Motor-driven advection competes with crowding to drive spatiotemporally heterogeneous transport in cytoskeleton composites.

The cytoskeleton-a composite network of biopolymers, molecular motors, and associated binding proteins-is a paradigmatic example of active matter. Particle transport through the cytoskeleton can range from anomalous and heterogeneous subdiffusion to superdiffusion and advection. Yet, recapitulating and understanding these properties-ubiquitous to the cytoskeleton and other out-of-equilibrium soft matter systems-remains challenging. Here, we combine light sheet microscopy with differential dynamic microscopy and single-particle tracking to elucidate anomalous and advective transport in actomyosin-microtubule composites. We show that particles exhibit multi-mode transport that transitions from pronounced subdiffusion to superdiffusion at tunable crossover timescales. Surprisingly, while higher actomyosin content increases the range of timescales over which transport is superdiffusive, it also markedly increases the degree of subdiffusion at short timescales and generally slows transport. Corresponding displacement distributions display unique combinations of non-Gaussianity, asymmetry, and non-zero modes, indicative of directed advection coupled with caged diffusion and hopping. At larger spatiotemporal scales, particles in active composites exhibit superdiffusive dynamics with scaling exponents that are robust to changing actomyosin fractions, in contrast to normal, yet faster, diffusion in networks without actomyosin. Our specific results shed important new light on the interplay between non-equilibrium processes, crowding and heterogeneity in active cytoskeletal systems. More generally, our approach is broadly applicable to active matter systems to elucidate transport and dynamics across scales.

Topological tuning of DNA mobility in entangled solutions of supercoiled plasmids.

Ring polymers in dense solutions are among the most intriguing problems in polymer physics. Because of its natural occurrence in circular form, DNA has been extensively used as a proxy to study the fundamental physics of ring polymers in different topological states. Yet, torsionally constrained-such as supercoiled-topologies have been largely neglected so far. The applicability of existing theoretical models to dense supercoiled DNA is thus unknown. Here, we address this gap by coupling large-scale molecular dynamics simulations with differential dynamic microscopy of entangled supercoiled DNA plasmids. We find that, unexpectedly, larger supercoiling increases the size of entangled plasmids and concomitantly induces an enhancement in DNA mobility. These findings are reconciled as due to supercoiling-driven asymmetric and double-folded plasmid conformations that reduce interplasmid entanglements and threadings. Our results suggest a way to topologically tune DNA mobility via supercoiling, thus enabling topological control over the (micro)rheology of DNA-based complex fluids.

DNA Conformation Dictates Strength and Flocculation in DNA-Microtubule Composites.

Polymer topology has been shown to play a key role in tuning the dynamics of complex fluids and gels. At the same time, polymer composites, ubiquitous in everyday life, have been shown to exhibit emergent desirable mechanical properties not attainable in single-species systems. Yet, how topology impacts the dynamics and structure of polymer composites remains poorly understood. Here, we create composites of rigid rods (microtubules) polymerized within entangled solutions of flexible linear and ring polymers (DNA) of equal length. We couple optical tweezers microrheology with confocal microscopy and scaled particle theory to show that composites with linear DNA exhibit a strongly nonmonotonic dependence of elasticity and stiffness on microtubule concentration due to depletion-driven polymerization and flocculation of microtubules. In contrast, composites containing ring DNA show a much more modest monotonic increase in elastic strength with microtubule concentration, which we demonstrate arises from the decreased conformational size and increased miscibility of rings.

Correction: Anomalous and heterogeneous DNA transport in biomimetic cytoskeleton networks.

Correction for 'Anomalous and heterogeneous DNA transport in biomimetic cytoskeleton networks' by Jonathan Garamella et al., Soft Matter, 2020, DOI: 10.1039/d0sm00544d.

Anomalous and heterogeneous DNA transport in biomimetic cytoskeleton networks.

The cytoskeleton, a complex network of protein filaments and crosslinking proteins, dictates diverse cellular processes ranging from division to cargo transport. Yet, the role the cytoskeleton plays in the intracellular transport of DNA and other macromolecules remains poorly understood. Here, using single-molecule conformational tracking, we measure the transport and conformational dynamics of linear and relaxed circular (ring) DNA in composite networks of actin and microtubules with variable types of crosslinking. While both linear and ring DNA undergo anomalous, non-Gaussian, and non-ergodic subdiffusion, the detailed dynamics are controlled by both DNA topology (linear vs. ring) and crosslinking motif. Ring DNA swells, exhibiting heterogeneous subdiffusion controlled via threading by cytoskeleton filaments, while linear DNA compacts, exhibiting transport via caging and hopping. Importantly, while the crosslinking motif has little effect on ring DNA, linear DNA in networks with actin-microtubule crosslinking is significantly less ergodic and shows more heterogeneous transport than with actin-actin or microtubule-microtubule crosslinking.

Topology-dependent anomalous dynamics of ring and linear DNA are sensitive to cytoskeleton crosslinking.

Cytoskeletal crowding plays a key role in the diffusion of DNA molecules through the cell, acting as a barrier to effective intracellular transport and conformational stability required for processes such as transfection, viral infection, and gene therapy. Here, we elucidate the transport properties and conformational dynamics of linear and ring DNA molecules diffusing through entangled and crosslinked composite networks of actin and microtubules. We couple single-molecule conformational tracking with differential dynamic microscopy to reveal that ring and linear DNA exhibit unexpectedly distinct transport properties that are influenced differently by cytoskeleton crosslinking. Ring DNA coils are swollen and undergo heterogeneous and biphasic subdiffusion that is hindered by crosslinking. Conversely, crosslinking actually facilitates the single-mode subdiffusion that compacted linear chains exhibit. Our collective results demonstrate that transient threading by cytoskeleton filaments plays a key role in the dynamics of ring DNA, whereas the mobility of the cytoskeleton dictates transport of linear DNA.

Filament Rigidity Vies with Mesh Size in Determining Anomalous Diffusion in Cytoskeleton.

The diffusion of microscopic particles through the cell, important to processes such as viral infection, gene delivery, and vesicle transport, is largely controlled by the complex cytoskeletal network, comprised of semiflexible actin filaments and rigid microtubules, that pervades the cytoplasm. By varying the relative concentrations of actin and microtubules, the cytoskeleton can display a host of different structural and dynamic properties that, in turn, impact the diffusion of particles through the composite network. Here, we couple single-particle tracking with differential dynamic microscopy to characterize the transport of microsphere tracers diffusing through composite in vitro networks with varying ratios of actin and microtubules. We analyze multiple complementary metrics for anomalous transport to show that particles exhibit anomalous subdiffusion in all networks, which our data suggest arises from caging by networks. Further, subdiffusive characteristics are markedly more pronounced in actin-rich networks, which exhibit similarly more prominent viscoelastic properties compared to microtubule-rich composites. While the smaller mesh size of actin-rich composites compared to microtubule-rich composites plays an important role in these results, the rigidity of the filaments comprising the network also influences the anomalous characteristics that we observe. Our results suggest that as microtubules in our composites are replaced with actin filaments, the decreasing filament rigidity competes with increasing network connectivity to drive anomalous transport.

An Adaptive Synthetic Cell Based on Mechanosensing, Biosensing, and Inducible Gene Circuits.

The bottom-up assembly of synthetic cell systems capable of recapitulating biological functions has become a means to understand living matter by construction. The integration of biomolecular components into active, cell-sized, genetically programmed compartments remains, however, a major bottleneck for building synthetic cells. A primary feature of real cells is their ability to actively interact with their surroundings, particularly in stressed conditions. Here, we construct a synthetic cell equipped with an inducible genetic circuit that responds to changes in osmotic pressure through the mechanosensitive channel MscL. Liposomes loaded with an E. coli cell-free transcription-translation (TXTL) system are induced with IPTG when exposed to hypo-osmotic solution, resulting in the expression of a bacterial cytoskeletal protein MreB. MreB associates with the membrane to generate a cortex-like structure. Our work provides the first example of molecular integration that couples mechanosensitivity, gene expression, and self-assembly at the inner membrane of synthetic cells.

TXTL-based approach to synthetic cells.

Cell-free transcription-translation (TXTL) has recently emerged as a versatile technology to engineer biological systems. In this chapter, we show how an all E. coli TXTL system can be used to build synthetic cell prototypes. We describe methods to encapsulate TXTL reactions in cell-sized liposomes, with an emphasis on the composition of the external solution and lipid bilayer. Cell-free expression is quantitatively described in bulk reactions and liposomes for three proteins: the soluble reporter protein eGFP, the membrane proteins alpha-hemolysin (AH) from Staphylococcus aureus, and the mechanosensitive channel of large conductance (MscL) from E. coli.

Engineering DNA nanotubes for resilience in an E. coli TXTL system.

Deoxyribonucleic acid (DNA) nanotechnology is a growing field with potential intracellular applications. In this work, we use an Escherichia coli cell-free transcription-translation (TXTL) system to assay the robustness of DNA nanotubes in a cytoplasmic environment. TXTL recapitulates physiological conditions as well as strong linear DNA degradation through the RecBCD complex, the major exonuclease in E. coli. We demonstrate that chemical modifications of the tiles making up DNA nanotubes extend their viability in TXTL for more than 24 h, with phosphorothioation of the sticky end backbone being the most effective. Furthermore, we show that a Chi-site double-stranded DNA, an inhibitor of the RecBCD complex, extends DNA nanotube lifetime significantly. These complementary approaches are a first step toward a systematic prototyping of DNA nanostructures in active cell-free cytoplasmic environments and expand the scope of TXTL utilization for bioengineering.

Cell-sized mechanosensitive and biosensing compartment programmed with DNA.

The bottom-up construction of cell-sized compartments programmed with DNA that are capable of sensing the chemical and physical environment remains challenging in synthetic cell engineering. Here, we construct mechanosensitive liposomes with biosensing capability by expressing the E. coli channel MscL and a calcium biosensor using cell-free expression.

The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology.

We report on and provide a detailed characterization of the performance and properties of a recently developed, all Escherichia coli, cell-free transcription and translation system. Gene expression is entirely based on the endogenous translation components and transcription machinery provided by an E. coli cytoplasmic extract, thus expanding the repertoire of regulatory parts to hundreds of elements. We use a powerful metabolism for ATP regeneration to achieve more than 2 mg/mL of protein synthesis in batch mode reactions, and more than 6 mg/mL in semicontinuous mode. While the strength of cell-free expression is increased by a factor of 3 on average, the output signal of simple gene circuits and the synthesis of entire bacteriophages are increased by orders of magnitude compared to previous results. Messenger RNAs and protein degradation, respectively tuned using E. coli MazF interferase and ClpXP AAA+ proteases, are characterized over a much wider range of rates than the first version of the cell-free toolbox. This system is a highly versatile cell-free platform to construct complex biological systems through the execution of DNA programs composed of synthetic and natural bacterial regulatory parts.