Periodic Reporting for period 1 - FA x Force (Reconstitution and Structural Analysis of a Minimal Mechanosensitive Focal Adhesion Complex)
Reporting period: 2019-09-01 to 2021-08-31
The Action “Reconstitution and Structural Analysis of a Minimal Mechanosensitive Focal Adhesion Complex” investigates the regulation of protein complexes connecting cells to their surrounding environment. These complexes play critical roles in cell shape, differentiation, movement, and division, and contribute to inside-out and outside in signaling between cells and their environment. These complexes are made up of hundreds of proteins, can be formed and disassembled rapidly in response to different cellular cues, and are tightly regulated spatially and temporally. The ability to properly form, stabilize, or disengage the connections mediated by focal assemblies is crucial to proper cell function. As such, mis-regulation and dysfunction of focal adhesions and individual focal adhesion proteins is linked to many human pathologies, including multiple immune disorders and many different types of cancers.
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.
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.
Phase separation in a cell results in “condensates”, or droplets with much higher concentrations of specific molecules, while excluding others. It is becoming clear that these assemblies occur in a broad range of cellular processes, and is likely a common mechanism of cell organization. Phase separation of focal adhesion proteins explains their dynamic nature, ability to form and disassemble rapidly, and other distinctive characteristics. In addition, condensates are believed to be prime targets for future therapeutics, leading a burst of start-ups based around just that concept in the last few years, including DewPoint Therapeutics, Faze Medicines, Transition Bio, Inc., and Neurid Therapeutics. Condensate biology has been especially implicated in cancer pathology. As such, the work carried out through this Action could prove invaluable to development of oncology therapeutics in the future.