Interdisciplinary Initiatives Program Round 12 - 2024
Project Investigators:
Polly Fordyce, Bioengineering and Genetics
Alex Dunn, Chemical Engineering
Abstract:
Living cells are exquisitely responsive to mechanical cues such as vibration, stretch, fluid flow, and the physical properties of their surroundings. However, our understanding of the physical aspects of cellular function lags far behind other aspects of cell biology, due largely to a lack of suitable tools. Nowhere is this more apparent than in the rapidly developing area of high-throughput systems biology. Recent work, including contributions from our team, makes it possible to assay thousands to millions of protein-protein interactions. However, these measurements take place in solution, in the absence of force. Here, we will fill this gap by creating the first high-throughput mechanobiology assay. As a model system, we will examine how mechanical force influences the interactions of PDZ proteins with their ligands. There are over 250 PDZ domains in the human proteome, many of which are associated with cancer and developmental disorders. Due to their health relevance and tractability, PDZ domain-peptide interactions have been studied for decades, but their mechanosensitivity has been completely overlooked. Recently, the Dunn lab discovered that PDZ function may be critically determined by how these proteins behave under force: at least one PDZ domain unexpectedly strengthens binding to its cognate peptide ligands under force. Given that many PDZ domain-peptide interactions experience cytoskeletally generated forces in vivo, their mechanosensitive properties are likely crucial for their function. However, how many PDZ domains are mechanosensitive and how mechanosensitivity is encoded in PDZ domain-peptide interactions is unknown. Here, we will develop a novel method for high-throughput single-molecule force spectroscopy (HT-SMFS) and use this platform to systematically screen 100s to 1000s of protein-protein interactions under force to understand how mechanosensitivity is encoded in PDZ-mediated protein-protein interactions. Developing HT-SMFS will require the Fordyce lab’s expertise in microfluidic instrumentation and the Dunn lab’s expertise in single-molecule biophysics. The results of these studies will reveal the design rules for engineering mechanosensitivity into proteins and therapeutics and mark the beginning of a new interdisciplinary field: high-throughput mechanobiology.
