Kinase-dependent regulation of plant endocytosis upon temperature stress.

How do plants regulate their cellular processes in response to temperature fluctuations? Liquid-liquid phase-separation has emerged as a crucial mechanism to organise and drive essential cellular processes in a temperature-dependent fashion and excess phase separation has been linked to protein inactivation. Recently I discovered that two essential subunits of the Arabidopsis TSET/TPLATE endocytic adaptor complex, AtEH1/Pan1 and AtEH2/Pan1 undergo temperature-dependent phase-separation. I also found that induced high temperature phase-separation of AtEH/Pan1 proteins is quickly reversed, indicating that plants have evolved a mechanism to counteract this process. AtEH1/Pan1 is differentially phosphorylated upon high temperature and I identified the U+2018dual specificity tyrosine-phosphorylation-regulated kinaseU+2019 (DYRK) family as potential regulators. Using the model plant Arabidopsis thaliana, I will establish Total Internal Reflection Fluorescence (TIRF) microscopy coupled to a microfluidic heating chamber to study how temperature increase influences endocytosis. Next, I will use a variety of cutting-edge approaches (CRISPR, proximity biotinylation, in vitro kinase assays, live-cell microscopy) to address how DYRKs directly regulate phase-separation of AtEH1/Pan1 to act as temperature regulators of endocytosis. The identification of a new mechanism how plants adapt to temperature fluctuations will be ground-breaking and pave the way to designing temperature resilient crops.