# Designing a new class of fluorescent reporters for imaging dynamic cell signaling in live animals

> **NIH NIH R35** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2020 · $582,392

## Abstract

Abstract
Proteases and kinases control a broad range of cellular processes. Dysregulation of protease and kinase
activities causes many diseases including cancer. Furthermore, temporal dynamics of kinase signaling can
elicit distinct cellular responses. To image dynamics of protease and kinase activity in live animals, genetically
encoded fluorescent reporters are ideal because they require no exogenous molecule and are non-invasive.
Although green fluorescent protein (GFP)-based Förster resonance energy transfer (FRET) reporters can be
used to image dynamics of kinase signaling in cultured cells where they can achieve subcellular resolution and
compartmentalized kinase signaling, in vivo use of the FRET-based kinase (and protease) reporters is difficult
because of small magnitude of the fluorescence changes of the fluorophores. We seek to design, demonstrate
and apply new classes of fluorescent reporters including the GFP-based reporters that, upon kinase activation,
phase separate and form highly concentrated droplets via multivalent protein-protein interactions. Our
approach is inspired by recent work showing that multivalent interactions drive protein phase separation to
form protein droplets that concentrate the protein ~10 fold. The phase separation-based kinase reporter has
both large dynamic range and high brightness because fluorophores concentrate in discrete punctae. This
punctal signal pattern is distinctive and easily detectable in whole animals and robust for in vivo detection of
kinase activity. The reporter also achieves fast kinetics at the second-to-minute timescale and is reversible,
with no apparent toxicity in transgenic animals. To further demonstrate the new design principle, we aim to
design and apply phase separation-based kinase reporters to visualize dynamics of several key kinases during
animal development and disease. We will also develop fluorogenic protease reporters to visualize dynamic
apoptosis signaling in live animals. Furthermore, we will combine the green fluorescent kinase reporters with
the infrared fluorogenic caspase reporters to simultaneously visualize kinase signaling and apoptosis and
investigate how they are correlated to morphogenetic processes during embryogenesis and tissue
homeostasis. We will also apply these reporters to investigate dynamic cell signaling network during brain
tumorigenesis.

## Key facts

- **NIH application ID:** 9930622
- **Project number:** 5R35GM131766-02
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** Xiaokun Shu
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $582,392
- **Award type:** 5
- **Project period:** 2019-06-01 → 2024-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9930622

## Citation

> US National Institutes of Health, RePORTER application 9930622, Designing a new class of fluorescent reporters for imaging dynamic cell signaling in live animals (5R35GM131766-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9930622. Licensed CC0.

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