Structure and dynamics of motor-driven microtubule bundles.

Connecting the large-scale emergent behaviors of active cytoskeletal materials to the microscopic properties of their constituents is a challenge due to a lack of data on the multiscale dynamics and structure of such systems. We approach this problem by studying the impact of depletion attraction on bundles of microtubules and kinesin-14 molecular motors. For all depletant concentrations, kinesin-14 bundles generate comparable extensile dynamics. However, this invariable mesoscopic behavior masks the transition in the microscopic motion of microtubules. Specifically, with increasing attraction, we observe a transition from bi-directional sliding with extension to pure extension with no sliding. Small-angle X-ray scattering shows that the transition in microtubule dynamics is concurrent with a structural rearrangement of microtubules from an open hexagonal to a compressed rectangular lattice. These results demonstrate that bundles of microtubules and molecular motors can display the same mesoscopic extensile behaviors despite having different internal structures and microscopic dynamics. They provide essential information for developing multiscale models of active matter.

Spatial patterning of energy metabolism during tissue morphogenesis.

Biophysical signaling organizes forces to drive tissue morphogenesis, a process co-opted during disease progression. The systematic buildup of forces at the tissue scale is energetically demanding. Just as mechanical forces, gene expression, and concentrations of morphogens vary spatially across a developing tissue, there might similarly be spatial variations in energy consumption. Recent studies have started to uncover the connections between spatial patterns of mechanical forces and spatial patterns of energy metabolism. Here, we define and review the concept of energy metabolism during tissue morphogenesis. We highlight experiments showing spatial variations in energy metabolism across several model systems, categorized by morphogenetic motif, including convergent extension, branching, and migration. Finally, we discuss approaches to further enable quantitative measurements of energy production and consumption during morphogenesis.

Plasticity in airway smooth muscle differentiation during mouse lung development.

It has been proposed that smooth muscle differentiation may physically sculpt airway epithelial branches in mammalian lungs. Serum response factor (SRF) acts with its co-factor myocardin to activate the expression of contractile smooth muscle markers. In the adult, however, smooth muscle exhibits a variety of phenotypes beyond contractile, and these are independent of SRF/myocardin-induced transcription. To determine whether a similar phenotypic plasticity is exhibited during development, we deleted Srf from the mouse embryonic pulmonary mesenchyme. Srf-mutant lungs branch normally, and the mesenchyme displays mechanical properties indistinguishable from controls. scRNA-seq identified an Srf-null smooth muscle cluster, wrapping the airways of mutant lungs, which lacks contractile smooth muscle markers but retains many features of control smooth muscle. Srf-null embryonic airway smooth muscle exhibits a synthetic phenotype, compared with the contractile phenotype of mature wild-type airway smooth muscle. Our findings identify plasticity in embryonic airway smooth muscle and demonstrate that a synthetic smooth muscle layer promotes airway branching morphogenesis.

Dissipation and energy propagation across scales in an active cytoskeletal material.

Living systems are intrinsically nonequilibrium: They use metabolically derived chemical energy to power their emergent dynamics and self-organization. A crucial driver of these dynamics is the cellular cytoskeleton, a defining example of an active material where the energy injected by molecular motors cascades across length scales, allowing the material to break the constraints of thermodynamic equilibrium and display emergent nonequilibrium dynamics only possible due to the constant influx of energy. Notwithstanding recent experimental advances in the use of local probes to quantify entropy production and the breaking of detailed balance, little is known about the energetics of active materials or how energy propagates from the molecular to emergent length scales. Here, we use a recently developed picowatt calorimeter to experimentally measure the energetics of an active microtubule gel that displays emergent large-scale flows. We find that only approximately one-billionth of the system's total energy consumption contributes to these emergent flows. We develop a chemical kinetics model that quantitatively captures how the system's total thermal dissipation varies with ATP and microtubule concentrations but that breaks down at high motor concentration, signaling an interference between motors. Finally, we estimate how energy losses accumulate across scales. Taken together, these results highlight energetic efficiency as a key consideration for the engineering of active materials and are a powerful step toward developing a nonequilibrium thermodynamics of living systems.

Engineering stability, longevity, and miscibility of microtubule-based active fluids.

Microtubule-based active matter provides insight into the self-organization of motile interacting constituents. We describe several formulations of microtubule-based 3D active isotropic fluids. Dynamics of these fluids is powered by three types of kinesin motors: a processive motor, a non-processive motor, and a motor which is permanently linked to a microtubule backbone. Another modification uses a specific microtubule crosslinker to induce bundle formation instead of a non-specific polymer depletant. In comparison to the already established system, each formulation exhibits distinct properties. These developments reveal the temporal stability of microtubule-based active fluids while extending their reach and the applicability.

Active Microphase Separation in Mixtures of Microtubules and Tip-Accumulating Molecular Motors.

Mixtures of filaments and molecular motors form active materials with diverse dynamical behaviors that vary based on their constituents' molecular properties. To develop a multiscale of these materials, we map the nonequilibrium phase diagram of microtubules and tip-accumulating kinesin-4 molecular motors. We find that kinesin-4 can drive either global contractions or turbulentlike extensile dynamics, depending on the concentrations of both microtubules and a bundling agent. We also observe a range of spatially heterogeneous nonequilibrium phases, including finite-sized radial asters, 1D wormlike chains, extended 2D bilayers, and system-spanning 3D active foams. Finally, we describe intricate kinetic pathways that yield microphase separated structures and arise from the inherent frustration between the orientational order of filamentous microtubules and the positional order of tip-accumulating molecular motors. Our work reveals a range of novel active states. It also shows that the form of active stresses is not solely dictated by the properties of individual motors and filaments, but is also contingent on the constituent concentrations and spatial arrangement of motors on the filaments.

Self-straining of actively crosslinked microtubule networks.

Cytoskeletal networks are foundational examples of active matter and central to self-organized structures in the cell. In vivo, these networks are active and densely crosslinked. Relating their large-scale dynamics to the properties of their constituents remains an unsolved problem. Here, we study an in vitro active gel made from aligned microtubules and XCTK2 kinesin motors. Using photobleaching, we demonstrate that the gel's aligned microtubules, driven by motors, continually slide past each other at a speed independent of the local microtubule polarity and motor concentration. This phenomenon is also observed, and remains unexplained, in spindles. We derive a general framework for coarse graining microtubule gels crosslinked by molecular motors from microscopic considerations. Using microtubule-microtubule coupling through a force-velocity relationship for kinesin, this theory naturally explains the experimental results: motors generate an active strain rate in regions of changing polarity, which allows microtubules of opposite polarities to slide past each other without stressing the material.