GFP Reconstitution Across Synaptic Partners (GRASP) defines cell contacts and synapses in living nervous systems.
The identification of synaptic partners is challenging in dense nerve bundles, where many processes occupy regions beneath the resolution of conventional light microscopy. To address this difficulty, we have developed GRASP, a system to label membrane contacts and synapses between two cells in living animals. Two complementary fragments of GFP are expressed on different cells, tethered to extracellular domains of transmembrane carrier proteins. When the complementary GFP fragments are fused to ubiquitous transmembrane proteins, GFP fluorescence appears uniformly along membrane contacts between the two cells. When one or both GFP fragments are fused to synaptic transmembrane proteins, GFP fluorescence is tightly localized to synapses. GRASP marks known synaptic contacts in C. elegans, correctly identifies changes in mutants with altered synaptic specificity, and can uncover new information about synaptic locations as confirmed by electron microscopy. GRASP may prove particularly useful for defining connectivity in complex nervous systems.
Feinberg
Evan H
EH
Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10065, USA.
Vanhoven
Miri K
MK
Bendesky
Andres
A
Wang
George
G
Fetter
Richard D
RD
Shen
Kang
K
Bargmann
Cornelia I
CI
D000818:Animals; D030801:Animals, Genetically Modified; D001522:Behavior, Animal; D017173:Caenorhabditis elegans; D029742:Caenorhabditis elegans Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D008854:Microscopy, Electron / Q000379:methods; D008954:Models, Biological; D009154:Mutation / Q000502:physiology; D009420:Nervous System / Q000166:cytology*; D009474:Neurons / Q000502:physiology* / Q000648:ultrastructure; D013569:Synapses / Q000502:physiology* / Q000648:ultrastructure
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D049452:Green Fluorescent Proteins