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

The overall goal of the following experiment is to detect the subcellular localization of endogenous protein protein interactions at the drosophila larva NMJ. This is achieved by performing immunofluorescence using a pair of oligonucleotide conjugated secondary antibodies termed PLA probes, which bind to the primary antibodies against the two proteins of interest. Next, the gap between the PLA probes is bridged through hybridization of the two connector oligonucleotides.

Only when the two proteins are in close proximity to each other and a closed circular DNA molecule is formed upon in situ ligation, then in situ rolling circle amplification is performed in order to amplify sequences within the closed circular DNA molecule, which are subsequently detected with complementary fluorescently tagged oligonucleotides. The resulting fluorescent signal generated from each amplification product is thus easily visible as a distinct bright spot when viewed using confocal microscopy. So the main advantage of this technique over other existing methods such as E two hybrid precipitation and threat, is that PLA is capable of detecting within the tissue the subcellular localization of endogenous proteins that are localized in close proximity to each other and likely forming a complex.

Though this method provide insight into the protein-protein interaction networks that occur specifically at de larva and mj. It can also be applied to other jolo systems as well. Suggest the epithelial tissues in embryos and imaginal discs.

Demonstrating the procedure will be hel yu and Ha king two technicians from our laboratory. After incubating drosophila cultures at 25 degrees Celsius for five to six days, use fine forceps to pick crawling third instar larvae from the vials or bottles to wash the larvae. Transfer them into a small Petri dish containing PBS.

Then place one of the washed larvae into a S cigar disc and immerse it in several drops of ice cold PBS to stunt it, thus making it easier to manipulate. Position the larvae with its dorsal side facing up so that the two tracheal tracks are visible under a dissecting microscope. Then using the forceps to grasp a mnuchin pin, pin the larvae down at the posterior end near the spirals with another pin pierce through the cuticle at the anterior end near the mouth hooks and gently stretch the larvae lengthwise before pinning it down.

Next, using micro dissection, 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. Then placing the tip of the bottom blade of the scissors into the incision, and while pointing the scissor blades up slightly to avoid damaging the underlying muscles, cut along the entire length of the dorsal midline between the two tracheal tracts. Now make a small horizontal incision just through the cuticle, slightly anterior to the posteriorly placed pin.

Then make another similar incision slightly posterior to the anteriorly placed pin. After adding a few forceful drops of PBS to the larval body, use the forceps to carefully clean out the internal organs. Avoid poking the larval body as this will cause damage to the body.

Wall muscles unfurl the larval body open and to pin the corners down, stretch the body wall both horizontally and vertically to form an evenly tensioned rectangle. Taking care not to tear the body wall muscles in the process to fix the tissue. Immerse the pinned body walls in several drops of boan solution and incubate on ice for 15 minutes following the fixation.

Use PBT to rinse the tissue three times. Then to save on reagents and to ensure that all of the body walls are treated equally during the assay. Cut the corners of each genotype differently to distinguish them.

And after removing the pins with fine forceps, place them all in the same siliconized, 0.65 milliliter micro centrifuge tube. To permeate the tissue, use PBT to wash the body walls three times for 10 minutes each. After removing the final wash, add 1%BSA to block the samples and incubate for one hour.

Next to immunostain, the body walls add mouse and rabbit primary antibodies against the two proteins of interest and incubate for two hours. In this case, one to 10 mouse anti DLG and one to 250 rabbit anti HTS in 1%BSA is used. Antibodies against markers can also be added after the incubation.

Use PBT to wash the body walls three times for 10 minutes each. Then add fluoro four labeled secondary antibodies against any markers in 1%BSA, and incubate in the dark at four degrees Celsius overnight the following morning. Use PBT to wash the body walls three times for 10 minutes each.

To begin the assay at a one to five dilution in at least 200 microliters. Total volume of both PLA probe, anti mouse minus and PLA probe anti rabbit plus in 1%BSA to the body walls incubate for two hours at 37 degrees Celsius after using wash buffer A to wash the body walls two times for five minutes each at a one to 40 dilution of Ligase in Ligase solution, incubate for one hour at 37 degrees Celsius following the incubation. Use wash buffer A to wash the body walls two times for two minutes each before adding a one to 80 dilution of polymerase in amplification solution.

Incubate for two hours at 37 degrees Celsius to prepare the samples for imaging. Use wash buffer B to wash the body walls twice for 10 minutes each. Then use a 100 x dilution of wash buffer B to do a final wash for one minute.

Equilibrate the body walls in a few drops of mounting solution for at least 30 minutes using fine forceps. Carefully transfer the body walls onto a platform slide with their cuticle spacing down. Position them in rows in the same orientation within a drop or two of mounting solution.

Place a 22 millimeter by 40 millimeter cover slip over the preparation, taking care not to generate air bubbles. Then use clear nail polish to seal the slide. Store the slides at negative 20 degrees Celsius until ready for confocal imaging.

As shown here in wild type larval njs, DLG is predominantly found at the postsynaptic membrane of type one glutamatergic bhuton with levels being more pronounced in type one B than type one S bs HTS is found throughout the muscle, but concentrates at the postsynaptic membrane with levels being equal in both type one Bhuton, DLG and HTS largely overlap at the postsynaptic region to determine if DLG and HTS exist in a complex. At the N-M-J-P-L-A was performed with anti DLG and anti HTS antibodies as seen here. PLA signal between the two proteins is observed specifically at the wild type NMJ, the signal mostly localizes circumferentially to the presynaptic membrane marked by HRP.

Thus, DLG and HTS are in close proximity and likely form a complex at the post-synaptic region. The result is specific as a negative control involving HTS mutants that lack. HTS Immunoreactivity shows no observable PLA signal high magnification views of the bns show that DLG and HTS Immunoreactivity grossly overlap at the post-synaptic membrane.

In contrast, PLA between the two proteins result in discrete PUNCTA indicating that only a subset of the total DLG and HTS proteins are in complex Once mastered. This technique takes no longer than a standard immunohistochemical procedure to complete. After watching this video, you should have a good understanding of how PLA is used to detect the subcellular localization of endogenous protein protein interaction that occur at the larva NMJ.