title: Imaging trans-cellular neurexin-neuroligin interactions by enzymatic probe ligation.
abstract: Neurexin and neuroligin are transmembrane adhesion proteins that play an important role in organizing the neuronal synaptic cleft. Our lab previously reported a method for imaging the trans-synaptic binding of neurexin and neuroligin called BLINC (Biotin Labeling of INtercellular Contacts). In BLINC, biotin ligase (BirA) is fused to one protein while its 15-amino acid acceptor peptide substrate (AP) is fused to the binding partner. When the two fusion proteins interact across cellular junctions, BirA catalyzes the site-specific biotinylation of AP, which can be read out by staining with streptavidin-fluorophore conjugates. Here, we report that BLINC in neurons cannot be reproduced using the reporter constructs and labeling protocol previously described. We uncover the technical reasons for the lack of reproducibilty and then re-design the BLINC reporters and labeling protocol to achieve neurexin-neuroligin BLINC imaging in neuron cultures. In addition, we introduce a new method, based on lipoic acid ligase instead of biotin ligase, to image trans-cellular neurexin-neuroligin interactions in human embryonic kidney cells and in neuron cultures. This method, called ID-PRIME for Interaction-Dependent PRobe Incorporation Mediated by Enzymes, is more robust than BLINC due to higher surface expression of lipoic acid ligase fusion constructs, gives stronger and more localized labeling, and is more versatile than BLINC in terms of signal readout. ID-PRIME expands the toolkit of methods available to study trans-cellular protein-protein interactions in living systems.
authors: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
authors: Daniel S
authors: DS
authors: Liu
authors: Ken H
authors: KH
authors: Loh
authors: Stephanie S
authors: SS
authors: Lam
authors: Katharine A
authors: KA
authors: White
authors: Alice Y
authors: AY
authors: Ting
mesh_terms: D020410:Activated-Leukocyte Cell Adhesion Molecule / Q000032:analysis* / Q000378:metabolism / Q000648:ultrastructure; D000818:Animals; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D002478:Cells, Cultured; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007365:Intercellular Junctions / Q000378:metabolism* / Q000648:ultrastructure; D008025:Ligases / Q000032:analysis / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000378:metabolism; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods
chemical_list: D020410:Activated-Leukocyte Cell Adhesion Molecule; D005456:Fluorescent Dyes; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D001710:Biotin; D008025:Ligases