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

Area of Science:

• Biochemistry
• Molecular Biology
• Neuroscience

Background:

• Self-associating split fluorescent proteins (FPs) are crucial tools for protein labeling and cellular analysis.
• Existing split FPs often exhibit suboptimal fluorescence after complementation, limiting their utility.
• Recent advancements expanded the range of self-associating FPs, but brightness remains a challenge.

Purpose of the Study:

• To investigate split FP complementation and identify strategies for improving fluorescence signal.
• To develop enhanced split FP variants with superior brightness and functionality.
• To create novel tools for visualizing biological processes, such as neuronal synapses.

Main Methods:

• Investigated split FP complementation mechanisms.
• Employed SpyTag/SpyCatcher interaction to assist FP complementation.
• Utilized directed evolution to generate improved split FP variants.
• Developed a new trans-synaptic marker (NLG-1 CLASP) based on enhanced sfCherry3.

Main Results:

• Demonstrated two effective strategies to enhance split FP performance: SpyTag/SpyCatcher assistance and directed evolution.
• Developed two split sfCherry3 variants with significantly improved brightness.
• Successfully tagged endogenous proteins using gene editing with the new variants.
• Created and validated Neuroligin-1 sfCherry3 Linker Across Synaptic Partners (NLG-1 CLASP) for neuronal synapse visualization.

Conclusions:

• The developed split sfCherry3 variants offer substantial improvements in brightness, facilitating endogenous protein tagging.
• NLG-1 CLASP provides a powerful new tool for multiplexed visualization of neuronal synapses in living organisms.
• These advancements broaden the applications of self-associating split FPs in biological research.