Intracellular pressure generation and the control of mesenchymal and epithelial dynamics
Authors:   , Jones Tia M. , Petrie Ryan J 
Published: March 2022

Cytoplasmic pressure, generated by water flow across the plasma membrane and actomyosin contractility can drive cell dynamics, including cell morphology, protrusion identity, and mode of migration. Recently, it has been discovered that mesenchymal cells migrating in highly cross-linked three-dimensional (3D) matrices regulate their intracellular pressure to achieve migratory plasticity switching from low pressure lamellipodial protrusions to high pressure lobopodial protrusions. However, the ability of cells to sense and integrate mechanical cues from the extracellular matrix for intracellular pressure generation remains unknown. Migratory plasticity can also be achieved when epithelial cells transition to mesenchymal cells through a process known as epithelial-mesenchymal transition. Yet, it remains unclear the role of intracellular pressure during this process. In the first part of my thesis, I investigated the molecular mechanism of migratory plasticity during 3D migration when activating the nuclear piston mechanism. I found that during activation of the nuclear piston there is a significant increase in nuclear mechanical stress as measured by nuclear deformation and pressure. Yet, it is not sufficient to activate the piston. However, phospho-proteomics analysis identified a potential molecular regulatory mechanism for nuclear piston activation through the Ras-MAPK signaling pathway. Upon modulation of ERK1/2 activity in primary fibroblasts migrating in 3D, I found that ERK1/2 acts as a negative regulator of the piston mechanism by suppressing intracellular pressure though non-muscle myosin II (NMII) activity in normal and aberrant cells. Critically, I found that upon piston activation by protease inhibition, endogenous ERK1/2 activity is downregulated, suggesting ERK1/2 activity is downstream of proteases. Finally, I show that Ras transformed fibroblasts promote lamellipodial migration indicating the Ras-MAPK pathway as a potential molecular switch for migratory plasticity. In the second part of my dissertation, I investigated the role of intracellular pressure in epithelial cells. I found that epithelial cells maintain a significantly higher intracellular pressure than that of mesenchymal cells on two-dimensional surfaces. This high intracellular pressure functions to maintain tissue integrity and promote barrier function. Furthermore, I show that the induction of epithelial to mesenchymal transition by hepatocyte growth factor requires intracellular pressure to decrease. This allows for the formation of lamellipodial protrusions through the actin nucleating protein, Arp2/3. Thus, reduction in cytoplasmic pressure facilitates lamellipodia formation and motility. Collectively, these data reveal the unique mechanism and role of intracellular pressure generation during 2D and 3D migration of both mesenchymal and epithelial cells. Intracellular pressure regulation functions to dynamically alter cellular migration and provides insights into the mechanism of migratory plasticity in 3D matrices.