Name: detection of in situ protein-protein complexes at the drosophila larval neuromuscular junction using proximity ligation assay
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Description: 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

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 ...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" width="630">
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			<td height="20" style="height:20px;width:211px;">Name of Material/Equipment</td>
			<td style="width:120px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:191px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:211px;">Forceps (fine #5)</td>
			<td style="width:120px;">Almedic</td>
			<td>A10-704</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="133">
			<td height="133" style="height:133px;width:211px;">Sylgard Disc</td>
			<td style="width:120px;">World Precision Instruments</td>
			<td>SYLG184</td>
			<td style="width:191px;">Mix elastomer base and curing agent in a 10:1 ratio. Set for 30 min. Pour into a mold (e.g. use a 12-well cell culture plate). Let cure for at least 24 hr. Adhere to the lid of a 60x15mm petri dish lid when dissecting.</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Minutien Pins (0.0125 mm tip diameter)</td>
			<td style="width:120px;">Fine Science Tools</td>
			<td>26002-10</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">Microdissection Scissors (ultra fine)</td>
			<td style="width:120px;">Fine Science Tools</td>
			<td style="width:111px;">15200-00</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="95">
			<td height="95" style="height:95px;width:211px;">Platform Slides</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">Glue two 22x22mm coverslips onto a microscope slide with clear nail polish, leaving a &#60;20mm gap in between for sample mounting.</td>
		</tr>
		<tr height="95">
			<td height="95" style="height:95px;width:211px;">w1118</td>
			<td style="width:120px;">Bloomington Drosophila Stock Center</td>
			<td style="width:111px;">3605</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="95">
			<td height="95" style="height:95px;width:211px;">hts01103</td>
			<td style="width:120px;">Bloomington Drosophila Stock Center</td>
			<td style="width:111px;">10989</td>
			<td style="width:191px;">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 height="76">
			<td height="76" style="height:76px;width:211px;">1x PBS (Phosphate Buffered Saline): 3mM NaH2PO4, 7mM Na2HPO4, 130mM NaCl, pH 7.0</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">NaH2PO4 (Caledon Laboratories - 8180-1), Na2HPO4 (Caledon - 8120-1), NaCl (Caledon - 7560-1)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Bouin&#39;s Solution</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">HT10132</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="114">
			<td height="114" style="height:114px;width:211px;">4% PFA (Paraformaldehyde): 4% PFA in 1x PBS</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">PFA (Anachemia Science - 66194-300). See doi:10.1101/pdb.rec9959 Cold Spring Harb Protoc 2006 for instructions on how to make the solution.&#160;</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">1x PBT (Phosphate Buffered Saline with Triton): 1x PBS with 0.01% Triton</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">Triton X-100 (Sigma-Aldrich - T8787)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">1% BSA (Bovine Serum Albumin): 1% BSA in 1x PBT</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">BSA (Bioshop Canada - ALB001). Store at 4 &#176;C.</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">mouse anti-Dlg (Discs large)</td>
			<td style="width:120px;">Developmental Studies Hybridoma Bank</td>
			<td>4F3</td>
			<td style="width:191px;">Use at a 1:10 dilution in 1% BSA.</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">rabbit anti-HtsM (Hu-li tai shao)</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">Provided by Dr. Lynn Cooley (Yale University). Use at a 1:250 dilution in 1% BSA.</td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:211px;">rabbit anti-Pak (p21-activated kinase)</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">Provided by Dr. Nicholas Harden (Simon Fraser University). Use at a 1:500 dilution in 1% BSA.</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">mouse anti-Wg (Wingless)</td>
			<td style="width:120px;">Developmental Studies Hybridoma Bank</td>
			<td style="width:111px;">4D4</td>
			<td style="width:191px;">Use at a 1:5 dilution in 1% BSA.</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">goat anti-Hrp (Horseradish peroxidase)</td>
			<td style="width:120px;">Jackson&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; ImmunoResearch</td>
			<td style="width:111px;">123-065-021</td>
			<td style="width:191px;">Use at a 1:200 dilution in 1% BSA.</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">FITC-conjugated donkey anti-goat</td>
			<td style="width:120px;">Jackson&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; ImmunoResearch</td>
			<td style="width:111px;">705-095-003</td>
			<td style="width:191px;">Use at a 1:200 dilution in 1% BSA.</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Duolink In Situ PLA Probe anti-mouse MINUS</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO92004</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Duolink In Situ PLA Probe anti-rabbit PLUS</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO92002</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Duolink In Situ Detection Reagents Red</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td>DUO92008</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:211px;">1x Wash Buffer A: 0.01M Tris, 0.15M NaCl, 0.05% Tween 20, pH 7.4</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO82049</td>
			<td style="width:191px;">Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1), Tween 20 (Fisher Scientific - BP337)</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">1x Wash Buffer B: 0.2M Tris, 0.1M NaCl, pH 7.5</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO82049</td>
			<td style="width:191px;">Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1)</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">0.01x Wash Buffer B: 2mM Tris, 1mM NaCl, pH 7.5</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO82049</td>
			<td style="width:191px;">Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Duolink In Situ Mounting Medium with DAPI</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO82040</td>
			<td style="width:191px;"></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 ...

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Neuroscience

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