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Plos One : Imaging Trans-cellular Neurexin-neuroligin Interactions by Enzymatic Probe Ligation, Volume 7

By Rao, Jianghong

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Book Id: WPLBN0003939086
Format Type: PDF eBook :
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Reproduction Date: 2015

Title: Plos One : Imaging Trans-cellular Neurexin-neuroligin Interactions by Enzymatic Probe Ligation, Volume 7  
Author: Rao, Jianghong
Volume: Volume 7
Language: English
Subject: Journals, Science, Medical Science
Collections: Periodicals: Journal and Magazine Collection (Contemporary)
Historic
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Publisher: Plos

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Rao, J. (n.d.). Plos One : Imaging Trans-cellular Neurexin-neuroligin Interactions by Enzymatic Probe Ligation, Volume 7. Retrieved from http://netlibrary.net/


Description
Description : 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.

 

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