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>

<ol>
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O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+tumourigenesis+by+the+Scribble/Dlg/Lgl+polarity+module.">Control of tumourigenesis by the Scribble/Dlg/Lgl polarity module.</a> Oncogene. 27, 6888-6907 (2008).</li><li>Lahey, T., Gorczyca, M., Jia, X. 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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 border="0" cellpadding="0" cellspacing="0" width="630">
	<tbody>
		<tr height="20">
			<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 ...

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

Research

JoVE Journal

Neuroscience

13.0K Görüntüleme.  Simon Fraser University.  Bu protokol Proximity ligasyon Testi Drosophila larva nöromüsküler kavşakta yerinde protein-protein etkileşimleri tespit etmek için nasıl kullanılabileceğini gösteriyor. Bu teknikle, Diskler, büyük ve Hu-li tai shao postsinaptik bölgede bir kompleks, daha önce birlikte immunoprecipitation ile tespit dernek kurma gösterilmiştir. 

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

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The Drosophila larval neuromuscular junction (NMJ), a well-established model for glutamatergic synapses, has been extensively studied for decades. Identification of mutations causing NMJ morphological defects revealed a repertoire of genes that regulate synapse development and function. Many of these were identified in large-scale studies that focused on qualitative approaches to detect morphological abnormalities of the Drosophila NMJ. A drawback of qualitative analyses is that many subtle players contributing to NMJ morphology likely remain unnoticed. Whereas quantitative analyses are required to detect the subtler morphological differences, such analyses are not yet commonly performed because they are laborious. This protocol describes in detail two image analysis algorithms "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics", available as Fiji-compatible macros, for quantitative, accurate and objective morphometric analysis of the Drosophila NMJ. This methodology is developed to analyze NMJ terminals immunolabeled with the commonly used markers Dlg-1 and Brp. Additionally, its wider application to other markers such as Hrp, Csp and Syt is presented in this protocol. The macros are able to assess nine morphological NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the NMJ terminal.

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Two image analysis algorithms, "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics" were created, to automatically quantify nine morphological features of the Drosophila neuromuscular junction (NMJ).

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The well characterized larval neuromuscular junction is often used for such studies. However, rapid larval development followed by muscle histolysis and nervous system remodeling during metamorphosis makes this model problematic for the study of slow age-dependent degenerative changes like those occurring in amyotrophic lateral sclerosis. Alternatively, adult flies live for 90 days and the adult leg can be used to study motor neuron changes over the course of adult lifespan using in vivo fluorescent imaging through the cuticle. Here, we describe a leg dissection technique coupled with immunocytochemistry, which allows for the study of molecular changes at the neuromuscular junction of identified adult leg motor neurons. These techniques can be coupled with a myriad of antibodies labeling both pre- and post-synaptic structures. Together these procedures allow a more complete characterization of slow age-dependent changes in adult flies and can be applied across multiple motor neuron disease models.

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

We describe a dissection technique that preserves the architecture of the neuromuscular junction and enables a detailed immunocytochemical study of motor neurons in the adult Drosophila leg.

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The ...