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The spontaneous formation of contractile asters is ubiquitous in reconstituted active materials composed of biopolymers and molecular motors. Asters are radially oriented biopolymers or biopolymer bundles with a dense motor-rich core. The microscopic origins of their material properties and their stability are unknown. Recent efforts highlighted how motor-filament and filament-filament interactions control the formation of asters composed of microtubules and kinesin motors. However, the impact of motor-motor interactions is less understood, despite growing evidence that molecular motors often spontaneously aggregate, both and . In this article, we combine experiments and simulations to reveal the origin of the arrested coarsening, aging, and stability of contractile asters composed of microtubules, clusters of adenosine triphosphate (ATP)-powered kinesin-1 motors, and a depletant. Asters coalesce into larger asters upon collision. We show that the spontaneous aggregation of motor clusters drives the solidification of aster cores, arresting their coalescence. We detect aggregation of motor clusters at the single microtubule level, where the uncaging of additional ATP drives the delayed but sudden detachment of large motor aggregates from isolated microtubules. Computer simulations of cytoskeletal assemblies demonstrate that decreasing the motors' unbinding rate slows down the aster's coalescence. Changing the motors' binding rate did not impact the aster's coalescence dynamics. Finally, we show that the aggregation of motor clusters and aster aging result from the combined effects of depletion forces and nonspecific binding of the clusters to themselves. We propose alternative formulations that mitigate these effects, and prevent aster aging. The resulting self-organized structures have a finite lifetime, which reveals that motor aggregation is crucial for maintaining aster's stability. Overall, these experiments and simulations enhance our understanding of how to rationally design long-lived and stable contractile materials from cytoskeletal proteins. Published by the American Physical Society2025 

Active nematic liquid crystals have the remarkable ability to spontaneously deform and flow in the absence of any external driving force. While living materials with orientational order, such as the mitotic spindle, can self-assemble in quiescent active phases, reconstituted active systems often display chaotic, periodic, or circulating flows under confinement. Quiescent active nematics are, therefore, quite rare, despite the prediction from active hydrodynamic theory that confinement between two parallel plates can suppress flows. This spontaneous flow transition—named the active Fréedericksz transition by analogy with the conventional Fréedericksz transition in passive nematic liquid crystals under a magnetic field—has been a cornerstone of the field of active matter. Here, we report experimental evidence that confinement in spherical droplets can stabilize the otherwise chaotic dynamics of a 3D extensile active nematics, giving rise to a quiescent—yet still out-of-equilibrium—nematic liquid crystal. The active nematics spontaneously flow when confined in larger droplets. The composite nature of our model system composed of extensile bundles of microtubules and molecular motors dispersed in a passive colloidal liquid crystal allows us to demonstrate how the interplay of activity, nematic elasticity, and confinement impacts the spontaneous flow transition. The critical diameter increases when motor concentration decreases or nematic elasticity increases. Experiments and simulations also demonstrate that the critical confinement depends on the confining geometry, with the critical diameter in droplets being larger than the critical width in channels. Biochemical assays reveal that neither confinement nor nematic elasticity impacts the energy-consumption rate, confirming that the quiescent active phase is the stable out-of-equilibrium phase predicted theoretically. Further experiments in dense arrays of monodisperse droplets show that fluctuations in the droplet composition can smooth the flow transition close to the critical diameter. In conclusion, our work provides experimental validation of the active Fréedericksz transition in 3D active nematics, with potential applications in human health, ecology, and soft robotics. Published by the American Physical Society2024 

Microtubules and molecular motors are essential components of the cellular cytoskeleton, driving fundamental processes in vivo, including chromosome segregation and cargo transport. When reconstituted in vitro, these cytoskeletal proteins serve as energy-consuming building blocks to study the self-organization of active matter. Cytoskeletal active gels display rich emergent dynamics, including extensile flows, locally contractile asters, and bulk contraction. However, it is unclear how the protein–protein interaction kinetics set their contractile or extensile nature. Here, we explore the origin of the transition from extensile bundles to contractile asters in a minimal reconstituted system composed of stabilized microtubules, depletant, adenosine 5′-triphosphate (ATP), and clusters of kinesin-1 motors. We show that the microtubule-binding and unbinding kinetics of highly processive motor clusters set their ability to end-accumulate, which can drive polarity sorting of the microtubules and aster formation. We further demonstrate that the microscopic time scale of end-accumulation sets the emergent time scale of aster formation. Finally, we show that biochemical regulation is insufficient to fully explain the transition as generic aligning interactions through depletion, cross-linking, or excluded volume interactions can drive bundle formation despite end-accumulating motors. The extensile-to-contractile transition is well captured by a simple self-assembly model where nematic and polar aligning interactions compete to form either bundles or asters. Starting from a five-dimensional organization phase space, we identify a single control parameter given by the ratio of the different component concentrations that dictates the material-scale organization. Overall, this work shows that the interplay of biochemical and mechanical tuning at the microscopic level controls the robust self-organization of active cytoskeletal materials. 

Abstract How active stresses generated by molecular motors set the large-scale mechanics of the cell cytoskeleton remains poorly understood. Here, we combine experiments and theory to demonstrate how the emergent properties of a biomimetic active crosslinked gel depend on the properties of its microscopic constituents. We show that an extensile nematic elastomer exhibits two distinct activity-driven instabilities, spontaneously bending in-plane or buckling out-of-plane depending on its composition. Molecular motors play a dual antagonistic role, fluidizing or stiffening the gel depending on the ATP concentration. We demonstrate how active and elastic stresses are set by each component, providing estimates for the active gel theory parameters. Finally, activity and elasticity were manipulated in situ with light-activable motor proteins, controlling the direction of the instability optically. These results highlight how cytoskeletal stresses regulate the self-organization of living matter and set the foundations for the rational design and optogenetic control of active materials.