Graduate student Michigan Technological University houghton, Michigan, United States
Introduction: Many studies have shown an increasing traction in response to increasing substrate stiffness (1-3). To explain this stiffness-dependent differential traction, a ‘molecular clutch’ model has been implemented (4). Myosin II contractility has been the main source of the retrograde flow where myosin’s muscle-like behavior explains the stiffness-dependent differential traction (5, 6). Given myosin as a ‘result’ of mechano-signaling, identifying how cells transmit differential force without myosin would shed light on the very initial stiffness sensing event by a cell without complication by myosin’s effect. In this abstract, we show experimental evidence that myosin-independent tractions are still stiffness-dependent and it comes from the actin retrograde flow powered by actin polymerization and its two main mediators.
Materials and
Methods: A. Soft substrates and traction force microscopy High-refractive index, soft silicone gels (Q-gel, CHT) of different stiffness, i.e., 0.6, 1.3, 2.7, 6, and 12.7 kPa, were fabricated through mixing of Q-gel in different ratios, which is coated with 40 nm diameter fluorescent beads as done previously (7). After imaging beads on the gel before and after 3T3 fibroblasts release, the images of the beads were processed for traction field via MATLAB-based TFM software (8).
Results, Conclusions, and Discussions: From experiments, we found that Arp2/3 inhibition in addition to myosin reduced traction magnitude by ~4-fold (Fig. 1A) compared to the traction by cells with myosin-only inhibition while minimally reducing the F-actin flow (Fig. 1B). The original molecular clutch model has failed to explain this behavior because the actin filaments were assumed to be rigid (9). To resolve this difficulty, we developed a novel molecular clutch model where F-actin’s viscoelasticity contributes to the transfer of the actin motion to the traction force. For example, an addition of a new F-actin unit network with higher rigidity can push the clutch molecule harder than those with softer F-actin, thus transmitting larger traction while preserving similar actin flow velocity even when cells are on the substrates with the same ECM rigidity. Via collaboration with Dr. Alex Cartagena-Rivera (NIH, NIBIB), we found that indeed Arp2/3 and formin contribute to the elasticity of the F-actin network (9).
Together, our data and simulation suggest Arp2/3 and formin enhance stiffness sensitivity by mechanically reinforcing the F-actin network, thereby facilitating more effective transmission of flow-induced forces.
Acknowledgements (Optional): ACKNOWLEDGEMENTS This work was funded by NIH 2R15GM135806.
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