HybriWell™ hybridization chambers are the ideal tool for conserving precious or costly reagents in imaging experiments. HybriWells™ are inexpensive, disposable devices that create a chamber whose ports allow liquid exchange via microfluidic passive pumping [2], as demonstrated in this video:
Seitz et, al. recently applied the method to develop and test fluorescence-based genetically-encoded biosensors to study kinase activation dynamics in living tissue.
Mitogen-activated protein kinases (MAPKs) regulates many biological processes in plants, including growth, development, and stress responses. Fluorescence biosensors are useful tools to study MAPKs-mediated pathways in living plant tissues. The researchers developed two different biosensors: A MAPK FRET biosensor and a translocation biosensor.
The MAPK FRET biosensor, featuring two fluorophores, a linker, a phosphoacceptor site, and the phosphoamino acid-binding domain optimized to produce a strong FRET change in response to phosphorylation. The addition of a suitable Arabidopsis MAPK docking domain was sufficient to allow the biosensor to be efficiently phosphorylated by plant MAPKs, thus demonstrating that used to rapidly screen in vitro for amino acid sequences that specifically interact with chosen plant MAPK isoforms.
Domain structure of the MAPK FRET biosensor. YPet is a yellow fluorescent protein, FHA is the FHA1 phosphopeptide binding domain of yeast RAD53, EV linker is a flexible linker domain, P.A. is the phosphoacceptor domain, MKP1 is an 80 amino acid region of the Arabidopsis MKP1 protein, and Turquoise GL is a blue fluorescent protein.
The translocation biosensor, contains a “nuclear localization signal (NLS) and a nuclear exclusion signal (NES) module, with several phosphorylation sites scattered throughout. The phosphorylation status of these sites impacts the activities of these localization signals, directing the biosensor to either localize or be excluded from the nuclei of cells in a phosphorylation-dependent manner. By attaching a docking domain targeted by a kinase of interest to this type of biosensor, the nucleocytoplasmic shuttling of the sensor can serve as a visual indicator of kinase activity”.
The FRET and translocation biosensors, described in this study, were demonstrated to be valuable tools to study MAPK signaling in Arabidopsis.
References
- Seitz K, Krysan PJ. Expanding the Toolkit of Fluorescent Biosensors for Studying Mitogen Activated Protein Kinases in Plants. Int J Mol Sci. 2020;21(15):5350. Published 2020 Jul 28. doi:10.3390/ijms21155350
- SVang S, Seitz K, Krysan PJ. A simple microfluidic device for live cell imaging of Arabidopsis cotyledons, leaves, and seedlings. Biotechniques. 2018 Jun;64(6):255-261. doi: 10.2144/btn-2018-0044. PMID: 29939090.
