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Published August 20, 2015 | Supplemental Material + Accepted Version
Journal Article Open

Genetically Encoded Spy Peptide Fusion System to Detect Plasma Membrane-Localized Proteins In Vivo


Membrane proteins are the main gatekeepers of cellular state, especially in neurons, serving either to maintain homeostasis or instruct response to synaptic input or other external signals. Visualization of membrane protein localization and trafficking in live cells facilitates understanding the molecular basis of cellular dynamics. We describe here a method for specifically labeling the plasma membrane-localized fraction of heterologous membrane protein expression using channelrhodopsins as a case study. We show that the genetically encoded, covalent binding SpyTag and SpyCatcher pair from the Streptococcus pyogenes fibronectin-binding protein FbaB can selectively label membrane-localized proteins in living cells in culture and in vivo in Caenorhabditis elegans. The SpyTag/SpyCatcher covalent labeling method is highly specific, modular, and stable in living cells. We have used the binding pair to develop a channelrhodopsin membrane localization assay that is amenable to high-throughput screening for opsin discovery and engineering.

Additional Information

© 2015 Elsevier Ltd. Received: February 10, 2015. Revised: May 19, 2015. Accepted: June 15, 2015. Published: July 23, 2015. We thank the Gradinaru and Arnold labs for helpful discussions, and Han Wang of the Sternberg lab for assistance with behavioral assays. We also thank Sheri McKinney for assistance with cultured neurons, and Dr. John Bedbrook for critical reading of the manuscript. This work was funded by the NIH/NINDS New Innovator (NIH IDP20D017782-01), the Beckman Institute for Optogenetics and CLARITY, the Gordon and Betty Moore Foundation through Grant GBMF2809 to the Caltech Programmable Molecular Technology Initiative (to V.G. and F.H.A.); by NIH 1R21MH103824-01 (to V.G. and F.H.A.); and by the Institute for Collaborative Biotechnologies through grant W911NF-09-0001 from the U.S. Army Research Office (to F.H.A.). The content of the information does not necessarily reflect the position or the policy of the Government, and no official endorsement should be inferred. P.W.S. is an investigator with the HHMI, which supported this research. C.N.B. acknowledges support from the NIMH of the NIH for the NRSA fellowship under Award Number F31MH102913.

Attached Files

Accepted Version - nihms711085.pdf

Supplemental Material - mmc1.pdf

Supplemental Material - mmc2.mp4


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