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

Summary

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.

Abstract

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.

Introduction

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. On....

Protocol

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.

1. Dissection
   1. Raise fly stocks and crosses at 25 °C for five to six days using standard procedures ¹³.
   2. Pick crawling third instar larvae from vials or bottles using fine forceps.
   3. Wash the larvae in a small Petri dish containing Phosphate Buffer Saline (PBS) to remove any food particles.
   4. Plac....

Results

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') ^8,17. Note tha.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Forceps (fine #5)	Almedic	A10-704
Sylgard Disc	World Precision Instruments	SYLG184	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 60 x 15 mm Petri dish lid when dissecting.
Minutien Pins (0.0125 mm tip diameter)	Fine Science Tools	26002-10
Microdissection Scissors (ultra fine)	Fine Science Tools	15200-00
Platform Slides			Glue two 22 x 22 mm2 coverslips onto a microscope slide with clear nail polish, leaving a <20 mm gap in between for sample mounting.
w1118	Bloomington Drosophila Stock Center	3605
hts01103	Bloomington Drosophila Stock Center	10989	Stock was re-balanced over a GFP balancer so that homozygous mutants can be selected based on the absence of GFP signal.
1x PBS (Phosphate Buffered Saline): 3 mM NaH2PO4, 7 mM Na2HPO4, 130 mM NaCl, pH 7.0			NaH2PO4 (Caledon Laboratories - 8180-1), Na2HPO4 (Caledon - 8120-1), NaCl (Caledon - 7560-1)
Bouin's Solution	Sigma-Aldrich	HT10132
4% PFA (Paraformaldehyde): 4% PFA in 1x PBS			PFA (Anachemia Science - 66194-300). See doi:10.1101/pdb.rec9959 Cold Spring Harb Protoc 2006 for instructions on how to make the solution.
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1x PBT (Phosphate Buffered Saline with Triton): 1x PBS with 0.01% Triton			Triton X-100 (Sigma-Aldrich - T8787)
1% BSA (Bovine Serum Albumin): 1% BSA in 1x PBT			BSA (Bioshop Canada - ALB001). Store at 4 °C.
mouse anti-Dlg (Discs large)	Developmental Studies Hybridoma Bank	4F3	Use at a 1:10 dilution in 1% BSA.
rabbit anti-HtsM (Hu-li tai shao)			Provided by Dr. Lynn Cooley (Yale University). Use at a 1:250 dilution in 1% BSA.
rabbit anti-Pak (p21-activated kinase)			Provided by Dr. Nicholas Harden (Simon Fraser University). Use at a 1:500 dilution in 1% BSA.
mouse anti-Wg (Wingless)	Developmental Studies Hybridoma Bank	4D4	Use at a 1:5 dilution in 1% BSA.
goat anti-Hrp (Horseradish peroxidase)	Jackson              ImmunoResearch	123-065-021	Use at a 1:200 dilution in 1% BSA.
FITC-conjugated donkey anti-goat	Jackson             ImmunoResearch	705-095-003	Use at a 1:200 dilution in 1% BSA.
Duolink In Situ PLA Probe anti-mouse MINUS	Sigma-Aldrich	DUO92004
Duolink In Situ PLA Probe anti-rabbit PLUS	Sigma-Aldrich	DUO92002
Duolink In Situ Detection Reagents Red	Sigma-Aldrich	DUO92008
1x Wash Buffer A: 0.01 M Tris, 0.15 M NaCl, 0.05% Tween 20, pH 7.4	Sigma-Aldrich	DUO82049	Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1), Tween 20 (Fisher Scientific - BP337)
1x Wash Buffer B: 0.2 M Tris, 0.1 M NaCl, pH 7.5	Sigma-Aldrich	DUO82049	Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1)
0.01x Wash Buffer B: 2 mM Tris, 1 mM NaCl, pH 7.5	Sigma-Aldrich	DUO82049	Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1)
Duolink In Situ Mounting Medium with DAPI	Sigma-Aldrich	DUO82040