogenesis, these organelles undergo dramatic movements powered by actin-based cytoplasmic streaming and motor-driven trafficking along microtubules. The development of a multitude of small, organelle-specific fluorescent probes (FPs) makes it possible to visualize a wide range of different lipid-containing structures in any genotype, allowing live imaging without requiring a genetically encoded fluorophore. This protocol shows how to inject vital dyes and molecular probes into Drosophila embryos to monitor the trafficking of specific organelles by live imaging. This approach is demonstrated by labeling lipid droplets (LDs) and following their bulk movement by particle image velocimetry (PIV). This protocol provides a strategy amenable to the study of other organelles, including lysosomes, mitochondria, yolk vesicles, and the ER, and for tracking the motion of individual LDs along microtubules. Using commercially available dyes brings the benefits of separation into the violet/blue and far-red regions of the spectrum. By multiplex co-labeling of organelles and/or cytoskeletal elements via microinjection, all the genetic resources in Drosophila are available for trafficking studies without the need to introduce fluorescently tagged proteins. Unlike genetically encoded fluorophores, which have low quantum yields and bleach easily, many of the available dyes allow for rapid and simultaneous capture of several channels with high photon yields.

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

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.

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of ...

Enhancing Protein Visualization with Antibody-Mediated Live Imaging

Visualizing Low-Abundance Proteins and Post-Translational Modifications in Living Drosophila Embryos via Fluorescent Antibody Injection

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein localization, dynamics, and function. Compared to immunofluorescence, live imaging more accurately reflects protein localization without potential artifacts arising from tissue fixation. Importantly, live imaging enables quantitative and temporal characterization of protein levels and localization, crucial for understanding dynamic biological processes such as cell movement or division. However, a major limitation of fluorescent tagging approaches is the need for sufficiently high protein expression levels to achieve successful visualization. Consequently, many endogenously tagged fluorescent proteins with relatively low expression levels cannot be detected. On the other hand, ectopic expression using viral promoters can sometimes lead to protein mislocalization or functional alterations in physiological contexts. To address these limitations, an approach is presented that utilizes highly sensitive antibody-mediated protein detection in living embryos, essentially performing immunofluorescence without the need for tissue fixation. As proof of principle, endogenously GFP-tagged Notch receptor that is barely detectable&#160;in living embryos can be successfully visualized after antibody injection. Furthermore, this approach was adapted to visualize post-translational modifications (PTMs) in living embryos, allowing the detection of temporal changes in tyrosine phosphorylation patterns during early embryogenesis and revealing a novel subpopulation of phosphotyrosine (p-Tyr) underneath apical membranes. This approach can be modified to accommodate other protein-specific, tag-specific, or PTM-specific antibodies and should be compatible with other injection-amenable model organisms or cell lines. This protocol opens new possibilities for live imaging of low-abundance proteins or PTMs that were previously challenging to detect using traditional fluorescent tagging methods.

Author Spotlight: Unveiling the Dynamics of Mechanical and Biochemical Signals in Animal Morphogenesis Research 

This protocol describes the customized antibody-based fluorescence labeling and injection into early Drosophila embryos to enable live imaging of low-abundance proteins or post-translational modifications that are challenging to detect using traditional GFP/mCherry-tag approaches.

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein ...

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 &deg;C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

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

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

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

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.

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...

Detection of Protein-Protein Interactions in Drosophila Larvae Using a Proximity Ligation Assay

Source: Wang, S., et al. <a href="https://app.jove.com/v/52139">Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay.</a> J. Vis. Exp. (2015).
This video demonstrates the visualization of protein-protein interactions at the Drosophila neuromuscular junction (NMJ) using Proximity Ligation Assay (PLA). After isolating the body walls, interacting proteins are probed with primary antibodies and PLA-conjugated secondary antibodies. The proximity of the interacting proteins facilitates the ligation of probes with connector oligonucleotides, forming circular DNA, which is then amplified by rolling circle amplification. Fluorescent probes bind to the amplified DNA, allowing the visualization of protein-protein interactions at the NMJ.

Source: Wang, S., et al. Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay. J. ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

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Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

In-vivo Centrifugation of Drosophila Embryos

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

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

Visualizing Low-Abundance Proteins and Post-Translational Modifications in Living Drosophila Embryos via Fluorescent Antibody Injection

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

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

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Detection of Protein-Protein Interactions in Drosophila Larvae Using a Proximity Ligation Assay