Contractility Dominates Adhesive Ligand Density in Regulating Cellular De-adhesion and Retraction Kinetics

Cells that are enzymatically detached from a solid substrate rapidly round up as the tensile prestress in the cytoskeleton is suddenly unopposed by cell–ECM adhesions. We recently showed that this retraction follows sigmoidal kinetics with time constants that correlate closely with cortical stiffness values. This raises the promising prospect that these de-adhesion measurements may be used for high-throughput screening of cell mechanical properties; however, an important limitation to doing so is the possibility that the retraction kinetics may also be influenced and potentially rate-limited by the time needed to sever matrix adhesions. In this study, we address this open question by separating contributions of contractility and adhesion to cellular de-adhesion and retraction kinetics. We first develop serum-free conditions under which U373 MG glioma cells can be cultured on substrates of fixed fibronectin density without direct matrix contributions from the medium. We show that while spreading area increases with ECM protein density, cortical stiffness and the time constants of retraction do not. Conversely, addition of lysophosphatidic acid (LPA) to stimulate cell contractility strongly speeds retraction, independent of the initial matrix protein density and LPA’s contributions to spreading area. All of these trends hold in serum-rich medium commonly used in tissue culture, with the time constants of retraction much more closely tracking cortical stiffness than adhesive ligand density or cell spreading. These results support the use of cellular de-adhesion measurements to track cellular mechanical properties

Contractile Force and Shape Regulation in Single Cells

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

Contributions of talin-1 to glioma cell–matrix tensional homeostasis

The ability of cells to adapt their mechanical properties to those of the surrounding microenvironment (tensional homeostasis) has been implicated in the progression of a variety of solid tumours, including the brain tumour glioblastoma multiforme (GBM). GBM tumour cells are highly sensitive to extracellular matrix (ECM) stiffness and overexpress a variety of focal adhesion proteins, such as talin. While talin has been shown to play critical early roles in integrin-based force-sensing in non-tumour cells, it remains unclear whether this protein contributes to tensional homeostasis in GBM cells. Here, we investigate the role of the talin isoform talin-1 in enabling human GBM cells to adapt to ECM stiffness. We show that human GBM cells express talin-1, and we use RNA interference to suppress talin-1 expression without affecting levels of talin-2, vinculin or phosphorylated focal adhesion kinase. Knockdown of talin-1 strongly reduces both cell spreading area and random migration speed but does not significantly affect overall focal adhesion size distributions. Most strikingly, atomic force microscopy indentation reveals that talin-1 suppression compromises adaptation of cell stiffness to changes in ECM stiffness. Together, these data support a role for talin-1 in the maintenance of tensional homeostasis in GBM and suggest a functional role for enriched talin expression in this tumour

Tensional Homeostasis in Single Fibroblasts

Adherent cells generate forces through acto-myosin contraction to move, change shape, and sense the mechanical properties of their environment. They are thought to maintain defined levels of tension with their surroundings despite mechanical perturbations that could change tension, a concept known as tensional homeostasis. 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. Here, we directly test the ability of single fibroblast cells to regulate tension when subjected to mechanical displacements in the absence of changes to spread area or substrate elasticity. We use a feedback-controlled atomic force microscope to measure and modulate forces and displacements of individual contracting cells as they spread on a fibronectin-patterned atomic-force microscope cantilever and coverslip. We find that the cells reach a steady-state contraction force and height that is insensitive to stiffness changes as they fill the micropatterned areas. Rather than maintaining a constant tension, the fibroblasts altered their contraction force in response to mechanical displacement in a strain-rate-dependent manner, leading to a new and stable steady-state force and height. This response is influenced by overexpression of the actin crosslinker α-actinin, and rheology measurements reveal that changes in cell elasticity are also strain- rate-dependent. Our finding of tensional buffering, rather than homeostasis, allows cells to transition between different tensional states depending on how they are displaced, permitting distinct responses to slow deformations during tissue growth and rapid deformations associated with injury

Deconvolution of the Cellular Force-Generating Subsystems that Govern Cytokinesis Furrow Ingression

<div><p>Cytokinesis occurs through the coordinated action of several biochemically-mediated stresses acting on the cytoskeleton. Here, we develop a computational model of cellular mechanics, and using a large number of experimentally measured biophysical parameters, we simulate cell division under a number of different scenarios. We demonstrate that traction-mediated protrusive forces or contractile forces due to myosin II are sufficient to initiate furrow ingression. Furthermore, we show that passive forces due to the cell's cortical tension and surface curvature allow the furrow to complete ingression. We compare quantitatively the furrow thinning trajectories obtained from simulation with those observed experimentally in both wild-type and <em>myosin II</em> null <em>Dictyostelium</em> cells. Our simulations highlight the relative contributions of different biomechanical subsystems to cell shape progression during cell division.</p> </div

Simulations considered.

<p>Simulations considered.</p