Competing instabilities reveal how to rationally design and control active crosslinked gels

Active matter, composed of motile units converting free energy into mechanical work, exhibits hydrodynamic instabilities absent in passive materials. Living cells, as prime examples, self-organize using molecular machinery, showcasing transitions between non-equilibrium states by regulating molecular building blocks. Understanding the connection between microscopic forces and macroscopic dynamics remains challenging due to the nonlinear collective organization of force-generating units. This limits the predictive assembly of active matter with controllable properties, despite advances in machine learning and light-harvesting molecular motors. While transitions between active gels and fluids have been described in contractile actomyosin networks, the behavior of extensile motor-polymer networks, like microtubules (MTs) and kinesin motors, is less understood. This research aims to explore spontaneous deformations in a minimal in vitro system of cytoskeletal proteins to reveal the rational design and control of active biomimetic materials.

Previous research on active gels and fluids has focused primarily on contractile actomyosin networks, demonstrating that the emergent properties (fluid-like or solid-like) are controlled by active stresses and network connectivity. However, less is known about extensile motor-polymer networks. Studies on in-plane and out-of-plane instabilities in active nematics have been reported separately, but a unifying understanding of the factors determining which instability dominates has been lacking. Recent work has explored ATP-dependent gelation transitions in MT gels, but connecting macroscopic properties to microscopic constituents remained a challenge. This study builds upon these findings by leveraging the competition between different instability modes to connect macroscopic mechanics to microscopic components.

The study used an in vitro system consisting of rod-like MTs, kinesin molecular motors, and crosslinkers (either a depletant or PRC1). The active material was confined in a thin microfabricated channel, and its behavior was observed after an initial shear flow alignment. The experiments varied ATP concentration, motor cluster concentration, crosslinker concentration, and MT length. Widefield and confocal microscopy were used to observe the in-plane bending and out-of-plane buckling instabilities. A blurriness coefficient (B) was quantified to characterize the directionality of the instability. A hydrodynamic model of a thin active elastomer was developed to understand how the balance between activity and elasticity controls the instability. A reaction kinetics model based on Michaelis-Menten enzyme kinetics was used to connect the concentrations of cytoskeletal proteins to the macroscopic material parameters (activity and shear modulus). Finally, light-dimerizable kinesin-1 motors were used to achieve in situ optogenetic control of the network's activity and elasticity.

The experiments revealed that the extensile active network spontaneously deformed either in-plane (bending) or out-of-plane (buckling), depending on its molecular composition. High ATP concentrations and low crosslinker density favored in-plane bending, while low ATP concentrations and high crosslinker density favored out-of-plane buckling. A transition from out-of-plane buckling to in-plane bending occurred with increasing ATP concentration, decreasing crosslinker concentration, increasing motor cluster concentration (at low ATP), or decreasing MT length. The hydrodynamic model demonstrated that the direction of the instability is set by the ratio of active stresses to passive elastic stresses (ζ/μ). The reaction kinetics model successfully connected the molecular composition (ATP, motor, crosslinker concentrations, and MT length) to the macroscopic material parameters (activity and shear modulus), providing a quantitative estimate of active and elastic stresses. Optogenetic control using light-dimerizable motors allowed for in situ control of the instability direction by varying light intensity and pulse period.

This study provides a multiscale framework linking microscopic protein interactions to macroscopic material properties and emergent dynamics in active gels. The findings illustrate how the balance between active and elastic stresses determines the type of instability observed. The results differ from observations in actomyosin gels, where active stresses stiffen the network, highlighting fundamental differences between extensile and contractile active matter. The ability to predict macroscopic properties from microscopic constituents and the demonstrated optogenetic control represent significant advances in the rational design and control of active materials. The framework developed has implications for understanding cytoskeletal mechanics and self-organization in living systems.

This research successfully connected the microscopic composition of an active crosslinked gel to its macroscopic mechanical properties and emergent instabilities. The development of a predictive multiscale model allows for the rational design of active materials with targeted mechanical properties. The demonstration of optogenetic control further advances the ability to manipulate the behavior of these materials in situ. Future research could explore more complex active systems and investigate the role of other cytoskeletal proteins in determining the emergent properties of active materials.

The model used several simplifications, including the scaling of shear modulus in nematic elastomers and the use of a simplified Michaelis-Menten kinetics model. These simplifications might affect the quantitative accuracy of the predictions. The study focused on a specific in vitro system and the generalizability to other active systems needs further investigation. The optogenetic control demonstrated is not fully reversible, likely due to the presence of persistent crosslinkers.