Reconstructing and deconstructing intracellular signaling at the membrane-cytosol interface

PROJECT SUMMARY (See instructions): In receptor signal transduction, a major goal is to understand how a complex network of biochemical interactions at the cell membrane is spatially coordinated to generate specific cellular responses. It is generally thought that this level of complexity is essential for the tight regulation of information processing in cells, as defects in signaling have been linked to numerous diseases. However, the complexity has created a gap between reconstituted systems and living cells, limiting progress toward a mechanistic understanding of signal transduction. This proposal addresses this challenge by developing a total reconstitution approach that substantially advances our ability to reconstruct complex signaling networks, approaching those in cells, while retaining the experimental strengths of an in vitro approach. We propose a hybrid approach, introducing undiluted cell extracts into model membrane systems, to overcome the fundamental limitations of traditional reconstitution methods and reconstitute cellular complexity. This innovation enables the reconstitution of intracellular signaling as an integrated entity rather than isolated compartments, thus clarifying how signals are amplified, sustained, or attenuated at the membrane. The R00 phase has two aims: 1) reconstituting the receptor tyrosine kinase (RTK) pathway and 2) dissecting the spatiotemporal mechanisms in the RTK pathway. Specific emphasis is placed on using the reconstituted system to study the pressing question of how RTK condensates regulate Ras GTPase activation. The findings are expected to contribute to a quantitative model of RTK signaling, establishing a paradigm for studying other signaling pathways. Such efforts are bringing us beyond a descriptive understanding of signal transduction to a mechanistic framework, offering quantitative insights into abnormal signaling in disease and alternative strategies for therapeutics.