The development and utilization of high-throughput molecular mechanotyping: a RAD-ical approach

Abstract

Mechanical forces have rapidly emerged as key regulators for many cellular processes from stem cell differentiation to immune cell activation. Rather than being passive responders to mechanical force, cells are active participants within a continuous game of tug-of-war with their surroundings. This exchange of mechanical force has emerged as essential to cellular function; specific forces alter protein conformation and function and act as regulatory mechanisms for many signaling cascades. Intriguingly, aberrant force transmission occurs upon disease onset and can contribute to the progression of diseases such as cancer and fibrosis. Currently, the understanding of cell generated force in cell function and disease remains incredibly limited due to the nascency of the field as a whole, yet cellular forces appear to play a role in functionally all phenomena so far probed. Molecular tension sensors (MTS) are essential tools for studying these events. MTSs allow for quantification of forces transmitted through or by specific proteins of interest. The use of MTSs has seen rapid expansion due to the development of DNA based MTSs. DNA based MTS report specific force transmission by rupturing once a critical force is achieved and is an inherently accessible and modular platform to expand the understanding of mechanobiology. Unfortunately, the potential of these tools outweighs the actual use due to their limited throughput and restrictive use cases. In this thesis, I present my work developing a high throughput method to quantify DNA based MTS rupture of single cells. Furthermore, I provide a clear method in how to use this method and how to overcome potential challenges. I also demonstrate how the incorporation of established methods and new strategies allows DNA based MTSs to be used in conditions inaccessible to traditional methods. This work establishes an accessible method to assign mechanical phenotype (mechanotype) to cells on the basis of their force transmission through adhesion molecules. I demonstrate how molecular mechanotype can be used for drug development and discus mechanotypes potential use from basic science to use as a diagnostic platform.