# Dissecting signaling in vivo via precise control and visualization of protein activity

> **NIH NIH R35** · UNIV OF NORTH CAROLINA CHAPEL HILL · 2021 · $789,940

## Abstract

ABSTRACT
Modern microscopy and image analysis, together with fluorescent probe technology, has evolved to quantify
signaling in living cells and animals with seconds and microns resolution. More recently, optogenetics and
chemogenetics have made it possible to control signaling in vivo, and thereby explore causal relationships
among signaling molecules as they are regulated by spatio-temporal dynamics. We propose here to combine
protein visualization and control in the same cell, for unprecedented quantitative accuracy in studying how Rho
GTPase signals are coordinated by feed-back and feed-forward relationships. To generate proteins controlled
by light or small molecules, we will use novel approaches that provide ready access to many different
structures and minimize perturbation of living cells. These include dye-based biosensors of endogenous
protein conformation, engineered allosteric control for inhibition or activation by light, and the use of
photoresponsive protein analogs that can serve as substitutes for endogenous proteins. We will study
‘frustrated phagocytosis’, a system where the complex dynamics driving phagocytosis are preserved, but are
restricted to two dimensions and occur in precise geometries generated by patterned substrates. We will
examine communication between spatially restricted zones of signaling using single molecule microscopy of
protein conformational change. Precise control of activation gradients, kinetics and localization will be used to
inform mathematical models examining how precisely segregated signaling domains are maintained. In a
second project, we will work with our collaborators Eric Betzig and Leong Chew of Janelia Farm to adapt
biosensor and optogenetic technologies to lattice light sheet microscopy, for visualization and control of the
complex morphological changes megakaryocytes undergo as they produce platelets. There our ultimate goal
will be optogenetic modification of signaling to enhance platelet production. We will focus on enabling
technologies to generate minimally perturbing biosensors and optogenetic tools that can be applied by other
researchers in a wide range of fields.

## Key facts

- **NIH application ID:** 10137260
- **Project number:** 5R35GM122596-05
- **Recipient organization:** UNIV OF NORTH CAROLINA CHAPEL HILL
- **Principal Investigator:** Klaus M. Hahn
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $789,940
- **Award type:** 5
- **Project period:** 2017-04-01 → 2022-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10137260

## Citation

> US National Institutes of Health, RePORTER application 10137260, Dissecting signaling in vivo via precise control and visualization of protein activity (5R35GM122596-05). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10137260. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*