Visualizing Dynamics of Cell Signaling In Vivo with a Phase Separation-Based Kinase Reporter

Abstract

SUMMARY Visualizing dynamics of kinase activity in living animals is essential for mechanistic understanding of cell and developmental biology. We describe GFP-based kinase reporters that phase-separate upon kinase activation via multivalent protein-protein interactions, forming intensively fluorescent droplets. Called SPARK (separation of phases-based activity reporter of kinase), these reporters have large dynamic range (fluorescence change), high brightness, fast kinetics, and are reversible. The SPARK-based protein kinase A (PKA) reporter reveals oscillatory dynamics of PKA activities upon G protein-coupled receptor activation. The SPARK-based extracellular signal-regulated kinase (ERK) reporter unveils transient dynamics of ERK activity during tracheal metamorphosis in live Drosophila. Because of intensive brightness and simple signal pattern, SPARKs allow easy examination of kinase signaling in living animals in a qualitative way. The modular design of SPARK will facilitate development of reporters of other kinases.

Kinase-mediated RAS signaling via membraneless cytoplasmic protein granules

“…Our findings prompted a new model in which clustering of an RTK in membraneless cytoplasmic protein granules is sufficient to organize activation of RAS/MAPK signaling. To directly test this hypothesis, we utilized the HOtag method recently developed for forced protein granule formation through multivalent interactions [32] (Fig. S17).…”

Visualizing cellular heterogeneity by quantifying the dynamics of MAPK activity in live mammalian cells with synthetic fluorescent biosensors

“…However, due to relative small dynamic change and high background noise, neither KTR nor FRET is sufficient to monitor kinase activity within a broad range of samples. Recently, the SPARK reporters demonstrated large dynamic changes allowing the monitoring of PKA and ERK activity in diverse samples 26 . Although displaying a clear visual readout for MAPK activity, the quantification of kinase kinetics is more challenging due to a nonuniform spatial readout, which unfortunately hinders its application for the automated quantification of a large population of single cells.…”

“…In yeast cells, the SKARS (Synthetic Kinase Activity Relocation Sensor) has been engineered to monitor MAPK activity in the mating and cell wall integrity pathways 25 . More recently, a new strategy based on protein aggregation has been presented 26 , where the phosphorylation of a synthetic substrate by the kinase leads to the formation of bright fluorescent foci. One key advantage of these reporters is that they occupy a single fluorescent channel in the microscope.…”

Applying Multivalent Biomolecular Interactions for Biosensors

“…[29] More recently,c ellular liquidliquid phase transitions, which are driven by multivalent protein interactions and subsequenta ssembly induced condensation, are also successfully used to monitork inase activities in live cells (Figure 4b). [30] Upon cellular kinase activation, multivalent kinase substrates are phosphorylated and interact with multivalent phosphor-protein binding proteins, which leads to the formation of liquid protein droplets. Simple fluorescent protein fusion to the multivalent kinase substrate allows the robust detection of kinase reaction by observing bright fluorescent droplets in live cells.…”