Mechanical signals from the extracellular microenvironment such as substrate stiffness and compressive or tensional strains have been shown to influence a wide range of biological processes including cell fate, differentiation, migration and tissue organization. Adherent cells generate forces through actomyosin contraction to sense the stiffness of their microenvironment, and they sense compressive or tensional strains through deformations of the actomyosin network. The dynamic nature of these mechanical signals signifies the importance of adaptation and internal regulation of the cell to maintain normal function. This dissertation aims to elucidate the mechanisms by which a single cell regulates its actomyosin contraction, or tension, as well as its shape in the presence of dynamic mechanical perturbations.

First, we investigated a mode of tension regulation in single fibroblast cells that has been proposed in literature, known as `tensional homeostasis'. This concept was first coined to describe the tendency of cell aggregates to maintain a set point tension despite mechanical perturbations that could transiently change their tension. Misregulation of tensional homeostasis has been proposed to drive disorganization of tissues and promote progression of diseases such as cancer. However, whether tensional homeostasis operates at the single cell level is unclear. We directly tested the ability of single fibroblast cells to regulate tension when subjected to mechanical displacements imposed by atomic force microscopy (AFM). We found that fibroblast tension is not held constant but rather is regulated through a strain-rate dependent buffering mechanism, which is in turn mediated by organization of the actomyosin network.

Secondly, we used the AFM to study transcellular tunnel formation in human umbilical vein endothelial cells (HUVECs). These tunnels form in the presence of bacterial toxins, EDIN and C3, that disrupt the actomyosin network and are morphologically similar to tunnels formed during leukocyte diapedesis. Whether the actomyosin network is the only barrier against tunnel formation in both cases remains unclear. We addressed this question by applying localized forces on HUVECs with the AFM while performing TIRF imaging of the cell membrane to determine whether a tunnel will form when membranes are brought into close apposition by force. We found that mechanical forces were sufficient to induce the formation of transcellular tunnels in HUVECs in the absence of toxin, and we observed localization of the I-BAR domain of MIM and actin around the edges of the transcellular tunnel, similar to toxin-induced tunnels. We also found that force-induced tunnels in cells expressing EDIN required less work than wild type cells, consistent with mechanical property measurements of these cells. Moreover, we observed transient actin enrichment in wild type cells that were partially indented with the AFM, indicating an active response of the network. Our study suggests that the actomyosin network plays a critical role not only controlling overall cell shape but also keeping the inner leaflets of plasma membranes apart to prevent contact and fusion when the cell is challenged by external forces.

In conclusion, these findings demonstrate the versatile design of the actomyosin network, which functions as a cellular tension buffer through its crosslinking architecture, and a physical barrier against shape change and spontaneous plasma membrane fusion through its mechanics and active response to deformation.