# Single Cell Analysis of MAPK Signaling Dynamics during Tissue Homeostasis

> **NIH NIH R35** · JOHNS HOPKINS UNIVERSITY · 2020 · $5,918

## Abstract

Project Summary
Our main goal is to understand how signaling network dynamics controls cell fate. Mitogen Activated Protein
Kinases, as well as the entire metabolism signaling network (i.e. AKT, AMPK, mTORC1), are clinically relevant
signaling molecules that orchestrate cellular responses to a diverse array of stimuli. There are three major
MAPK signaling cascades (ERK, p38 and JNK) that are highly interconnected with the metabolism signaling
network and control critical cellular decisions such as survival/apoptosis or proliferation/senescence. Our
current understanding of how signaling controls cell fate is incomplete because: (i) a lack of integrated
methods to quantify the dynamics of the network as a whole and (ii) the use of cell population assays that
average unsynchronized single cell behaviors. To address this need, my laboratory has pioneered a new
generation of biosensors that allow simultaneous quantification of multiple kinase activities in thousands of live
single cells. These biosensors convert phosphorylation into a nucleocytoplasmic shuttling event that can be
easily measured by fluorescent microscopy.
Eukaryotic cells constantly adjust metabolic fluxes in response to environmental demands via the mammalian
Target of Rapamycin (mTOR). Over the past 20 years, the topology of the mTOR signaling network has been
dissected using classic biochemical and genetic approaches. However, recent advances in single cell analysis
have demonstrated that cell populations often behave qualitatively different than individual cells. Thus, we
recently generated a single cell biosensor to measure mTOR complex 1 activity in live single cells (mTORC1-
KTR). Surprisingly, our preliminary data shows striking oscillations of mTORC1 activity occurring out-of-phase
between neighboring cells of epithelial monolayers. This finding is in agreement with a recent preprint
suggesting that individual cells undergo autonomous cycles in energetic balance under normal conditions.
However, the cause and the consequence of these oscillatory patterns are not understood. Our lab has
previously shown that oscillatory or sustained MAPK activity differentially regulate cell fate highlighting the role
of temporal dynamics in regulating biological functions. Taken together, we hypothesize that the temporal
patterns of mTOR activity differentially regulate metabolism to fine tune energetic demands. To address this
hypothesis we divided the proposal in three aims:(i) Aim 1 will address the molecular mechanisms responsible
for mTORC1 oscillatory dynamics, (ii) Aim 2 will implement a new optogenetic tool to modulate mTORC1
activity using light, and (iii) Aim 3 will measure the physiological consequences of mTORC1 dynamics at the
transcriptome and metabolome levels.

## Key facts

- **NIH application ID:** 10145877
- **Project number:** 3R35GM133499-01S1
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Sergi Regot
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $5,918
- **Award type:** 3
- **Project period:** 2019-08-07 → 2024-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10145877

## Citation

> US National Institutes of Health, RePORTER application 10145877, Single Cell Analysis of MAPK Signaling Dynamics during Tissue Homeostasis (3R35GM133499-01S1). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10145877. Licensed CC0.

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