Direct visualization of trans-synaptic neurexin-neuroligin interactions during synapse formation.
Neurexins and neuroligins are synaptic cell-adhesion molecules that are essential for normal synapse specification and function and are thought to bind to each other trans-synaptically, but such interactions have not been demonstrated directly. Here, we generated neurexin-1β and neuroligin-1 and neuroligin-2 fusion proteins containing complementary "split" GFP fragments positioned such that binding of neurexin-1β to neuroligin-1 or neuroligin-2 allowed GFP reconstitution without dramatically changing their binding affinities. GFP fluorescence was only reconstituted from split-GFP-modified neurexin-1β and neuroligin-1 if and after neurexin-1β bound to its neuroligin partner; reassociation of the split-GFP components with each other did not mediate binding. Using trans-cellular reconstitution of GFP fluorescence from split-GFP-modified neurexin-1β and neuroligins as an assay, we demonstrate that trans-synaptic neurexin/neuroligin binding indeed occurred when mouse hippocampal neurons formed synapses onto non-neuronal COS-7 cells expressing neuroligins or when mouse hippocampal neurons formed synapses with each other. This visualization of synapses by neurexin/neuroligin binding prompted us to refer to this approach as "SynView." Our data demonstrate that neurexin-1β forms a trans-synaptic complex with neuroligin-1 and neuroligin-2 and that this interaction can be used to label synapses in a specific fashion in vivo.
Department of Molecular and Cellular Physiology and tsetsenis@gmail.com tcs1@stanford.edu.
Theodoros
T
Tsetsenis
Department of Molecular and Cellular Physiology and.
Antony A
AA
Boucard
Department of Molecular and Cellular Physiology and Howard Hughes Medical Institute, Stanford University, Stanford, California 94305.
Demet
D
Araç
Department of Molecular and Cellular Physiology and Howard Hughes Medical Institute, Stanford University, Stanford, California 94305.
Axel T
AT
Brunger
Department of Molecular and Cellular Physiology and Howard Hughes Medical Institute, Stanford University, Stanford, California 94305 tsetsenis@gmail.com tcs1@stanford.edu.
Thomas C
TC
Südhof
D000818:Animals; D019556:COS Cells; D015816:Cell Adhesion Molecules, Neuronal / Q000378:metabolism*; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D057809:HEK293 Cells; D006624:Hippocampus / Q000166:cytology / Q000254:growth & development; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D055495:Neurogenesis; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D011485:Protein Binding; D013569:Synapses / Q000378:metabolism*
D015816:Cell Adhesion Molecules, Neuronal; D009419:Nerve Tissue Proteins; C093438:neuroligin 1; C086959:neurexin Ibeta
SynView; microscopy; neurexin; neuroligin; split-GFP; synapses