Engineering life in synthetic systems.

The second EMBO-EMBL Symposium 'Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture' was held virtually in March 2021, with participants from all over the world joining from the comfort of their sofas to discuss synthetic morphogenesis at large. Leading scientists from a range of disciplines, including developmental biology, physics, chemistry and computer science, covered a gamut of topics from the principles of cell and tissue organization, patterning and gene regulatory networks, to synthetic approaches for exploring evolutionary and developmental biology principles. Here, we describe some of the high points.

Active forces shape the metaphase spindle through a mechanical instability.

The metaphase spindle is a dynamic structure orchestrating chromosome segregation during cell division. Recently, soft matter approaches have shown that the spindle behaves as an active liquid crystal. Still, it remains unclear how active force generation contributes to its characteristic spindle-like shape. Here we combine theory and experiments to show that molecular motor-driven forces shape the structure through a barreling-type instability. We test our physical model by titrating dynein activity in Xenopus egg extract spindles and quantifying the shape and microtubule orientation. We conclude that spindles are shaped by the interplay between surface tension, nematic elasticity, and motor-driven active forces. Our study reveals how motor proteins can mold liquid crystalline droplets and has implications for the design of active soft materials.

Rethinking embryology in vitro: A synergy between engineering, data science and theory.

Pluripotent stem cells, in the recent years, have been demonstrated to mimic different aspects of metazoan embryonic development in vitro. This has led to the establishment of synthetic embryology: a field that makes use of in vitro stem cell models to investigate developmental processes that would be otherwise inaccessible in vivo. Currently, a plethora of engineering-inspired techniques, including microfluidic devices and bioreactors, exist to generate and culture organoids at high throughput. Similarly, data analysis and deep learning-based techniques, that were established in in vivo models, are now being used to extract quantitative information from synthetic systems. Finally, theory and data-driven in silico modeling are starting to provide a system-level understanding of organoids and make predictions to be tested with further experiments. Here, we discuss our vision of how engineering, data science and theoretical modeling will synergize to offer an unprecedented view of embryonic development. For every one of these three scientific domains, we discuss examples from in vivo and in vitro systems that we think will pave the way to future developments of synthetic embryology.

Arrested coalescence of multicellular aggregates.

Multicellular aggregates are known to exhibit liquid-like properties. The fusion process of two cell aggregates is commonly studied as the coalescence of two viscous drops. However, tissues are complex materials and can exhibit viscoelastic behaviour. It is known that elastic effects can prevent the complete fusion of two drops, a phenomenon known as arrested coalescence. Here we study this phenomenon in stem cell aggregates and provide a theoretical framework which agrees with the experiments. In addition, agent-based simulations show that active cell fluctuations can control a solid-to-fluid phase transition, revealing that arrested coalescence can be found in the vicinity of an unjamming transition. By analysing the dynamics of the fusion process and combining it with nanoindentation measurements, we obtain the effective viscosity, shear modulus and surface tension of the aggregates. More generally, our work provides a simple, fast and inexpensive method to characterize the mechanical properties of viscoelastic materials.

A Brownian Ratchet Model Explains the Biased Sidestepping of Single-Headed Kinesin-3 KIF1A.

The kinesin-3 motor KIF1A is involved in long-ranged axonal transport in neurons. To ensure vesicular delivery, motors need to navigate the microtubule lattice and overcome possible roadblocks along the way. The single-headed form of KIF1A is a highly diffusive motor that has been shown to be a prototype of a Brownian motor by virtue of a weakly bound diffusive state to the microtubule. Recently, groups of single-headed KIF1A motors were found to be able to sidestep along the microtubule lattice, creating left-handed helical membrane tubes when pulling on giant unilamellar vesicles in vitro. A possible hypothesis is that the diffusive state enables the motor to explore the microtubule lattice and switch protofilaments, leading to a left-handed helical motion. Here, we study the longitudinal rotation of microtubules driven by single-headed KIF1A motors using fluorescence-interference contrast microscopy. We find an average rotational pitch of ≃1.5µm, which is remarkably robust to changes in the gliding velocity, ATP concentration, microtubule length, and motor density. Our experimental results are compared to stochastic simulations of Brownian motors moving on a two-dimensional continuum ratchet potential, which quantitatively agree with the fluorescence-interference contrast experiments. We find that single-headed KIF1A sidestepping can be explained as a consequence of the intrinsic handedness and polarity of the microtubule lattice in combination with the diffusive mechanochemical cycle of the motor.

Fluidization and Active Thinning by Molecular Kinetics in Active Gels.

We derive the constitutive equations of an active polar gel from a model for the dynamics of elastic molecules that link polar elements. Molecular binding kinetics induces the fluidization of the material, giving rise to Maxwell viscoelasticity and, provided that detailed balance is broken, to the generation of active stresses. We give explicit expressions for the transport coefficients of active gels in terms of molecular properties, including nonlinear contributions on the departure from equilibrium. In particular, when activity favors linker unbinding, we predict a decrease of viscosity with activity-active thinning-of kinetic origin, which could explain some experimental results on the cell cortex. By bridging the molecular and hydrodynamic scales, our results could help understand the interplay between molecular perturbations and the mechanics of cells and tissues.

Cooperative force generation of KIF1A Brownian motors.

KIF1A is a kinesin motor protein that can work processively in a monomeric (single-headed) form by using a noise-driven ratchet mechanism. Here, we show that the combination of a passive diffusive state and finite-time kinetics of adenosine triphosphate hydrolysis provides a powerful mechanism of cooperative force generation, implying for instance that ∼10 monomeric KIF1As can team up to become ∼100 times stronger than a single one. Consequently, we propose that KIF1A could outperform conventional (double-headed) kinesin collectively and thus explain its specificity in axonal trafficking. We elucidate the cooperativity mechanism with a lattice model that includes multiparticle transitions.

Single-cell Bayesian deconvolution.

Individual cells exhibit substantial heterogeneity in protein abundance and activity, which is frequently reflected in broad distributions of fluorescently labeled reporters. Since all cellular components are intrinsically fluorescent to some extent, the observed distributions contain background noise that masks the natural heterogeneity of cellular populations. This limits our ability to characterize cell-fate decision processes that are key for development, immune response, tissue homeostasis, and many other biological functions. It is therefore important to separate the contributions from signal and noise in single-cell measurements. Addressing this issue rigorously requires deconvolving the noise distribution from the signal, but approaches in that direction are still limited. Here, we present a non-parametric Bayesian formalism that performs such a deconvolution efficiently on multidimensional measurements, providing unbiased estimates of the resulting confidence intervals. We use this approach to study the expression of the mesodermal transcription factor Brachyury in mouse embryonic stem cells undergoing differentiation.

Osteoclast-mediated resorption primes the skeleton for successful integration during axolotl limb regeneration.

Early events during axolotl limb regeneration include an immune response and the formation of a wound epithelium. These events are linked to a clearance of damaged tissue prior to blastema formation and regeneration of the missing structures. Here, we report the resorption of calcified skeletal tissue as an active, cell-driven, and highly regulated event. This process, carried out by osteoclasts, is essential for a successful integration of the newly formed skeleton. Indeed, the extent of resorption is directly correlated with the integration efficiency, and treatment with zoledronic acid resulted in osteoclast function inhibition and failed tissue integration. Moreover, we identified the wound epithelium as a regulator of skeletal resorption, likely releasing signals involved in recruitment/differentiation of osteoclasts. Finally, we reported a correlation between resorption and blastema formation, particularly, a coordination of resorption with cartilage condensation. In sum, our results identify resorption as a major event upon amputation, playing a critical role in the overall process of skeletal regeneration.

Author Correction: Formation of helical membrane tubes around microtubules by single-headed kinesin KIF1A.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Physics of the Metaphase Spindle.

The assembly of the mitotic spindle and the subsequent segregation of sister chromatids are based on the self-organized action of microtubule filaments, motor proteins, and other microtubule-associated proteins, which constitute the fundamental force-generating elements in the system. Many of the components in the spindle have been identified, but until recently it remained unclear how their collective behaviors resulted in such a robust bipolar structure. Here, we review the current understanding of the physics of the metaphase spindle that is only now starting to emerge.

Autocatalytic microtubule nucleation determines the size and mass of Xenopus laevis egg extract spindles.

Regulation of size and growth is a fundamental problem in biology. A prominent example is the formation of the mitotic spindle, where protein concentration gradients around chromosomes are thought to regulate spindle growth by controlling microtubule nucleation. Previous evidence suggests that microtubules nucleate throughout the spindle structure. However, the mechanisms underlying microtubule nucleation and its spatial regulation are still unclear. Here, we developed an assay based on laser ablation to directly probe microtubule nucleation events in Xenopus laevis egg extracts. Combining this method with theory and quantitative microscopy, we show that the size of a spindle is controlled by autocatalytic growth of microtubules, driven by microtubule-stimulated microtubule nucleation. The autocatalytic activity of this nucleation system is spatially regulated by the limiting amounts of active microtubule nucleators, which decrease with distance from the chromosomes. This mechanism provides an upper limit to spindle size even when resources are not limiting.

Nonlinear amplitude dynamics in flagellar beating.

The physical basis of flagellar and ciliary beating is a major problem in biology which is still far from completely understood. The fundamental cytoskeleton structure of cilia and flagella is the axoneme, a cylindrical array of microtubule doublets connected by passive cross-linkers and dynein motor proteins. The complex interplay of these elements leads to the generation of self-organized bending waves. Although many mathematical models have been proposed to understand this process, few attempts have been made to assess the role of dyneins on the nonlinear nature of the axoneme. Here, we investigate the nonlinear dynamics of flagella by considering an axonemal sliding control mechanism for dynein activity. This approach unveils the nonlinear selection of the oscillation amplitudes, which are typically either missed or prescribed in mathematical models. The explicit set of nonlinear equations are derived and solved numerically. Our analysis reveals the spatio-temporal dynamics of dynein populations and flagellum shape for different regimes of motor activity, medium viscosity and flagellum elasticity. Unstable modes saturate via the coupling of dynein kinetics and flagellum shape without the need of invoking a nonlinear axonemal response. Hence, our work reveals a novel mechanism for the saturation of unstable modes in axonemal beating.

Formation of helical membrane tubes around microtubules by single-headed kinesin KIF1A.

The kinesin-3 motor KIF1A is in charge of vesicular transport in neuronal axons. Its single-headed form is known to be very inefficient due to the presence of a diffusive state in the mechanochemical cycle. However, recent theoretical studies have suggested that these motors could largely enhance force generation by working in teams. Here we test this prediction by challenging single-headed KIF1A to extract membrane tubes from giant vesicles along microtubule filaments in a minimal in vitro system. Remarkably, not only KIF1A motors are able to extract tubes but they feature a novel phenomenon: tubes are wound around microtubules forming tubular helices. This finding reveals an unforeseen combination of cooperative force generation and self-organized manoeuvreing capability, suggesting that the diffusive state may be a key ingredient for collective motor performance under demanding traffic conditions. Hence, we conclude that KIF1A is a genuinely cooperative motor, possibly explaining its specificity to axonal trafficking.

Cooperative action of KIF1A Brownian motors with finite dwell time.

We study in detail the cooperative action of small groups of KIF1A motors in its monomeric (single-headed) form within an arrangement relevant to vesicle traffic or membrane tube extraction. It has been recently shown that under these circumstances, the presence of a finite dwell time in the motor cycle contributes to remarkably enhance collective force generation [D. Oriola and J. Casademunt, Phys. Rev. Lett. 111, 048103 (2013)]. We analyze this mechanism in detail by means of a two-state noise-driven ratchet model with hard-core repulsive interactions. We obtain staircase-shaped velocity-force curves and show that motors self-organize in clusters with a nontrivial force distribution that conveys a large part of the load to the central motors. Under heavy loads, large clusters adopt a synchronic mode of totally asymmetric steps. We also find a dramatic increase of the collective efficiency with the number of motors. Finally, we complete the study by addressing different interactions that impose spatial constraints such as rigid coupling and raft-induced confinement. Our results reinforce the hypothesis that the specificity of KIF1A to axonal vesicular transport may be deeply related to its high cooperativity.