Bright split red fluorescent proteins for the visualization of endogenous proteins and synapses

Siyu Feng, Aruna Varshney, Doris Coto Villa, Cyrus Modavi, John Kohler, Fatima Farah, Shuqin Zhou, Nebat Ali, Joachim D. Müller, Miri K. Van Hoven, Bo Huang

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

Self-associating split fluorescent proteins (FPs) are split FPs whose two fragments spontaneously associate to form a functional FP. They have been widely used for labeling proteins, scaffolding protein assembly and detecting cell-cell contacts. Recently developments have expanded the palette of self-associating split FPs beyond the original split GFP1-10/11. However, these new ones have suffered from suboptimal fluorescence signal after complementation. Here, by investigating the complementation process, we have demonstrated two approaches to improve split FPs: assistance through SpyTag/SpyCatcher interaction and directed evolution. The latter has yielded two split sfCherry3 variants with substantially enhanced overall brightness, facilitating the tagging of endogenous proteins by gene editing. Based on sfCherry3, we have further developed a new red-colored trans-synaptic marker called Neuroligin-1 sfCherry3 Linker Across Synaptic Partners (NLG-1 CLASP) for multiplexed visualization of neuronal synapses in living C. elegans, demonstrating its broad applications.

Original languageEnglish (US)
Article number344
JournalCommunications biology
Volume2
Issue number1
DOIs
StatePublished - Dec 1 2019

Bibliographical note

Funding Information:
We thank Dr. David Alexander Brown and Dr. Yina Wang for extensive discussion on data analysis, Dr. Bin Yang for help in fluorescence light microscopy, Alejandro Ramirez for preparing reagents and supplies, Dr. Kari Herrington for help in FRAP experiment, Dr. Noelle L’Etoile for advice on NLG-1 CLASP experiments, Dr. Xiaokun Shu for sharing the BioSpectrum Imaging System, Dr. Joseph DeRisi for generously sharing the nucleofector device, and Jordan Mitchell for contributing to the SDS behavior experiment. This work is supported by the National Institutes of Health R21EB022798 and R01GM124334 (to B.H.), UCSF Program for Breakthrough Biomedical Research (Byers Award in Basic Science to B.H.), the National Institutes of Health R01GM064589 (to J.K. and J.D.M.), the National Institutes of Health R01NS087544 and SC3GM089595 (to M. V.), T34GM008253 MARC fellowship (to N.A.), and the National Science Foundation 1355202 (to M.V.). B.H. is a Chan Zuckerberg Biohub investigator.

Publisher Copyright:
© 2019, The Author(s).

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