Dynamic lane formation and long-range active nematic alignment are reported using a geometry in which kinesin motors are directly coupled to a lipid bilayer, allowing for in-plane motor diffusion during microtubule gliding. We use fluorescence microscopy to image protein distributions in and below the dense two-dimensional microtubule layer, revealing evidence of diffusion-enabled kinesin restructuring within the fluid membrane substrate as microtubules collectively glide above. We find that the lipid membrane acts to promote filament–filament alignment within the gliding layer, enhancing the formation of a globally aligned active nematic state. We also report the emergence of an intermediate, locally ordered state in which apolar dynamic lanes of nematically aligned microtubules migrate across the substrate. To understand this emergent behavior, we implement a continuum model obtained from coarse graining a collection of self-propelled rods, with propulsion set by the local motor kinetics. Tuning the microtubule and kinesin concentrations as well as active propulsion in these simulations reveals that increasing motor activity promotes dynamic nematic lane formation. Simulations and experiments show that, following fluid bilayer substrate mediated spatial motor restructuring, the total motor concentration becomes enriched below the microtubule lanes that they drive, with the feedback leading to more dynamic lanes. Our results have implications for membrane-coupled active nematics in vivo as well as for engineering dynamic and reconfigurable materials where the structural elements and power sources can dynamically colocalize, enabling efficient mechanical work.
more »
« less
Active liquid crystals powered by force-sensing DNA-motor clusters
Cytoskeletal active nematics exhibit striking nonequilibrium dynamics that are powered by energy-consuming molecular motors. To gain insight into the structure and mechanics of these materials, we design programmable clusters in which kinesin motors are linked by a double-stranded DNA linker. The efficiency by which DNA-based clusters power active nematics depends on both the stepping dynamics of the kinesin motors and the chemical structure of the polymeric linker. Fluorescence anisotropy measurements reveal that the motor clusters, like filamentous microtubules, exhibit local nematic order. The properties of the DNA linker enable the design of force-sensing clusters. When the load across the linker exceeds a critical threshold, the clusters fall apart, ceasing to generate active stresses and slowing the system dynamics. Fluorescence readout reveals the fraction of bound clusters that generate interfilament sliding. In turn, this yields the average load experienced by the kinesin motors as they step along the microtubules. DNA-motor clusters provide a foundation for understanding the molecular mechanism by which nanoscale molecular motors collectively generate mesoscopic active stresses, which in turn power macroscale nonequilibrium dynamics of active nematics.
more »
« less
- Award ID(s):
- 2011846
- PAR ID:
- 10277460
- Publisher / Repository:
- Proceedings of the National Academy of Sciences
- Date Published:
- Journal Name:
- Proceedings of the National Academy of Sciences
- Volume:
- 118
- Issue:
- 30
- ISSN:
- 0027-8424
- Page Range / eLocation ID:
- Article No. e2102873118
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
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 Society2025more » « less
-
Abstract The cytoskeleton is an active composite of filamentous proteins that dictates diverse mechanical properties and processes in eukaryotic cells by generating forces and autonomously restructuring itself. Enzymatic motors that act on the comprising filaments play key roles in this activity, driving spatiotemporally heterogeneous mechanical responses that are critical to cellular multifunctionality, but also render mechanical characterization challenging. Here, we couple optical tweezers microrheology and fluorescence microscopy with simulations and mathematical modeling to robustly characterize the mechanics of active composites of actin filaments and microtubules restructured by kinesin motors. It is discovered that composites exhibit a rich ensemble of force response behaviors–elastic, yielding, and stiffening–with their propensity and properties tuned by motor concentration and strain rate. Moreover, intermediate kinesin concentrations elicit emergent mechanical stiffness and resistance while higher and lower concentrations exhibit softer, more viscous dissipation. It is further shown that composites transition from well‐mixed interpenetrating double‐networks of actin and microtubules to de‐mixed states of microtubule‐rich aggregates surrounded by relatively undisturbed actin phases. It is this de‐mixing that leads to the emergent mechanical response, offering an alternate route that composites can leverage to achieve enhanced stiffness through coupling of structure and mechanics.more » « less
-
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.more » « less
-
The inside of a cell is a crowded space, full of proteins and other molecules. Yet, the molecular motors that transport some of those molecules within the cell move at the same speed as they would in pure water – about one micrometer per second. How the molecular motors could achieve such speeds in crowded cells was unclear. Nevertheless, Tjioe et al. suspected that the answer might be related to how multiple motors work together. Molecular motors move by walking along filaments inside the cell and pulling their cargo from one location to another. Other molecules that bind to the filaments should, in theory, act like “roadblocks” and impede the movement of the cargo. Tjioe et al. studied a motor protein called kinesin, which walks on filaments called microtubules. But instead of looking at these motors moving along microtubules inside a cell, Tjioe et al. used a simpler system where the cell was eliminated, and all parts were purified. Specifically, Tjioe et al. tethered purified motors to a piece of glass and then observed them under an extremely accurate microscope as they moved free-floating, fluorescently labelled microtubules. The microtubules, in this scenario, were acting like cargoes, where many kinesins could bind. Each kinesin motor also had a small chemical tag that could emit light. By following the movement of the lights, it was possible to calculate what each kinesin was doing and how the cargo moved. When more than one kinesin molecule was acting, the tension and speed of one kinesin affected the movement of the others. In any group of kinesins, about two-thirds of kinesin pulled the cargo, and unexpectedly, about one-third tended to resist and slow the cargo. These latter kinesins were moved along with the group without actually driving the cargo. These resisting kinesins did come off more rapidly than the driving kinesins, meaning the cargo should be able to quickly bypass roadblocks. This would help to keep the whole group travelling in the right direction at a steady pace.more » « less
An official website of the United States government
