Multiplexed labeling of cellular proteins with split fluorescent protein tags

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• Self-complementing split fluorescent proteins (split FP_1-10/11) are vital for live-cell protein imaging.
• Existing split FP systems have limitations in simultaneously labeling multiple proteins in single cells.

Purpose of the Study:

• To expand the number of orthogonal split FP systems with spectrally distinct colors.
• To enable multiplexed labeling of cellular proteins for advanced live-cell imaging.

Main Methods:

• Rational design and directed evolution were employed to expand the spectral color palette of split FP_1-10/11.
• Circularly permuted GFP was synthesized, creating β-strand 7, 8, or 10 systems.
• Orthogonal split GFP pairs were developed for multiplexed labeling.

Main Results:

• New orthogonal split FP systems with distinct spectral colors were successfully created.
• The developed systems are orthogonal to existing FP_1-10/11 pairs.
• Simultaneous imaging of four distinct proteins in single cells was demonstrated.

Conclusions:

• The novel multiplexing approach significantly expands multicolor live-cell imaging capabilities.
• The study reveals the nuclear localization of focal adhesion protein Zyxin through simultaneous imaging.
• This advancement provides a powerful tool for studying complex cellular processes.