# Reconstructing and deconstructing intracellular signaling at the membrane-cytosol interface

> **NIH NIH K99** · STANFORD UNIVERSITY · 2023 · $100,000

## Abstract

PROJECT ABSTRACT
In cellular signal transduction, the physical mechanism and the dynamical path of how signaling proteins in a
network transmit information remains poorly understood. The long-term goal is to construct a molecular model
that quantitatively describes intracellular signaling from receptor triggering to downstream activation, both in
health and in diseases. The objective of this proposal is to advance a novel type of reconstitution approach
integrating model membranes and cell extracts to study the membrane-cytosol coupling in the receptor tyrosine
kinase (RTK) signaling pathway. The central hypothesis is that the relevant regulation and kinetics of membrane
signal transduction is dependent on the cytosolic molecules and environment, which are generally not captured
by conventional membrane reconstitution. The rationale underlying this proposal is that such approach offers a
unique experimental advantage that complements live-cell studies in developing a quantitative description of
early signal transduction. Identification of kinetic bottleneck and feedbacks could provide viable therapeutic
targets. The central hypothesis will be pursued by four specific aims: 1) Optimize a robust membrane-cytosol
reconstitution protocol, 2) Compare the first-encounter rate of molecules in the cytosol versus membrane, 3)
Reconstitute and characterize the temporal regulation of the RTK-Ras-MAPK pathway, and 4) Dissect the
spatiotemporal coupling and dynamical path of the RTK-Ras-MAPK signaling. The membrane-cytosol
reconstitution represents a conceptually and technically innovative approach to interrogate intracellular signaling
at the membrane-cytosol interface. Preliminary data support the biochemical feasibility of this reconstitution
approach. In combination with advanced fluorescence microscopy, this platform enables control and
characterization of real-time signaling events, down to the single-molecule level. The significance of this research
program is the development of a mechanistic and dynamical framework of the RTK signaling pathway, which
acts as a paradigm for studying other signaling pathways. Such efforts could broadly impact our understanding
of the organizing principles of signal transduction, and transform our view on diseases and therapeutics.
Dr. Yuan-Chi Huang (William Y. C. Huang) is the principal investigator of this project. Dr. Huang's goal is to
become a leading expert in the biophysics of cellular signal transduction. Dr. Huang has extensive research
experience developing imaging-based membrane assays that map complex signaling reactions to quantifiable
reconstituted systems. This award enables Dr. Huang to integrate an additional imaging method, lattice light-
sheet microscopy, to resolve cytosolic dynamics, as well as acquire experimental training in single-cell imaging.
Dr. Huang is mentored by a leader in systems biology, Dr. James Ferrell, and is further supported by a strong
collaboration team, Dr. Steven Boxer, Dr. Ch...

## Key facts

- **NIH application ID:** 10640274
- **Project number:** 5K99GM143481-02
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** Yuan-Chi Huang
- **Activity code:** K99 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $100,000
- **Award type:** 5
- **Project period:** 2022-07-01 → 2024-02-29

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10640274

## Citation

> US National Institutes of Health, RePORTER application 10640274, Reconstructing and deconstructing intracellular signaling at the membrane-cytosol interface (5K99GM143481-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10640274. Licensed CC0.

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