Reconstructing and deconstructing intracellular signaling at the membrane-cytosol interface

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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. Christopher Garcia, and Dr. Joanna Wysocka. All of them are faculty members at Stanford University. Such arrangement demonstrates the exceptionally collaborative environment of Stanford University, and highlights the feasibility and effectiveness of the mentorship and collaboration.