PMID- 23457442
OWN - NLM
STAT- MEDLINE
DCOM- 20130826
LR  - 20211021
IS  - 1932-6203 (Electronic)
IS  - 1932-6203 (Linking)
VI  - 8
IP  - 2
DP  - 2013
TI  - Imaging trans-cellular neurexin-neuroligin interactions by enzymatic probe 
      ligation.
PG  - e52823
LID - 10.1371/journal.pone.0052823 [doi]
LID - e52823
AB  - 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.
FAU - Liu, Daniel S
AU  - Liu DS
AD  - Department of Chemistry, Massachusetts Institute of Technology, Cambridge, 
      Massachusetts, United States of America.
FAU - Loh, Ken H
AU  - Loh KH
FAU - Lam, Stephanie S
AU  - Lam SS
FAU - White, Katharine A
AU  - White KA
FAU - Ting, Alice Y
AU  - Ting AY
LA  - eng
GR  - DP1 OD003961/OD/NIH HHS/United States
GR  - P30 CA014051/CA/NCI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20130214
PL  - United States
TA  - PLoS One
JT  - PloS one
JID - 101285081
RN  - 0 (Activated-Leukocyte Cell Adhesion Molecule)
RN  - 0 (Fluorescent Dyes)
RN  - 0 (Nerve Tissue Proteins)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 6SO6U10H04 (Biotin)
RN  - EC 6.- (Ligases)
SB  - IM
MH  - Activated-Leukocyte Cell Adhesion Molecule/*analysis/metabolism/ultrastructure
MH  - Animals
MH  - Biotin/metabolism
MH  - Biotinylation
MH  - Cells, Cultured
MH  - Fluorescent Dyes/analysis/metabolism
MH  - HEK293 Cells
MH  - Humans
MH  - Intercellular Junctions/*metabolism/ultrastructure
MH  - Ligases/analysis/metabolism
MH  - Microscopy, Confocal/methods
MH  - Nerve Tissue Proteins/*analysis/metabolism
MH  - Neurons/*cytology/metabolism
MH  - Protein Interaction Mapping/*methods
MH  - Rats
MH  - Recombinant Fusion Proteins/analysis/metabolism
MH  - Staining and Labeling/methods
PMC - PMC3573046
COIS- Competing Interests: The authors have declared that no competing interests exist.
EDAT- 2013/03/05 06:00
MHDA- 2013/08/27 06:00
PMCR- 2013/02/14
CRDT- 2013/03/05 06:00
PHST- 2012/10/04 00:00 [received]
PHST- 2012/11/22 00:00 [accepted]
PHST- 2013/03/05 06:00 [entrez]
PHST- 2013/03/05 06:00 [pubmed]
PHST- 2013/08/27 06:00 [medline]
PHST- 2013/02/14 00:00 [pmc-release]
AID - PONE-D-12-30649 [pii]
AID - 10.1371/journal.pone.0052823 [doi]
PST - ppublish
SO  - PLoS One. 2013;8(2):e52823. doi: 10.1371/journal.pone.0052823. Epub 2013 Feb 14.