Summary Membrane fusion is an energy-consuming process that requires tight juxtaposition of two lipid bilayers. Little is known about how cells overcome energy barriers to bring their membranes together for fusion. Previously, we have shown that cell-cell fusion is an asymmetric process in which an “attacking” cell drills finger-like protrusions into the “receiving” cell to promote cell fusion. Here we show that the receiving cell mounts a Myosin II (MyoII)-mediated mechanosensory response to its invasive fusion partner. MyoII acts as a mechanosensor, which directs its force-induced recruitment to the fusion site, and the mechanosensory response of MyoII is amplified by cell adhesion molecule-initiated chemical signaling. The accumulated MyoII, in turn, increases cortical tension and promotes fusion pore formation. We propose that the protrusive and resisting forces from two fusion partners put the fusogenic synapse under high mechanical tension, which helps to overcome energy barriers for membrane apposition and drives cell membrane fusion.

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.

Current approaches to cancer treatment focus on targeting signal transduction pathways. Here, we develop an alternative system for targeting cell mechanics for the discovery of novel therapeutics. We designed a live-cell, high-throughput chemical screen to identify mechanical modulators. We characterized 4-hydroxyacetophenone (4-HAP), which enhances the cortical localization of the mechanoenzyme myosin II, independent of myosin heavy-chain phosphorylation, thus increasing cellular cortical tension. To shift cell mechanics, 4-HAP requires myosin II, including its full power stroke, specifically activating human myosin IIB (MYH10) and human myosin IIC (MYH14), but not human myosin IIA (MYH9). We further demonstrated that invasive pancreatic cancer cells are more deformable than normal pancreatic ductal epithelial cells, a mechanical profile that was partially corrected with 4-HAP, which also decreased the invasion and migration of these cancer cells. Overall, 4-HAP modifies nonmuscle myosin II-based cell mechanics across phylogeny and disease states and provides proof of concept that cell mechanics offer a rich drug target space, allowing for possible corrective modulation of tumor cell behavior. mechanical modulator | 3,4-dichloroaniline | 4-hydroxyacetophenone | myosin II | pancreatic cancer C ell shape change processes include cell growth, division, motility, and the formation of complex structures like tissues and organs, all of which are governed by the intersection of biochemistry, genetics, and mechanics. These three modules are integral not just for normal function in healthy cells but also in disease states. Pharmacological manipulation of some of these modules has already led to treatment strategies for inflammation and cancer (e.g., paclitaxel) (1, 2). However, many presently available therapies, which address only one aspect of cell shape change, typically either fail to abolish the disease completely or lead to compensatory regulatory changes, and therefore to drug resistance. Targeting cell mechanics remains an underused approach for drug development. In cancer, altered cell mechanics are a hallmark of metastatic efficiency: cell stiffness decreases up to 70% in many metastatic cancer cells (3-5). It is rational then that one therapeutic approach is to increase cellular elasticity, which would, in turn, reduce metastatic potential and act downstream of cancer-inducing genetic alterations.Known mechanical modulators (e.g., latrunculin, blebbistatin) are often lethal, have numerous off-site targets, and act to generate a softer and metastatic-like mechanical phenotype (6, 7). However, the field's ability to increase cellular elasticity on acute time scales is highly restricted. In an effort to close this gap and find modulators that stiffen cells, we leveraged our molecular and analytical understanding of cytokinesis, an evolutionarily conserved and highly mechanical cell shape change event, to establish an in vivo, large-scale, high-throughput chemical screen for small-molecule modulators of cell shap...

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 myosin II null Dictyostelium cells. Our simulations highlight the relative contributions of different biomechanical subsystems to cell shape progression during cell division.