Cells are subject to a wide variety of forces throughout their lifetimes. During epithelial morphogenesis, epithelial cells form sheets of cells that line the cavities and surfaces of organs. These cells protect the organs and are responsible for sensing and responding to mechanical forces. The ability of cells to respond and adapt to forces underlies the etiology of a variety of diseases including cardiovascular disease, cancer, and lung dysfunctions. Therefore, it is critical to understand how force affects cells.
Cells sense forces through cell surface adhesion receptors. These receptors trigger reinforcement of cell anchoring junctions to counter the applied force. Anchoring junctions occur in two functionally distinct forms. The first type, adherens junctions, mediate cell-cell adhesion while the second type, focal adhesions, bind cells to the extracellular matrix. Each anchoring junction contains specific transmembrane adhesion receptors. These receptors in adherens junctions are the Ca2+-dependent transmembrane adhesion proteins known as cadherins. In epithelia, the cadherins are denoted E-cadherin or epithelial cadherin. For the second type of cell anchoring junction, the transmembrane receptors are the integrins.
In response to force, epithelial cells reinforce the cell anchoring junctions and the actin cytoskeleton. Force on E-cadherin causes clustering of E-cadherins and stimulates the recruitment of β-catenin, α-catenin, and actin, thereby triggering growth of the adhesion complex. Force also stimulates a signaling pathway involving RhoA and phosphorylation of myosin light chain culminating in more actin stress fibers and an overall increase in actin polymerization. More specifically, force on E-cadherin actives AMPK. Active AMPK stimulates Abl activity to phosphorylate vinculin Y822. This phosphorylation event promotes RhoA-mediated cytoskeletal rearrangements, a process known as cell stiffening.
The ability of a cell to regulate its actin cytoskeleton is critical for maintaining the balance of force between itself and its surroundings. Disruptions in actin cytoskeletal rearrangements underlie the progression of many diseases, including cardiovascular disease, lung dysfunctions, and cancer. Therefore, elucidating the signaling pathway that culminates in changes to the actin cytoskeleton is critical to understand the basic mechanisms that go awry in these diseased states.
The second chapter of my thesis focuses on identifying a novel component of E-cadherin force transmission. I focus on the apoptotic regulator p21-activated kinase 2 (PAK2) as previous studies have implicated PAK2 in cell-cell junction mechanics. To examine its role in force transmission, I applied a variety of types of mechanical force to epithelial cells and monitored PAK2 activity. I observed that force results in activation of PAK2 and triggers its recruitment to the cadherin adhesion complex at cell-cell junctions. Furthermore, PAK2 is required for cell stiffening. Loss of PAK2 results in decreased E-cadherin and F-actin enrichment as well as loss of critical phosphorylation events in the E-cadherin force transmission signaling cascade. Finally, I demonstrated that PAK2 also plays a critical role in determining cell survival in response to force. Under normal physiological levels of force, AMPK binds PAK2 and protects it from caspase-3 mediated cleavage. As the amplitude of force increases, AMPK no longer binds PAK2 and PAK2 is cleaved. The cleaved product translocates to the nucleus where it promotes transcription of pro-apoptotic genes. This work provides a paradigm for how different amplitudes of force affect cell survival and calls for more in depth consideration of force applications in future studies.
The third chapter of my thesis focuses on identifying a negative regulator of E-cadherin force transmission. Evidence suggests that the protein tyrosine phosphatase, SHP-2, may be involved in E-cadherin negative regulation. SHP-2 localizes to cell-cell junctions, and cells expressing constitutively active SHP-2 have similar phenotypes to cells expressing a phospho-mutant Y822F vinculin. I demonstrate that vinculin Y822 phosphorylation is followed by SHP-2 phosphorylation and activation. Active SHP-2 de-phosphorylates vinculin Y822 directly, halting the cell stiffening response. This work is the first specific negative regulator of E-cadherin force transmission. Together, these findings reveal the importance of strict regulation of cell stiffening, and provide a framework for exploring how diseases involving contractile disturbances.