Active networks composed of biopolymers and motor proteins provide versatile biomimetic systems that have advanced active matter physics and deepened our understanding of cytoskeletal dynamics and self-organization under diverse stimuli. In these systems, activity arises in aqueous solutions where motor proteins cross-link biopolymers and generate active stress driving the emergent network behavior. Here, we establish the active network in the form of a sessile, multi-component droplet on a substrate and investigate how evaporation influences its dynamics. We focus on how mass loss and compositional changes in the droplet reshape the behavior of the active suspension. We show that capillary and Marangoni flows drive the self-organization of microtubules into a distinctive radial arrangement within the droplet. The cross-linking ability of motor proteins gives rise to a striking non-monotonic wetting behavior, where the extensile stresses generated by the motor proteins strongly affect the characteristic timescale of the contact-line retracting and subsequent expansion. Using a combined experimental and theoretical approach, we demonstrate the crucial role of crosslinking in evaporating microtubule networks, and explain how active stresses together with evaporation-induced flows govern the dynamics of reconstituted microtubule systems and their wetting behavior. Evaporating droplets have recently attracted significant attention in the scientific community, and the findings of the setup presented in this study can have broad implications, ranging from self-organization and mechanical pattern formation in biological systems to questions about the origin of life.
Cytoskeletal assemblies, in the form of microtubule networks, drive vital cellular processes such as intracellular cargo transport [1], cell guidance during migration [2,3], and play a crucial role in mechanical stability [4], cell morphology [5], and cell division [6]. These functions result from the self-organization of microtubules and motor proteins which, through interactions at the molecular scale, give rise to the system's large-scale emergent behavior. Examples of such self-organization, both in-vivo and in-vitro, have been demonstrated in numerous studies over the past decades, using both experimental and theoretical approaches [7][8][9][10][11][12][13].
A pivotal experimental system to investigate the self-organization of cytoskeleton components consists of a reconstituted in-vitro assay made of suspensions of microtubules and multi-headed kinesin motors [11].
These suspensions are driven out of equilibrium by kinesin motors, which bind to the microtubules and move along them, exerting active stresses during their stepping motion, powered by a continuous supply of adenosine triphosphate (ATP) in solution. In such in-vitro assays, the activity of the motors is enhanced by organizing the microtubules into bundles through the addition of depleting agents such as polyethylene glycol (PEG), which induces effective attractive interactions between randomly oriented microtubules via entropic forces [11,14,15]. Specifically, PEG acts as a depletion agent, bundling the microtubules and promoting kinesin motor binding, thereby enhancing force generation through their active motion along the filaments. Under this arrangement, the motors generate active forces that lead to spatial displacements of the microtubules and bending of the bundles at larger length scales, as well as to emergent behaviors such as the formation of microtubule asters, vortices, or nematic structures in 2D and 3D [8,9,11,16].
Suspensions of microtubules and kinesin motors have been studied in various contexts, most notably when confined at the interface between two fluids [11], or within channels with rigid boundaries [16][17][18].
Of particular interest is the influence of the external environment on such suspensions, especially how they respond to mechanical cues. Indeed, gliding assay experiments have shown that applying external stress can influence motor activity and tune the spatio-temporal distribution of the internal driving forces leading to the emergent behaviour of the system [19]. Several in-vitro experimental setups have been developed to investigate the response of reconstituted active cytoskeletal networks to externally applied stimuli. These include patterned surfaces, varying confinement geometries, and interactions at water/oil interfaces [17,20,21]. At fluid interfaces, microtubule-kinesin motor suspensions exhibit fascinating behaviors, such as active turbulence, characterised by the continuous creation and annihilation of defects in the microtubule orientation field [22], and defect ordering driven by changes in friction with the surrounding fluid [23].
Theoretical attempts to understand these phenomena include analyses of the dynamics of a nematic order parameter, representing the orientation of force-generating microtubules, coupled to a velocity field and the resulting instabilities that lead to the formation of flow patterns and defects [24][25][26][27]; the ordering of defects in the presence of friction with the external environment [28][29][30][31]; and the transition to flow in confined geometries [16,32,33].
Here we study the behaviour of these active suspensions of microtubules and kinesin motors when they are in a liquid droplet deposited on a substrate and evaporate in the environment. Previous studies reported the dynamics of active microtubule-kinesin network inside droplet-like compartments, namely water-in-oil droplets [34], lipid vesicles [35] and water/water phase separation (w/wPS) droplet composed of a Dextran-PEG mixture [36]. On the other hand, the droplet-like confinements mentioned above are immersed in a bulk aqueous solution and therefore present a liquid-liquid interface. The intriguing aspect of the active droplets we investigate is that they are not confined in a close experimental chambers with aqueous solution. They rather exhibit a dynamic liquid-vapor interface, across which the mass of the suspension changes over time due to evaporation. Droplet evaporation induces dynamic changes in the geometry of confinement, leading to outward capillary flows [37]. However, preferential evaporation can generate compositional gradients that-even in passive systems-drive inward Marangoni flows and exert mechanical stresses on the suspended constituents. The aggregation of colloids into ordered or disordered structures under such flows has been the subject of extensive studies [37][38][39][40][41][42][43]. In contrast, to the best of our knowledge, the behavior of an active evaporating droplet
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