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PreprintCited by 0Advanced Fluorescence Microscopy TechniquesBiochemical and Structural Characterizationbioluminescence and chemiluminescence research

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Intensity Modulation of Trichromatic Split Fluorescent Proteins for Live Cell Mapping

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Ishii, Mamoru, Kinjo, Tomoaki, Kondo, Yohei, Terai, Kenta, Aoki, Kazuhiro, Kuhlman, Brian, Matsuda, Michiyuki·2025·Cell Reports Methods
DOI 10.1101/2025.05.13.653616PMC PMC13107049

Preprint — published as 10.1016/j.crmeth.2026.101363

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Abstract

Multiplexed live cell-labeling technologies are essential tools for investigating complex biological systems, yet existing approaches face limitations in achieving reliable discrimination of multiple cell populations. Here, we present color and intensity modulation with split fluorescent proteins for cell labeling (Caterpie), a live cell labeling system that enables high-fidelity identification of multiple cell populations through rational design of split fluorescent proteins. Drawing inspiration from human trichromatic vision, we engineered optimized split fluorescent protein variants—split CFP2, split mNG3A, and split sfCherry3C—and developed a comprehensive library of the 11th β-strand (FP 11 ) tags comprising variable numbers of tandem repeats. Structure-guided protein engineering enhanced the performance of split mNG3A and split sfCherry3C through targeted modifications of their C-terminal sequences and binding interfaces, respectively. By systematically varying the composition and arrangement of FP 11 repeats, we selected 20 distinct FP 11 tags that enable robust cell population discrimination with 97% accuracy using conventional fluorescence microscopy. In these FP 11 tag-expressing cells, we expressed multiple EGF ligands and their receptors and cultured them in bulk to examine the effects of EGF signaling on chemotaxis within the cell population. The results showed that chemotaxis changes according to the intensity of EGFR signaling. The Caterpie system holds significant potential for applications requiring precise identification and tracking of multiple cell populations in complex biological systems.

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Metadata

Authors
Ishii, M., Kinjo, T., Kondo, Y., Terai, K., Aoki, K., Kuhlman, B., & Matsuda, M.
Affiliations
  • Kyoto University (JP)
  • University of North Carolina at Chapel Hill (US)
  • Nagoya University (JP)
  • Tokushima University (JP)
Corresponding author
  • Ishii, Mamoru
  • Matsuda, Michiyuki
ORCID
Venue
Cell Reports Methods
Publisher
openRxiv
Published
2025-05-14
Volume
6
Issue
4
Pages
101363-101363
ISSN
2667-2375 (ISSN-L)
DOI
10.1101/2025.05.13.653616
PMC
PMC13107049
OpenAlex
W4410430249
Cited by
0
References
1
FWCI
0.00
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Reports Methods (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • Carolina Digital Repository (University of North Carolina at Chapel Hill) (submittedVersion, cc-by)
Funders
  • Takeda Science Foundation
  • National Institutes of Health
  • Japan Society for the Promotion of Science
  • National Institute of General Medical Sciences (R35GM131923)
  • Moonshot Research and Development Program
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › bioluminescence and chemiluminescence research
Keywords
split fluorescent proteins, live cell labeling, intensity modulation, structure-guided protein engineering, EGF signaling, EGFR, cell chemotaxis
MeSH terms
Humans, Luminescent Proteins, Microscopy, Fluorescence, Green Fluorescent Proteins, HEK293 Cells

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