3D printing cytoskeletal networks: ROS-induced filament severing leads to surge in actin polymerization

The cytoskeletal protein actin forms a highly spatially organized biopolymer network which plays a central role in many cellular processes. This actin cytoskeleton needs to react quickly to intracellular cues and changes in local environments; to achieve this, it is controlled by a robust, but complex, regulatory machinery which still presents many open questions. Here we show that in experiments with purified actin in vitro, a surprisingly simple experimental setup is sufficient to precisely control both actin assembly and actin disassembly. To do so, we use photosensitive molecules such as fluorophores, as a tool to locally generate reactive oxygen species (ROS) upon light exposure. We see that ROS sever actin filaments, presumably via oxidation of actin molecules within the filaments. This increases the number of actin filaments and thus the number of polymerizing barbed ends. Given the nucleation-limited nature of actin assembly, continued exposure results in an exponential increase in F-actin. Tuning the degree of ROS generation allows us to switch between actin assembly and actin disassembly. By controlling light patterns, we control actin network morphology in our samples in 3D. This effect is scalable and even allows for "multi-color printing" as we demonstrate with an actin homolog. Our experimental data is accompanied by simulations using a kinetic model of actin polymerization, which reveal further details about the behavior. In cells, ROS are known to regulate the actin cytoskeleton, but the underlying mechanisms are mostly unknown. Our results suggest that ROS might directly affect actin reorganization in living cells.