Liese et al. reveal a genetically encoded fluorescent biosensor for CDPK conformational activation.
By Anja Liese1,2 and Tina Romeis1,2
1 Department for Biochemistry of Plant Interactions, Leibniz Institute of Plant Biochemistry, D-06120 Halle (Saale), Germany
2 Dahlem Centre of Plant Sciences, Freie Universität Berlin, D-14195 Berlin, Germany
Background: Changes in cytosolic calcium (Ca2+) concentration are among the earliest reactions in signaling in practically all aspects of eukaryotic life. Calcium dependent protein kinases (CDPKs) bind Ca2+ directly and translate the Ca2+-signal into protein phosphorylation patterns. CDPK-dependent translation of Ca2+-signals includes a conformational change of the protein itself as a requirement for kinase activity. However, elucidating isoform specificity in Ca2+ decoding via CDPKs remains a major challenge.
Question: How can we visualize the Ca2+-dependent conformational activation and inactivation of CDPKs?
Findings: We developed a genetically encoded fluorescent biosensor for CDPK conformational activation named CPKaleon. The regulatory Ca2+-binding domain of two Arabidopsis thaliana CDPKs (CPK21 and CPK23) was positioned between fluorescent proteins, allowing energy transfer (Förster resonance energy transfer, FRET) between these two fluorescent proteins. FRET measurements reported the Ca2+-dependent conformational change during the CDPK activation process. These fluorescence-based measurements recorded in vivo conformational activation and inactivation in tobacco pollen tubes and Arabidopsis guard cells in real time. We observed isoform-specific Ca2+-sensitivity and reversibility of the conformational change both in vitro and in vivo.
Next steps: We plan to use the CPKaleon approach to study additional CPDK isoforms. This would allow us to record Ca2+-decoding via CDPKs in real time and to uncover the function(s) of CDPKs in signaling pathways of interest.
Anja Liese, Bernadette Eichstädt, Sarah Lederer, Philipp Schulz, Jan Oehlschläger, Susanne Matschi, José A Feijó, Waltraud X. Schulze, Kai R. Konrad, and Tina Romeis. (2023). Imaging of plant calcium-sensor kinase conformation monitors real time calcium-dependent decoding in planta. https://doi.org/10.1093/plcell/koad196