Advisor: Andrés García, Ph.D. (Georgia Institute of Technology)

Committee:

Cheng Zhu, Ph.D. (Georgia Institute of Technology)

Wilbur Lam, Ph.D.(Georgia Institute of Technology)

John Blazeck, Ph.D.(Georgia Institute of Technology)

Jiangping Fu, Ph.D. (University of Michigan)

Title

Abstract

Dynamic cell-cell and cell-ECM (extracellular matrix) interactions regulate tissue morphogenesis and wound healing through modulating cellular processes such as differentiation and cell migration. Adhesive interactions function as the primary way cells turn upstream ECM cues into downstream cellular processes. Focal adhesions (FA), clusters of structural and signaling proteins, function as principal sites of force transfer and mechanotransduction. Previous studies in mechanobiology demonstrated that FAs are mechanosensitive, but the mechanism for how FAs are involved in mechanotransduction remains poorly defined. Recent studies have implicated FAs with yes associated protein (YAP), a transcriptional coactivator that can turn mechanical cues like substrate rigidity into changes in gene expression. These studies have shown that the inhibition of certain FA proteins has led to a reduction in YAP nuclear localization, and it was demonstrated that YAP also promotes the transcription of FA-related genes. This link needs to be explored; however, this can only be accomplished with an experimental platform with high spatiotemporal resolution. The objective of this project is to elucidate the mechanism by which cells take information at the cell periphery and communicate it to the nucleus. This proposal hypothesizes that FAs function as mechanosensors through focal adhesion kinase (FAK), where FAK impacts YAP nuclear localization by either modulating nuclear lamin phosphorylation or expression, therefore, altering nuclear stiffness or by modulating PLCγ1 activity, therefore, altering levels of phosphatidylinositol 4,5-bisphosphate (PIP2) which alters RAP2 activity, and that FAs utilize their quantity and spatial distribution across the cell to direct YAP nuclear localization. This hypothesis will be tested via three specific aims: (1) characterize the effect of FAK-talin-vinculin functionalities and interactions on YAP nuclear localization and YAP related transcriptional activity; (2) characterize the effects of FA number, area, and spatial distribution across the cell body on YAP nuclear localization and YAP related transcriptional activity; and (3) investigate whether FAK modulates YAP localization by regulating PLCγ1 activity leading to changes in RAP2 activity and YAP nuclear localization or by altering lamin expression or phosphorylation leading to changes in nuclear stiffness and nuclear pore organization which alters YAP nuclear localization. With these studies, an experimental platform with high spatiotemporal resolution will be generated; the molecular mechanism by which FAs and FAK impact YAP signaling will be explored; and new insights will be generated in mechanobiology.