This project focused on understanding the mechanisms regulating focal adhesion assembly. Focal adhesions have been the focus of intense study for over three decades, and though much has been learned about their composition and organization, an understanding of the regulatory “rules” controlling focal adhesion formation and dynamics are lacking. What are the basic units necessary to form these connections? How are they turned on and off? Through this project, we tried to replicate these structures at the most basic level outside the cell, in order to shed light on these questions. The better we understand how these connections work, the better equipped we will be to address problems that may arise, in the context of human disease.
Over the course of this action, the core structural components of focal adhesions were isolated and reconstituted in vitro. More specifically, we used the focal adhesion proteins talin and vinculin to form minimal, membrane-bound “focal adhesions” outside of the context of a cell, using purified proteins and synthetic membrane systems. We established a novel membrane-based reconstitution system which will serve as a much-needed platform for studying focal adhesion assembly and dynamics in the future. Additionally, these experiments elucidated the autoinhibitory mechanisms of talin and vinculin, and confirmed that membrane binding plays an important role in the activation of talin – likely a critical step in focal adhesion assembly. Notably, this work demonstrated that talin, vinculin, and actin can interact without the application of force, an important question which has been debated for years. These results were published in 2020 in eLIFE, in an article entitled “Phosphoinositides regulate force-independent interactions between talin, vinculin, and actin” and presented at the international meeting of the American Society of Cell Biology in 2019. A follow-up publication is currently nearing completion, which further investigates the importance of membrane binding to focal adhesion assembly and organization, in the context of liquid-liquid phase separation.
Liquid-liquid phase separation, by which different molecules form distinct phases in solution while retaining the properties of a liquid, has revolutionized how we think of cell biology.
