Bipartite Genetically Encoded Biosensors to Sense Calcium Ion Dynamics at Membrane–Membrane Contact Sites
Self-complementing bipartite fluorescent proteins (FPs) are useful tools for the detection of protein-protein proximity and for localizing fluorophores to membrane-membrane contact sites. Here, we report versions of circularly permuted green FP (GFP), red FP (RFP), and mNeonGreen (NG), which are split into a large fragment composed of nine β-strands and a small fragment composed of two β-strands. In each case, the large and small fragments can associate in live cells to form the complete 11-stranded FP β-barrel. We further converted each of these three self-complementing FPs into bipartite calcium ion (Ca<sup>2+</sup>) biosensors. We demonstrate that appropriately targeted versions of these split FPs, and split FP-based biosensors, can be functionally assembled at membrane-membrane contact sites. We employ the bipartite NG-based Ca<sup>2+</sup> biosensor for visualization of pharmacologically induced Ca<sup>2+</sup> release at mitochondria-endoplasmic reticulum contact sites (MERCs).
Authors
- Wenchao Zhu
- Giulia Dematteis (ORCID: https://orcid.org/0000-0002-6317-3182)
- Yusuke Nasu (ORCID: https://orcid.org/0000-0002-3193-9104)
- Yurong Wen (ORCID: https://orcid.org/0000-0001-8730-5197)
- Tito Calí (ORCID: https://orcid.org/0000-0002-8901-1659)
- Lucia Barazzuol (ORCID: https://orcid.org/0000-0002-7569-9704)
- Robert E. Campbell (ORCID: https://orcid.org/0000-0003-0604-092X)
- Mikhail Drobizhev (ORCID: https://orcid.org/0000-0001-9072-1300)
- Isamu Yamaguchi
- Dmitry Lim (ORCID: https://orcid.org/0000-0002-4316-2654)
Institutions
- Università degli Studi del Piemonte Orientale “Amedeo Avogadro” (IT)
- University of Padua (IT)
- National Taiwan University (TW)
- Montana State University (US)
- Biochemical Society (GB)
- German Center for Neurodegenerative Diseases (DE)
- Institute of Biological Chemistry, Academia Sinica (TW)
- Xenobe Research Institute (US)
- Institute for Neurodegenerative Disorders (US)
- First Affiliated Hospital of Xi'an Jiaotong University (CN)
- Institute for Medical Informatics and Biostatistics (CH)
- Université Laval (CA)
- The University of Tokyo (JP)
- Academia Sinica (TW)
Publication Details
- Journal
- Analytical Chemistry
- Published
- 2025-09-01
- DOI
- https://doi.org/10.1021/acs.analchem.5c03831
- Citations
- 1
- Primary Topic
- Advanced biosensing and bioanalysis techniques
- Type
- article
- Field-Weighted Citation Impact
- 0.81
Funders
- Nakatani Foundation for Advancement of Measuring Technologies in Biomedical Engineering