We thank the Bloomington Drosophila Stock Center for providing fly stocks. We also thank the Developmental Studies Hybridoma Bank and Dr. Lynn Cooley (Yale University) for providing antibodies. A special thanks goes to AhHyun Yoo for her help on the manuscript.&#160;This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (Krieger), the William and Ada Isabelle Steel Fund (Krieger), and the Canadian Institutes of Health Research (Harden).

1. Body Wall Preparation
NOTE: Preparation of third instar larval body walls (for study of the NMJs which innervate the body wall muscles) was performed as previously described in Brent et al. 10, or Ramachandran and Budnik 11,12, but with some modifications.
<ol>
	<li>Dissection
	<ol>
		<li>Raise fly stocks and crosses at 25 &#176;C for five to six days using standard procedures 13.</li>
		<li>Pick crawling third instar larvae from vials or bottles using fine forceps.</li>
		<li>Wash the larvae in a small Petri dish containing Phosphate Buffer Saline (PBS) to remove any food particles.</li>
		<li>Place a single larva onto a sylgard disc and immerse it in a few drops of ice-cold PBS. Using ice-cold PBS will help stun the larva making it easier to manipulate. Throughout the dissection, ensure that the preparation is always submerged in PBS to prevent it from drying.</li>
		<li>Position the larva with its dorsal side facing up so that the two tracheal tracts are visible under a dissecting microscope (Figure 2A). Using the forceps to grasp a minutien pin, pin the larva down at the posterior end near the spiracles (Figure 2B). With another pin, pierce through the cuticle at the anterior end near the mouth hooks. Gently stretch the larva out lengthwise, then pin it down (Figure 2B).</li>
		<li>Using microdissection scissors, pinch the posterior end near the pin to create a small opening. The incision should be superficial enough to just pass through the cuticle.</li>
		<li>Placing the tip of the bottom blade of the scissors into the incision, cut along the entire length of the dorsal midline between the two tracheal tracts (Figure 2C). Point the scissor blades slightly upwards when cutting to avoid damaging the ventral body wall muscles.</li>
		<li>Make a small horizontal incision slightly anterior to the posteriorly-placed pin (Figure 2C). Make another similar incision slightly posterior to the anteriorly-placed pin (Figure 2C). The incisions should just pass through the cuticle. 
		NOTE: The three incisions from steps 1.1.7 and 1.1.8 combined should resemble an &#34;<img fo:content-width="0.09in" src="/files/ftp_upload/52139/52139_letter_I.jpg" width="15px" />&#34; when completed, i.e., a left- and right-hand flap on the dorsal side of the larval body should be produced.</li>
		<li>Carefully clean out the internal organs with the forceps. Adding a few forceful drops of PBS will help displace the organs out of the larval body, thus making it easier to remove them. Avoid poking the larval body as it will cause damage to the body wall muscles.</li>
		<li>Unfurl the larval body open and pin the corners down (Figure 2D). When pinning, stretch the body wall both horizontally and vertically to form an evenly-tensioned rectangle (see Figure 2G for the shape), taking care not to tear the body wall muscles in the process.</li>
		<li>Finish removing any remaining internal organs (Figure 2E).</li>
	</ol>
	</li>
	<li>Fixation and Permeabilization
	<ol>
		<li>Immerse the pinned body walls in several drops of Bouin&#39;s Solution. Incubate for 15 min on ice. Alternatively, use 4% paraformaldehyde (PFA) as an alternative fixative; incubate for 30 min.</li>
		<li>Rinse three times with Phosphate Buffer Saline with Triton (PBT).</li>
		<li>Using fine forceps, carefully remove the pins and transfer the body walls by their corners into a siliconized 0.65 ml microcentrifuge tube.</li>
		<li>Store the body walls in PBT at 4 &#176;C until ready for PLA. For optimal results, start immunostaining the body walls within a day or two of dissection. 
		NOTE: To save on reagents and to ensure that all body walls are treated equally during the assay, different genotypes can be placed into a single tube. Genotypes can be distinguished by cutting the corners of the body walls differently (see Figure 2F for examples).</li>
	</ol>
	</li>
</ol>
2. Immunohistochemistry
NOTE: Immunostaining of third instar larval body walls was performed as previously described in Brent et al. 14 and Ramachandran and Budnik 11, but with some modifications 11,14. 
NOTE: Perform all steps at room temperature and with gentle agitation unless otherwise stated.
<ol>
	<li>Blocking
	<ol>
		<li>Wash the body walls with PBT three times for 10 min each.</li>
		<li>Block with 1% Bovine Serum Albumin (BSA) for 1 hr.</li>
	</ol>
	</li>
	<li>Immunostaining
	<ol>
		<li>Incubate the body walls with mouse and rabbit primary antibodies against the two proteins of interest (diluted in 1% BSA) for 2 hr at room temperature, or overnight at 4 &#176;C. In this case, use 1:10 mouse anti-Dlg and 1:250 rabbit anti-HtsM 15,16. Antibodies against markers that are not made in mouse or rabbit can also be included &#8212; e.g., use 1:200 goat anti-Hrp to delineate the neuronal membranes.</li>
		<li>Wash with PBT three times for 10 min each.</li>
		<li>Incubate with fluorophore-conjugated secondary antibodies to detect the markers (diluted in 1% BSA) for 2 hr at room temperature, or overnight at 4 &#176;C. As the PLA signal is later visualized with a red fluorophore, another fluorophore must be used to detect the marker &#8212; e.g., use 1:200 FITC-conjugated anti-goat to detect the goat anti-Hrp antibody. Due to the use of light-sensitive reagents, keep the tubes in the dark from this point onwards. 
		NOTE: The kit that is used allows for PLA to be performed between primary antibodies raised in mouse and rabbit, with the signal visualized on the red channel under confocal microscopy. If desired, other kits are available that allow the assay to be done with primary antibodies raised in other species, and the signal visualized on other channels.</li>
	</ol>
	</li>
</ol>
3. Proximity Ligation Assay
NOTE: Perform all steps at room temperature and with gentle agitation unless otherwise stated.

<ol>
	<li>PLA Probes
	<ol>
		<li>Wash the body walls with PBT three times for 10 min each.</li>
		<li>Incubate with PLA probes (1:5 dilution each in 1% BSA) for 2 hr at 37 &#176;C. In this case, use 40 &#181;l of PLA probe anti-mouse MINUS, 40 &#181;l of PLA probe anti-rabbit PLUS and 120 &#181;l of 1% BSA to ensure the proper immersion and mixing of 5-10 body walls. Up to a 1:25 dilution of the PLA probes can still result in an adequate signal-to-noise ratio (i.e., for this experiment).</li>
	</ol>
	</li>
	<li>Ligation
	<ol>
		<li>Wash the body walls with Wash Buffer A twice for 5 min each.</li>
		<li>Incubate with Ligation solution (1:40 dilution of Ligase in Ligation buffer) for 1 hr at 37 &#176;C. In this case, use 5 &#181;l of Ligase, 40 &#181;l of 5 Ligation buffer and 155 &#181;l of high purity water to ensure the proper immersion and mixing of 5-10 body walls.</li>
	</ol>
	</li>
	<li>Amplification
	<ol>
		<li>Wash the body walls with Wash Buffer A twice for 2 min each.</li>
		<li>Incubate with Amplification solution (1:80 dilution of Polymerase in Amplification buffer) for 2 hr at 37 &#176;C. In this case, use 2.5 &#181;l of Polymerase, 40 &#181;l of 5 Amplification buffer and 157.5 &#181;l of high purity water to ensure the proper immersion and mixing of 5-10 body walls.</li>
	</ol>
	</li>
	<li>Preparation for Imaging
	<ol>
		<li>Wash the body walls with Wash Buffer B twice for 10 min each.</li>
		<li>Wash with 0.01x Wash Buffer B once for 1 min.</li>
		<li>Equilibrate in a few drops of mounting solution for at least 30 min before mounting, or store overnight at 4 &#176;C.</li>
		<li>Using fine forceps, carefully transfer the body walls onto a platform slide with their cuticles facing down. Position the body walls in rows and in the same orientation within a drop or two of mountant. Place a 22 mm x 40 mm coverslip over the preparation taking care not to generate air bubbles, then seal the slide with clear nail polish.</li>
		<li>Store the slides in the dark at -20 &#176;C until ready for confocal imaging.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

This report demonstrates how PLA can be applied to the Drosophila larval NMJ. The assay is performed on larval body wall muscle preparations for the purpose of detecting endogenous protein-protein interactions present at the NMJ. With this technique, Dlg and Hts are shown to be in close proximity to each other, and thus exist in a complex, specifically at the postsynaptic region 27. In support of this result, a previous study has provided evidence of their association with the following data: 1) the i...

Drosophila Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family of scaffolding proteins that help orchestrate the assembly of large protein complexes at specific sites of the plasma membrane. Originally identified as a tumor suppressor protein, Dlg serves as an important determinant of epithelial apicobasal polarity 1,2,3. Dlg also serves as a major scaffolding module at the neuromuscular junction (NMJ) of glutamatergic motor neurons during larval development 4. Dlg plays diverse roles at the larval NMJ, and its pleiotropism relies on its ability to associate with multiple proteins 5,6. One such protein is Hu-li tai shao (Hts), a homologue to the mammalian adducins that have mainly been described in regards to their roles in regulating the actin-spectrin cytoskeleton 7. It has previously been shown that Dlg and Hts can form a complex with each other based on in vitro co-immunoprecipitation experiments involving whole adult fly lysates 8. One shortcoming of these results, however, is that they do not indicate where this complex forms. With the use of immunohistochemistry, the distributions of Dlg and Hts are observed to overlap at the postsynaptic membrane of larval NMJs, but are they in a complex in this region 8? As recently shown and detailed further here, Proximity Ligation Assay (PLA) is used to look for an in situ association between Dlg and Hts specifically at the larval NMJ 27.
PLA is a relatively new technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ 9. In this assay, primary antibodies against the two proteins of interest are detected with a pair of species-specific secondary antibodies, termed PLA probes, which are conjugated to oligonucleotides (Figure 1A, B). If the two proteins are in close proximity to each other (i.e. within a few tens of nanometers), the distance between the attached PLA probes can be bridged through hybridization of two additional connector oligonucleotides (Figure 1C). In this conformation, the free ends of the connector oligonucleotides are close enough to make contact with each other, and a closed circular DNA molecule can be formed upon in situ ligation (Figure 1D). The circular DNA molecule serves as a template for in situ rolling circle amplification, which is primed by one of the oligonucleotides conjugated to the PLA probes (Figure 1E). Sequences within the resulting amplified, concatemeric DNA product can then be visualized with fluorescently-labeled, complementary oligonucleotide probes (Figure 1F). Since the amplified DNA remains attached to one of the PLA probes, the subcellular localization of the protein-protein interaction within a tissue can be readily ascertained.
Several methods are commonly used to detect protein-protein interactions including in vitro techniques such as co-immunoprecipitation, pull-down assays and yeast two-hybrid screening, and in vivo techniques such as F&#246;rster Resonance Energy Transfer (FRET) and Bimolecular Fluorescence Complementation (BiFC). A pitfall of the in vitro techniques is that they do not identify where the interaction is endogenously occurring, while the aforementioned in vivo techniques involve the artificial expression of fusion proteins that may not reflect the native behavior of their endogenous counterparts. One major advantage of PLA is that it is capable of determining within a tissue the subcellular localization of endogenous protein interactors that are in close proximity to each other and likely forming a complex, with the degree of closeness required to generate a signal being comparable to FRET and BiFC. PLA can detect interactions with high specificity and sensitivity due to the coupling of antibody recognition and DNA amplification. Thus, the assay can generate discrete, bright signals in the form of puncta that reveal the exact position of the interaction. In addition, scarcely visible antigens can be detected. Finally, PLA is a relatively simple technique to perform, and it takes no longer than a standard immunohistochemical procedure to complete. Therefore, PLA provides a technical advantage over other protein-protein interaction assays that are often plagued with long preparation times and extensive troubleshooting.
This protocol demonstrates how PLA can be applied to the Drosophila larval NMJ for the purpose of detecting endogenous protein-protein interactions in situ. Here, PLA is performed on larval body wall muscle preparations where Dlg and Hts are shown to indeed exist in a complex at the postsynaptic region of NMJs. PLA has not been previously used to study the larval NMJ, and there are at present only a handful of published papers that have used this assay in Drosophila tissue. It is hoped that further exposure of PLA to the Drosophila community will result in its increased use as an additional tool to complement other, more commonly used protein-protein interaction assays.

<table><tbody><tr><td>Forceps (fine #5)</td><td>Almedic</td><td>A10-704</td><td></td></tr><tr><td>Sylgard Disc</td><td>World Precision Instruments</td><td>SYLG184</td><td>Mix elastomer base and curing agent in a 10:1 ratio. Set for 30 min. Pour into a mold (&lt;em&gt;e.g.&lt;/em&gt; use a 12-well cell culture plate). Let cure for at least 24 hr. Adhere to the lid of a 60 x 15 mm Petri dish lid when dissecting.</td></tr><tr><td>Minutien Pins (0.0125&amp;nbsp;mm tip diameter)</td><td>Fine Science Tools</td><td>26002-10</td><td></td></tr><tr><td>Microdissection Scissors (ultra fine)</td><td>Fine Science Tools</td><td>15200-00</td><td></td></tr><tr><td>Platform Slides</td><td></td><td></td><td>Glue two 22 x 22 mm&lt;sup&gt;2&lt;/sup&gt; coverslips onto a microscope slide with clear nail polish, leaving a &amp;lt;20 mm gap in between for sample mounting.</td></tr><tr><td>w&lt;sup&gt;1118&lt;/sup&gt;</td><td>Bloomington Drosophila Stock Center</td><td>3605</td><td></td></tr><tr><td>hts&lt;sup&gt;01103&lt;/sup&gt;</td><td>Bloomington Drosophila Stock Center</td><td>10989</td><td>Stock was re-balanced over a GFP balancer so that homozygous mutants can be selected based on the absence of GFP signal.</td></tr><tr><td>1x PBS (Phosphate Buffered Saline): 3&amp;nbsp;mM NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 7&amp;nbsp;mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 130&amp;nbsp;mM NaCl, pH 7.0</td><td></td><td></td><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (Caledon Laboratories - 8180-1), Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; (Caledon - 8120-1), NaCl (Caledon - 7560-1)</td></tr><tr><td>Bouin&#039;s Solution</td><td>Sigma-Aldrich</td><td>HT10132</td><td></td></tr><tr><td>4% PFA (Paraformaldehyde): 4% PFA in 1x PBS</td><td></td><td></td><td>PFA (Anachemia Science - 66194-300). See doi:10.1101/pdb.rec9959 Cold Spring Harb Protoc 2006 for instructions on how to make the solution.</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>1x PBT (Phosphate Buffered Saline with Triton): 1x PBS with 0.01% Triton</td><td></td><td></td><td>Triton X-100 (Sigma-Aldrich - T8787)</td></tr><tr><td>1% BSA (Bovine Serum Albumin): 1% BSA in 1x PBT</td><td></td><td></td><td>BSA (Bioshop Canada - ALB001). Store at 4 &amp;deg;C.</td></tr><tr><td>mouse anti-Dlg (Discs large)</td><td>Developmental Studies Hybridoma Bank</td><td>4F3</td><td>Use at a 1:10 dilution in 1% BSA.</td></tr><tr><td>rabbit anti-HtsM (Hu-li tai shao)</td><td></td><td></td><td>Provided by Dr. Lynn Cooley (Yale University). Use at a 1:250 dilution in 1% BSA.</td></tr><tr><td>rabbit anti-Pak (p21-activated kinase)</td><td></td><td></td><td>Provided by Dr. Nicholas Harden (Simon Fraser University). Use at a 1:500 dilution in 1% BSA.</td></tr><tr><td>mouse anti-Wg (Wingless)</td><td>Developmental Studies Hybridoma Bank</td><td>4D4</td><td>Use at a 1:5 dilution in 1% BSA.</td></tr><tr><td>goat anti-Hrp (Horseradish peroxidase)</td><td>Jackson&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; ImmunoResearch</td><td>123-065-021</td><td>Use at a 1:200 dilution in 1% BSA.</td></tr><tr><td>FITC-conjugated donkey anti-goat</td><td>Jackson&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; ImmunoResearch</td><td>705-095-003</td><td>Use at a 1:200 dilution in 1% BSA.</td></tr><tr><td>Duolink In Situ PLA Probe anti-mouse MINUS</td><td>Sigma-Aldrich</td><td>DUO92004</td><td></td></tr><tr><td>Duolink In Situ PLA Probe anti-rabbit PLUS</td><td>Sigma-Aldrich</td><td>DUO92002</td><td></td></tr><tr><td>Duolink In Situ Detection Reagents Red</td><td>Sigma-Aldrich</td><td>DUO92008</td><td></td></tr><tr><td>1x Wash Buffer A: 0.01&amp;nbsp;M Tris, 0.15&amp;nbsp;M NaCl, 0.05% Tween 20, pH 7.4</td><td>Sigma-Aldrich</td><td>DUO82049</td><td>Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1), Tween 20 (Fisher Scientific - BP337)</td></tr><tr><td>1x Wash Buffer B: 0.2&amp;nbsp;M Tris, 0.1&amp;nbsp;M NaCl, pH 7.5</td><td>Sigma-Aldrich</td><td>DUO82049</td><td>Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1)</td></tr><tr><td>0.01x Wash Buffer B: 2&amp;nbsp;mM Tris, 1&amp;nbsp;mM NaCl, pH 7.5</td><td>Sigma-Aldrich</td><td>DUO82049</td><td>Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1)</td></tr><tr><td>Duolink In Situ Mounting Medium with DAPI</td><td>Sigma-Aldrich</td><td>DUO82040</td><td></td></tr></tbody></table>

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.

In wild-type third instar larval NMJs, Dlg is predominately found at the postsynaptic membrane of type I glutamatergic boutons, with Dlg immunoreactivity levels being more pronounced in type Ib boutons than type Is boutons (Figure 3A) 4. Hts is present throughout the muscle but concentrates at the postsynaptic region with Hts immunoreactivity levels appearing equal in both type I boutons, and is also found presynaptically (Figure 3A&#39;) 8,17. Note...

Watch this Scientific Journal Video about Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay at JoVE.com

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.

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 ...

detection-situ-protein-protein-complexes-at-drosophila-larval

Research

JoVE Journal

Neuroscience

13.1K Views.  Simon Fraser University.  Dit protocol toont hoe Proximity Ligatie Assay kunnen worden toegepast voor aantonen in situ eiwit-eiwit interacties bij de Drosophila larven neuromusculaire junctie. Met deze techniek worden Schijven grote en Hu-li tai Shao aangetoond dat een complex in het postsynaptische gebied, een associatie eerder geïdentificeerd door middel co-immunoprecipitatie vormen. 

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

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

Biochemistry

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes such as development are affected. The Caenorhabditis elegans germline is a great model system for studying PPIs that are related to the regulation of stem cells, meiosis, and development. There are a variety of well-developed techniques that allow proteins of interest to be tagged for recognition by standard antibodies, making this system advantageous for proximity ligation assay (PLA) reactions. As a result, the PLA is able to show where PPIs occur in a spatial and temporal manner in germlines more effectively than alternative approaches. Described here is a protocol for the application and quantification of this technology to probe PPIs in the C. elegans germline.

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

This protocol demonstrates use of the proximity ligation assay to probe for protein-protein interactions in situ in the C. elegans germline.

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes ...

Developmental Biology

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, form a complex with Smad4 and translocate to the nucleus where they function as transcription factors to initiate BMP specific downstream effects 1. Traditional immuno-fluorescence techniques with antibodies against phospho-Smad peptides exhibit low sensitivity, high background and offer gross quantification as they rely on intensity of the antibody signal particularly if this is photosensitive fluorescent. In addition, phospho-Smads may not all be in complex with Smad4 and engaged in active transcription.
In situ PLA is a technology capable of detecting protein interactions with high specificity and sensitivity 2-4. This new technology couples antibody recognition with the amplification of DNA surrogate of the protein. It generates a localized, discrete signal in a form of spots revealing the exact position of the recognition event. The number of signals can be counted and compared providing a measurement. We applied in situ PLA, using the Duolink kit, with a combination of antibodies that allows the detection of the BMP signaling effectors phospho-Smad1/5/8 and Smad4 only when these are in proximity i.e. in a complex, which occurs only with signaling activation. This allowed for the first time, the visualization and measurement of endogenous BMP signaling with high specificity and sensitivity in a time course experiment under BMP4 stimulation.

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

Here we show how to use Proximity Ligation Assay (PLA), with a combination of antibodies to visualize Bone Morphogenetic Protein (BMP) signaling in fixed cells. This technique allowed us to follow the nuclear accumulation of endogenous BMP activated effector-complexes and quantify their levels over time under BMP4 stimulation.

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, ...

Biology

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.

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.

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

Viewing Microtubule Networks in Drosophila Neuromuscular Junctions and Muscle Cells

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

Author Spotlight: Understanding Microtubule Network in Drosophila Neuromuscular Junctions 

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with ...

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Here, we describe a procedure to visualize and quantify with high sensitivity the endogenous interactions between the endoplasmic reticulum and mitochondria in fixed cells. The protocol features an optimized in situ proximity ligation assay targeting the inositol 1,4,5-triphosphate receptor/glucose-regulated protein 75/voltage-dependent anion channel/cyclophilin D complex at the mitochondria-associated membrane interface.

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

Proximity Ligation Assay to Study In Situ Protein-Protein Interactions

Source: Wang, X., et al., <a href="https://app.jove.com/v/60982">In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay.</a>&#160;J. Vis. Exp.&#160; (2020)
This video describes the proximity ligation assay to study in situ protein-protein interactions in nematode cells. The close proximity of the proteins of interest generates the circular DNA template with the help of a PLA probe attached to a secondary antibody. The probe is further amplified using the amplification solution, which produces a long single-stranded DNA followed by the hybridization of the detector probe, enabling the localization of protein-protein interactions in cells.

Source: Wang, X., et al., In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay.&#160;J. Vis. ...

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

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