title: Detection of in situ protein-protein complexes at the Drosophila larval neuromuscular junction using proximity ligation assay.
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.
authors: Department of Molecular Biology and Biochemistry, Simon Fraser University.
authors: Simon
authors: S
authors: Wang
authors: Department of Molecular Biology and Biochemistry, Simon Fraser University.
authors: SooHyun
authors: S
authors: Yoo
authors: Department of Molecular Biology and Biochemistry, Simon Fraser University.
authors: Hae-Yoon
authors: HY
authors: Kim
authors: Department of Biomedical Physiology and Kinesiology, Simon Fraser University.
authors: Mannan
authors: M
authors: Wang
authors: Department of Biomedical Physiology and Kinesiology, Simon Fraser University.
authors: Clare
authors: C
authors: Zheng
authors: Department of Biomedical Physiology and Kinesiology, Simon Fraser University.
authors: Wade
authors: W
authors: Parkhouse
authors: Department of Biomedical Physiology and Kinesiology, Simon Fraser University.
authors: Charles
authors: C
authors: Krieger
authors: Department of Molecular Biology and Biochemistry, Simon Fraser University; nharden@sfu.ca.
authors: Nicholas
authors: N
authors: Harden
mesh_terms: D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D004330:Drosophila / Q000254:growth & development / Q000378:metabolism*; D029721:Drosophila Proteins / Q000032:analysis / Q000378:metabolism*; D051528:Guanylate Kinases / Q000378:metabolism; D007150:Immunohistochemistry; D007814:Larva / Q000737:chemistry / Q000378:metabolism; D009469:Neuromuscular Junction / Q000737:chemistry / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods; D025521:Tumor Suppressor Proteins / Q000032:analysis / Q000378:metabolism*
chemical_list: D002148:Calmodulin-Binding Proteins; D029721:Drosophila Proteins; D025521:Tumor Suppressor Proteins; C108208:hts protein, Drosophila; C070350:dlg1 protein, Drosophila; D051528:Guanylate Kinases
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