Cell-sized Confinement Research Articles

Cell-Sized Confinements Alter Molecular Diffusion in Concentrated Polymer Solutions Due to Length-Dependent Wetting of Polymers.

Living cells are characterized by the micrometric confinement of various macromolecules at high concentrations. Using droplets containing binary polymer blends as artificial cells, we previously showed that cell-sized confinement causes phase separation of the binary polymer solutions because of the length-dependent wetting of the polymers. Here, we demonstrate that the confinement-induced heterogeneity of polymers also emerges in single-component polymer solutions. The resulting structural heterogeneity also leads to a slower transport of small molecules at the center of cell-sized droplets than that in bulk solutions. Coarse-grained molecular simulations support this confinement-induced heterogeneous distribution by polymer length and demonstrate that the effective wetting of the shorter chains at the droplet surface originates from the length-dependent conformational entropy. Our results suggest that cell-sized confinement functions as a structural regulator for polydisperse polymer solutions that specifically manipulates the diffusion of molecules, particularly those with sizes close to the correlation length of the polymer chains.

Impact of cell-sized confinement on particle transport within a motor-driven cytoskeletal network.

Intracellular transport of small particles and macromolecules is affected by the degree of crowding, confinement, and motor-driven activity within the cell. In vitro studies have examined how transport dynamics depend on each of these factors. But studies have primarily focused on these factors in isolation. Previously, we showed how crowding and confinement synergistically couple to slow the diffusion of DNA molecules. Here, we explore the combined effects of crowding by a composite network of actin and microtubule filaments, activity driven by molecular motors, and confinement by a lipid membrane to cell-sized volumes. We quantify transport of tracer particles within this tunable in vitro environment using differential dynamic microscopy (DDM) and single particle tracking. We simultaneously measure the dynamics and spatial organization of the composite network to link transport dynamics to network properties. To what degree tracers exhibit subdiffusive motion due to caging within the network mesh or superdiffusive motion due to motor activity depends on network composition, motor concentration, and confinement size.

Cell-Sized Confinement Initiates Phase Separation of Polymer Blends and Promotes Fractionation upon Competitive Membrane Wetting

Cell-Sized Confinement Initiates Phase Separation of Polymer Blends and Promotes Fractionation upon Competitive Membrane Wetting

Functional DNA-based cytoskeletons for synthetic cells

The cytoskeleton is an essential component of a cell. It controls the cell shape, establishes the internal organization, and performs vital biological functions. Building synthetic cytoskeletons that mimic key features of their natural counterparts delineates a crucial step towards synthetic cells assembled from the bottom up. To this end, DNA nanotechnology represents one of the most promising routes, given the inherent sequence specificity, addressability and programmability of DNA. Here we demonstrate functional DNA-based cytoskeletons operating in microfluidic cell-sized compartments. The synthetic cytoskeletons consist of DNA tiles self-assembled into filament networks. These filaments can be rationally designed and controlled to imitate features of natural cytoskeletons, including reversible assembly and ATP-triggered polymerization, and we also explore their potential for guided vesicle transport in cell-sized confinement. Also, they possess engineerable characteristics, including assembly and disassembly powered by DNA hybridization or aptamer–target interactions and autonomous transport of gold nanoparticles. This work underpins DNA nanotechnology as a key player in building synthetic cells.

Membrane Surface Modulates Slow Diffusion in Small Crowded Droplets.

Membranes are ubiquitous structures in cells. The effects of membranes on various functional molecules have been reported, but their behaviors under macromolecular crowding and cell-sized confinement have not fully been understood. In this study, we model an intracellular environment by crowding micrometer-sized droplets and investigate the effects of membrane properties on molecular diffusion. The molecular diffusion inside small droplets covered with a lipid layer of phosphatidylcholine (PC) becomes slower compared with that of the corresponding bulk solutions under a crowding condition of polysaccharide dextran but not of its monomer unit, glucose. The addition of a poly(ethylene glycol) conjugated lipid (PEGylated lipid) to the PC membrane significantly alters the degree of slow diffusion observed inside small droplets of concentrated dextran. Interestingly, the change is not monotonic against dextran concentration; that is, the PEGylated membrane increases and decreases the degree of slow diffusion with increasing dextran concentration. We explain the nonmonotonic alternation from the increase in effective dextran concentration and the hindered temporal adsorption of dextran to the membrane. Because diffusion alteration by adding PEGylated lipid is observed for condensed small droplets of linear polymer PEG and hydrophilic protein bovine serum albumin, the phenomenon is general for other polymer systems as well. Furthermore, our findings may facilitate the understanding of intracellular molecular behaviors based on membrane effects as well as the development of numerous applications using polymer droplets.

Tug-of-war between actomyosin-driven antagonistic forces determines the positioning symmetry in cell-sized confinement

Symmetric or asymmetric positioning of intracellular structures including the nucleus and mitotic spindle steers various biological processes such as cell migration, division, and embryogenesis. In typical animal cells, both a sparse actomyosin meshwork in the cytoplasm and a dense actomyosin cortex underneath the cell membrane participate in the intracellular positioning. However, it remains unclear how these coexisting actomyosin structures regulate the positioning symmetry. To reveal the potential mechanism, we construct an in vitro model composed of cytoplasmic extracts and nucleus-like clusters confined in droplets. Here we find that periodic centripetal actomyosin waves contract from the droplet boundary push clusters to the center in large droplets, while network percolation of bulk actomyosin pulls clusters to the edge in small droplets. An active gel model quantitatively reproduces molecular perturbation experiments, which reveals that the tug-of-war between two distinct actomyosin networks with different maturation time-scales determines the positioning symmetry.

Cell-sized confinement controls generation and stability of a protein wave for spatiotemporal regulation in cells.

The Min system, a system that determines the bacterial cell division plane, uses changes in the localization of proteins (a Min wave) that emerges by reaction-diffusion coupling. Although previous studies have shown that space sizes and boundaries modulate the shape and speed of Min waves, their effects on wave emergence were still elusive. Here, by using a microsized fully confined space to mimic live cells, we revealed that confinement changes the conditions for the emergence of Min waves. In the microsized space, an increased surface-to-volume ratio changed the localization efficiency of proteins on membranes, and therefore, suppression of the localization change was necessary for the stable generation of Min waves. Furthermore, we showed that the cell-sized space strictly limits parameters for wave emergence because confinement inhibits both the instability and excitability of the system. These results show that confinement of reaction-diffusion systems has the potential to control spatiotemporal patterns in live cells.

Geometric Effect for Biological Reactors and Biological Fluids.

As expressed “God made the bulk; the surface was invented by the devil” by W. Pauli, the surface has remarkable properties because broken symmetry in surface alters the material properties. In biological systems, the smallest functional and structural unit, which has a functional bulk space enclosed by a thin interface, is a cell. Cells contain inner cytosolic soup in which genetic information stored in DNA can be expressed through transcription (TX) and translation (TL). The exploration of cell-sized confinement has been recently investigated by using micron-scale droplets and microfluidic devices. In the first part of this review article, we describe recent developments of cell-free bioreactors where bacterial TX-TL machinery and DNA are encapsulated in these cell-sized compartments. Since synthetic biology and microfluidics meet toward the bottom-up assembly of cell-free bioreactors, the interplay between cellular geometry and TX-TL advances better control of biological structure and dynamics in vitro system. Furthermore, biological systems that show self-organization in confined space are not limited to a single cell, but are also involved in the collective behavior of motile cells, named active matter. In the second part, we describe recent studies where collectively ordered patterns of active matter, from bacterial suspensions to active cytoskeleton, are self-organized. Since geometry and topology are vital concepts to understand the ordered phase of active matter, a microfluidic device with designed compartments allows one to explore geometric principles behind self-organization across the molecular scale to cellular scale. Finally, we discuss the future perspectives of a microfluidic approach to explore the further understanding of biological systems from geometric and topological aspects.

Thermo-Responsive Behavior of Polymer Solutions Coated with Phospholipids

Various kinds of cell model systems have been utilized for fundamental studies on physical properties of cells. Water-in-Oil (W/O) droplet system coated with phospholipids is one of the beneficial simple model systems of living cells. For instance, a biomimetic phase-separated membrane was constructed in the W/O systems with phospholipid mixture (Hamada et al., J. Phys. Chem. Lett., 2010) since phase separation in lipid membrane system corresponds to the separation between lipid raft domains and other soft domains in the living cell membranes. Phase transition of a polymer has been studied for many years, such as coil-globule transition (Nishio et al., Nature, 1979), and helix-coil transition (Tanaka et al., J. Chem. Phys., 1973). In addition to the phase transition of polymers, phase separation in a binary polymer solution was also reported (Yanagisawa et al., J. Mol. Liq., 2014). However, cell-sized confinement effects on polymer solutions have been poorly confirmed. To clarify the cell-sized confinement effects on polymer solutions, we previously observed the dynamic behavior of polymer droplets (Yoshida et al., Polymers, 2017). The previous study revealed that droplet size influences the start time of phase separation in the case of temperature change from 10 to 65 degrees Celsius. In this study, we observed the dynamic behavior of hydroxypropyl cellulose (HPC) solution droplets in response to temperature change from 10 to 65 degrees Celsius using a fluorescence microscope. The droplets were coated with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE). As a control, we observed non-coated water droplets. DPPC and DPPE are widely used for studies on model cell membranes. Using time-lapse images, we confirmed the water/HPC distribution using a spatial autocorrelation function. The results revealed that water-rich region appeared in the case of lipid coated droplets with increase in temperature while such phenomenon was not observed in the case of non-coated droplets.

Confinement Effects on Polymer Dynamics: Thermo-Responsive Behaviours of Hydroxypropyl Cellulose Polymers in Phospholipid-Coated Droplets (Water-in-Oil Emulsion).

In order to construct the artificial cells and to understand the physicochemical properties of living cells, it is important to clarify the cell-sized confinement effect on the behaviours of bio-inspired polymers. We report the dynamic behaviours of aqueous hydroxypropyl cellulose (HPC) solution coated with phospholipids in oil (water-in-oil droplets, W/O droplets), accompanied by an increase in the temperature. We directly observed the beginning of phase separation of HPC solution using a fluorescence microscope and confirmed the dependence of such phenomena on droplet size. The results indicate that the start time of phase separation is decreased with an increase in droplet size. The experimental results suggest that the confinement situation accelerates the phase separation of aqueous HPC solutions.

Actin Macromolecules and Bundles in Cell-Sized Confinement: From Collective Behavior to Emerging Networks

Microfluidics-based experiments provide an opportunity to study the complexity of hierarchical dynamics and structural assembly in complex chemical as well as biological processes. The precise control of external parameters and the possibility to generate gradients on the nano- and micrometer length scale allows for investigations of intermediates and transitional states as well as dynamic and kinetic properties of the studied systems. Using a straightforward microfluidics system to achieve step-by-step reaction sequences, we explore the rich dynamics and emergent properties of actin with an emphasis on assembling networks of actin bundles under appropriate stimuli, mimicking these processes in living cells. Due to the sharing of filaments within different bundles, long actin filaments form networks by percolation, when induced by specific bundling agents in micro-confinements. Using this bottom-up approach, we observe the dynamics of hierarchical assembly and disassembly processes and find exceptional attributes such as spindle-like structure formation and internal stress generation in networks. Furthermore, our research suggests that such percolated networks are very much likely to exist within living cells in a dynamic fashion with a long-range connectivity resulting in enhanced responsiveness to internal and external signals.

Alignment of nematic and bundled semiflexible polymers in cell-sized confinement

The finite size of cells poses severe spatial constraints on the network of semiflexible filaments called the cytoskeleton, a main determinant of cell shape. At the same time, the high packing density of cytoskeletal filaments poses mutual packing constraints. Here we investigate the competition between excluded volume interactions in the bulk and surface packing constraints on the orientational ordering of confined actin filaments as a function of filament density and the presence of crosslinks. We grow fluorescently labeled actin filaments in shallow (thickness dz 3 μm), rectangular microchambers with a systematically varied length (dy between 5 and 100 μm) and in-plane aspect ratio (dx/dy between 1 and 10). We determine the nematic director field by image analysis of fluorescence confocal images. We find that high-density (nematic) solutions respond sensitively to changes in the size and aspect ratio of the chambers. In small chambers (dy ≤ 20 μm), filaments align parallel to the long walls as soon as the aspect ratio is ≥1.5, indicating that surface-induced ordering dominates. In larger chambers, the filaments instead align along the chamber diagonal, indicating that bulk packing constraints dominate. The nematic order parameter is maximal in small and highly anisometric chambers. In contrast to the nematic solutions, low-density (isotropic) solutions are rather insensitive to confinement. Bundled actin solutions behave similarly to nematic solutions, but are less well-ordered. Our observations imply that the orientational order of actin filaments in flat confining geometries is primarily determined by a balance between bulk and surface packing constraints with a minimal effect of the enthalpic cost of filament bending. Our assay provides an interesting platform for the future reconstitution of more complex, active cytoskeletal systems with actively treadmilling filaments or molecular motors.

Cell-Sized confinement in microspheres accelerates the reaction of gene expression

Cell-sized water-in-oil droplet covered by a lipid layer was used to understand how lipid membranes affect biochemical systems in living cells. Here, we report a remarkable acceleration of gene expression in a cell-sized water-in-oil droplet entrapping a cell-free translation system to synthesize GFP (green fluorescent protein). The production rate of GFP (VGFP) in each droplet remained almost constant at least for on the order of a day, which implies 0th-order reaction kinetics. Interestingly, VGFP was inversely proportional to radius of droplets (R) when R is under 50 μm, and VGFP in droplets with R ∼ 10 μm was more than 10 times higher than that in the bulk. The acceleration rates of GFP production in cell-sized droplets strongly depended on the lipid types. These results demonstrate that the membrane surface has the significant effect to facilitate protein production, especially when the scale of confinement is on the order of cell-size.

Self-organized patterns of actin filaments in cell-sized confinement

Cells use actin filaments to define and maintain their shape and to exert forces on the surrounding tissue. Accessory proteins like crosslinkers and motors organize these filaments into functional structures. However, physical effects also influence filament organization: steric interactions impose packing constraints at high filament density and spatially confine the filaments within the cell boundaries. Here we investigate the combined effects of packing constraints and spatial confinement by growing dense actin networks in cell-sized microchambers with nonadhesive walls. We show that the filaments spontaneously form dense, bundle-like structures above a threshold concentration of 1 mg ml−1, in contrast to unconfined networks, which are homogeneous and undergo a bulk isotropic-to-nematic phase transition above 5 mg ml−1. Bundling requires quasi-2D confinement in chambers with a depth comparable to the mean filament length (6 μm). The bundles curve along the walls and central bundles align along the chamber diagonal or, in elongated chambers, along the long axis. We propose that bundling is a result of the polydisperse length distribution of the filaments: filaments shorter than the chamber depth introduce an entropic depletion attraction between the longest filaments, which are confined in-plane. Bundle alignment reflects a competition between bulk liquid-crystalline ordering and alignment along the boundaries. This physical mechanism may influence intracellular organization of actin in combination with biochemical regulation and actin–membrane adhesion.