Tuning the dynamics of active cytoskeleton composites via microtubule stabilization

The cytoskeleton is a dynamic network of protein filaments such as semiflexible actin filaments and rigid microtubules, along with motor proteins, such as microtubule-associated kinesin, capable of generating forces and restructuring to perform key cellular processes. As such, in vitro systems of cytoskeletal proteins have been studied for decades as promising candidates for active and adaptable materials. However, most in vitro systems to date have been limited in the control, tunability and lifetime of restructuring and force generation. Here, we engineer in vitro composites of actin and microtubules, driven by kinesin motors, allowing us to control composite activity independent of viscoelasticity in order to optimize composites for sustained and controlled force-generation and dynamics. We use engineered clusters of kinesin dimers that can crosslink and pull on anti-aligned microtubules, similar to myosin motors acting on actin filaments. We investigate the role of microtubule stabilization and crosslinking on the composite structure and dynamics, using multi-spectral confocal microscopy to visualize microtubules and crosslinkers during activity. We show that composites in which microtubules are stabilized using taxol versus GMPCPP result in dramatically different structures that are sculpted by the presence or absence of actin filaments and microtubule crosslinker ASE1. To improve fatigue resistance and enable mechanical sensing, we incorporate synthetic hydrogel inclusions into composites. Active cytoskeleton composites offer a robust platform for engineering programmable, adaptive and responsive materials. The ability to tune their dynamics and optimize activity lifetime makes these composites a promising route toward next-generation autonomous materials engineering.