Cell/Development/Systems

Abstract

CS-20-4 - Fluorescent Sensors for Imaging MAP Kinase Activity in Arabidopsis

Monday, July 16
4:18 PM - 4:38 PM

MAP kinase cascades play central roles in a wide range of signaling pathways in plants.  Standard methods for studying MAP kinase signaling involve extracting protein from homogenized tissue and testing the activation status of the kinase in vitro.  Live-cell imaging methods offer an attractive alternative for studying signal transduction due to their ability to reveal spatial and temporal heterogeneity.  In order to study MAP kinase signaling using live-cell imaging methods, a fluorescent sensor that reports MAP kinase activity in plants is required.  We have developed such a FRET sensor for Arabidopsis that shifts from a low-FRET to a high-FRET state upon phosphorylation by MAP kinases.  We have used transgenic Arabidopsis lines expressing this sensor to study MAP kinase activation triggered by NaCl, flg22, and chitin treatment.  Our results have demonstrated that different stimuli and different cell types produce distinct MAP kinase activation profiles.  These findings highlighting how fluorescent sensor technology has the ability to reveal spatial and temporal heterogeneity in signaling pathways that would otherwise be obscured by methods that homogenize a sample prior to analysis.  


In addition to the FRET sensor described above, we have also developed an alternative type of fluorescent sensor for MAP kinase activity in Arabidopsis called a “kinase translocation reporter”. In cells that have not been stimulated, this reporter localizes to the nucleus.  Treatment with stimuli known to activate MAP kinase causes the reporter to relocate to the cytoplasm.  By measuring the ratio of nuclear to cytoplasmic fluorescence, one can infer the activation status of the MAP kinases in cells using live-cell imaging methods.  The kinetics of reporter translocation indicate that this sensor is able to report both kinase activation and inactivation on a time-scale consistent with published MAP kinase activation profiles. 


 

Co-Authors

Kati Seitz – University of Wisconsin-Madison; Najia Zaman – University of Wisconsin-Madison; Mohuddin Kabir – East West University

Patrick J. Krysan

Professor
University of Wisconsin-Madison

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CS-20-4 - Fluorescent Sensors for Imaging MAP Kinase Activity in Arabidopsis



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