Emergence of uniform linearly-arranged micro-droplets entrapping DNA and living cells through water/water phase-separation.

Living cells maintain their lives through self-organization in an environment crowded with a rich variety of biological species. Recently, it was found that micro-droplets containing biomacromolecules, which vary widely in size, are generated accompanied by water/water phase-separation by simple mechanical mixing of an aqueous solution with binary polymers. Here, we report that cell-sized droplets of nearly the same size are generated as a linear array within a glass capillary upon the introduction of a binary polymer solution of polyethylene glycol (PEG) and dextran (DEX). Interestingly, when DNA molecules are added to the polymer solution, stable droplets entrapping DNA molecules are obtained. Similarly, living cells are entrapped spontaneously for the linearly-arranged cell-sized droplets. This simple method for generating micro-droplets entrapping DNA and also living cells is expected to stimulate further study on the self-construction of protocells and micro organoids.

Magnetic cilia carpets with programmable metachronal waves.

Metachronal waves commonly exist in natural cilia carpets. These emergent phenomena, which originate from phase differences between neighbouring self-beating cilia, are essential for biological transport processes including locomotion, liquid pumping, feeding, and cell delivery. However, studies of such complex active systems are limited, particularly from the experimental side. Here we report magnetically actuated, soft, artificial cilia carpets. By stretching and folding onto curved templates, programmable magnetization patterns can be encoded into artificial cilia carpets, which exhibit metachronal waves in dynamic magnetic fields. We have tested both the transport capabilities in a fluid environment and the locomotion capabilities on a solid surface. This robotic system provides a highly customizable experimental platform that not only assists in understanding fundamental rules of natural cilia carpets, but also paves a path to cilia-inspired soft robots for future biomedical applications.

Compartments for Synthetic Cells: Osmotically Assisted Separation of Oil from Double Emulsions in a Microfluidic Chip.

Liposomes are used in synthetic biology as cell-like compartments and their microfluidic production through double emulsions allows for efficient encapsulation of various components. However, residual oil in the membrane remains a critical bottleneck for creating pristine phospholipid bilayers. It has been discovered that osmotically driven shrinking leads to detachment of the oil drop. Separation inside a microfluidic chip has been realized to automate the procedure, which allows for controlled continuous production of monodisperse liposomes.

Integrated approaches to unravel the impact of protein lipoxidation on macromolecular interactions.

Protein modification by lipid derived reactive species, or lipoxidation, is increased during oxidative stress, a common feature observed in many pathological conditions. Biochemical and functional consequences of lipoxidation include changes in the conformation and assembly of the target proteins, altered recognition of ligands and/or cofactors, changes in the interactions with DNA or in protein-protein interactions, modifications in membrane partitioning and binding and/or subcellular localization. These changes may impact, directly or indirectly, signaling pathways involved in the activation of cell defense mechanisms, but when these are overwhelmed they may lead to pathological outcomes. Mass spectrometry provides state of the art approaches for the identification and characterization of lipoxidized proteins/residues and the modifying species. Nevertheless, understanding the complexity of the functional effects of protein lipoxidation requires the use of additional methodologies. Herein, biochemical and biophysical methods used to detect and measure functional effects of protein lipoxidation at different levels of complexity, from in vitro and reconstituted cell-like systems to cells, are reviewed, focusing especially on macromolecular interactions. Knowledge generated through innovative and complementary technologies will contribute to comprehend the role of lipoxidation in pathophysiology and, ultimately, its potential as target for therapeutic intervention.

More from less - bottom-up reconstitution of cell biology.

The ultimate goal of bottom-up synthetic biology is recreating life in its simplest form. However, in its quest to find the minimal functional units of life, this field contributes more than its main aim by also offering a range of tools for asking, and experimentally approaching, biological questions. This Review focusses on how bottom-up reconstitution has furthered our understanding of cell biology. Studying cell biological processes in vitro has a long tradition, but only recent technological advances have enabled researchers to reconstitute increasingly complex biomolecular systems by controlling their multi-component composition and their spatiotemporal arrangements. We illustrate this progress using the example of cytoskeletal processes. Our understanding of these has been greatly enhanced by reconstitution experiments, from the first in vitro experiments 70 years ago to recent work on minimal cytoskeleton systems (including this Special Issue of Journal of Cell Science). Importantly, reconstitution approaches are not limited to the cytoskeleton field. Thus, we also discuss progress in other areas, such as the shaping of biomembranes and cellular signalling, and prompt the reader to add their subfield of cell biology to this list in the future.

Biologically Inspired Design of Nanoparticle Artificial Antigen-Presenting Cells for Immunomodulation.

Particles engineered to engage and interact with cell surface ligands and to modulate cells can be harnessed to explore basic biological questions as well as to devise cellular therapies. Biology has inspired the design of these particles, such as artificial antigen-presenting cells (aAPCs) for use in immunotherapy. While much has been learned about mimicking antigen presenting cell biology, as we decrease the size of aAPCs to the nanometer scale, we need to extend biomimetic design to include considerations of T cell biology-including T-cell receptor (TCR) organization. Here we describe the first quantitative analysis of particle size effect on aAPCs with both Signals 1 and 2 based on T cell biology. We show that aAPCs, larger than 300 nm, activate T cells more efficiently than smaller aAPCs, 50 nm. The 50 nm aAPCs require saturating doses or require artificial magnetic clustering to activate T cells. Increasing ligand density alone on the 50 nm aAPCs did not increase their ability to stimulate CD8+ T cells, confirming the size-dependent phenomenon. These data support the need for multireceptor ligation and activation of T-cell receptor (TCR) nanoclusters of similar sizes to 300 nm aAPCs. Quantitative analysis and modeling of a nanoparticle system provides insight into engineering constraints of aAPCs for T cell immunotherapy applications and offers a case study for other cell-modulating particles.

A novel approach for targeted delivery to motoneurons using cholera toxin-B modified protocells.

BACKGROUND: Trophic interactions between muscle fibers and motoneurons at the neuromuscular junction (NMJ) play a critical role in determining motor function throughout development, ageing, injury, or disease. Treatment of neuromuscular disorders is hindered by the inability to selectively target motoneurons with pharmacological and genetic interventions. NEW METHOD: We describe a novel delivery system to motoneurons using mesoporous silica nanoparticles encapsulated within a lipid bilayer (protocells) and modified with the atoxic subunit B of the cholera toxin (CTB) that binds to gangliosides present on neuronal membranes. RESULTS: CTB modified protocells showed significantly greater motoneuron uptake compared to unmodified protocells after 24h of treatment (60% vs. 15%, respectively). CTB-protocells showed specific uptake by motoneurons compared to muscle cells and demonstrated cargo release of a surrogate drug. Protocells showed a lack of cytotoxicity and unimpaired cellular proliferation. In isolated diaphragm muscle-phrenic nerve preparations, preferential axon terminal uptake of CTB-modified protocells was observed compared to uptake in surrounding muscle tissue. A larger proportion of axon terminals displayed uptake following treatment with CTB-protocells compared to unmodified protocells (40% vs. 6%, respectively). COMPARISON WITH EXISTING METHOD(S): Current motoneuron targeting strategies lack the functionality to load and deliver multiple cargos. CTB-protocells capitalizes on the advantages of liposomes and mesoporous silica nanoparticles allowing a large loading capacity and cargo release. The ability of CTB-protocells to target motoneurons at the NMJ confers a great advantage over existing methods. CONCLUSIONS: CTB-protocells constitute a viable targeted motoneuron delivery system for drugs and genes facilitating various therapies for neuromuscular diseases.

Self-Assembly of Bilayer Vesicles Made of Saturated Long Chain Fatty Acids.

Saturated long chain fatty acids (sLCFA, e.g., C14:0, C16:0, and C18:0) are potentially the greenest and cheapest surfactants naturally available. However, because aqueous sodium soaps of sLCFA are known to crystallize, the self-assembly of stable bilayer vesicles has not been reported yet. Here, by using such soaps in combination with guanidine hydrochloride (GuHCl), which has been shown recently to prevent crystallization, we were capable of producing stable bilayer vesicles made of sLCFA. The phase diagrams were established for a variety of systems showing that vesicles can form in a broad range of composition and pH. Both solid state NMR and small-angle neutron scattering allowed demonstrating that in such vesicles sLCFA are arranged in a bilayer structure which exhibits similar dynamic and structural properties as those of phospholipid membranes. We expect these vesicles to be of interest as model systems of protocells and minimal cells but also for various applications since fatty acids are potentially substitutes to phospholipids, synthetic surfactants, and polymers.

Synthetic biology. Programmable on-chip DNA compartments as artificial cells.

The assembly of artificial cells capable of executing synthetic DNA programs has been an important goal for basic research and biotechnology. We assembled two-dimensional DNA compartments fabricated in silicon as artificial cells capable of metabolism, programmable protein synthesis, and communication. Metabolism is maintained by continuous diffusion of nutrients and products through a thin capillary, connecting protein synthesis in the DNA compartment with the environment. We programmed protein expression cycles, autoregulated protein levels, and a signaling expression gradient, equivalent to a morphogen, in an array of interconnected compartments at the scale of an embryo. Gene expression in the DNA compartment reveals a rich, dynamic system that is controlled by geometry, offering a means for studying biological networks outside a living cell.

Reconstitution of cytoskeletal protein assemblies for large-scale membrane transformation.

Membranes determine two-dimensional and three-dimensional biochemical reaction spaces in living systems. Defining size and shape of surfaces and volumes encompassed by membrane is of key importance for cellular metabolism and homeostasis, and the maintenance and controlled transformation of membrane shapes are coordinated by a large number of different protein assemblies. The orchestration of spatial elements over distances orders of magnitudes larger than protein molecules, as required for cell division, is a particularly challenging task, requiring large-scale ordered protein filaments and networks. The structure and function of these networks, particularly of cytoskeletal elements, have been characterized extensively in cells and reconstituted systems. However, their co-reconstitution with membranes from the bottom-up under defined conditions, to elucidate their mode of action in detail, is still a relatively new field of research. In this short review, we discuss recent approaches and achievements with regard to the study of cytoskeletal protein assemblies on model membranes, with specific focus on contractile elements as those based on the bacterial division FtsZ protein and eukaryotic actomyosin structures.

Positive roles of compartmentalization in internal reactions.

Recently, many researchers have attempted to construct artificial cell models using a bottom-up approach in which various biochemical reactions that involve a defined set of molecules are reconstructed in cell-like compartments, such as liposomes and water-in-oil droplets. In many of these studies, the cell-like compartments have acted only as containers for the encapsulated biochemical reactions, whereas other studies have indicated that compartmentalization improves the rates and yields of these reactions. Here, we introduce two ways in which compartmentalization can improve internal reactions: the isolation effect and the condensation effect. These positive effects of compartmentalization might have played an important role in the genesis of the first primitive cell on early Earth.

Synthetic cellularity based on non-lipid micro-compartments and protocell models.

This review discusses recent advances in the design and construction of protocell models based on the self-assembly or microphase separation of non-lipid building blocks. We focus on strategies involving partially hydrophobic inorganic nanoparticles (colloidosomes), protein-polymer globular nano-conjugates (proteinosomes), amphiphilic block copolymers (polymersomes), and stoichiometric mixtures of oppositely charged biomolecules and polyelectrolytes (coacervates). Developments in the engineering of membrane functionality to produce synthetic protocells with gated responses and control over multi-step reactions are described. New routes to protocells comprising molecularly crowded, cytoskeletal-like hydrogel interiors, as well as to the construction of hybrid protocell models are also highlighted. Together, these strategies enable a wide range of biomolecular and synthetic components to be encapsulated, regulated and processed within the micro-compartmentalized volume, and suggest that the development of non-lipid micro-ensembles offers an approach that is complementary to protocell models based on phospholipid or fatty acid vesicles.

Emergence of photoautotrophic minimal protocell-like supramolecular assemblies, "Jeewanu" synthesied photo chemically in an irradiated sterilised aqueous mixture of some inorganic and organic substances.

Sunlight exposed sterilised aqueous mixture of ammonium molybdate, diammonium hydrogen phosphate, biological minerals and formaldehyde showed photochemical formation of self-sustaining biomimetic protocell-like supramolecular assemblies "Jeewanu" (Bahadur and Ranganayaki J Brit Interplanet Soc 23:813-829 1970). The structural and functional characteristics of Jeewanu suggests that in possible prebiotic atmosphere photosy nergistic collaboration of non-linear processes at mesoscopic level established autocatalytic pathways on mineral surfaces by selforganisation and self recognition and led to emergence of similar earliest energy transducing supramolecular assemblies which might have given rise to common universal ancestor on the earth or elsewhere.

Specific transformation of assembly with actin filaments and molecular motors in a cell-sized self-emerged liposome.

Eukaryotes, by the same combination of cytoskeleton and molecular motor, for example actin filament and myosin, can generate a variety of movements. For this diversity, the organization of biological machineries caused by the confinement and/or crowding effects of internal living cells, may play very important roles.

Protocell design through modular compartmentalization.

De novo synthetic biological design has the potential to significantly impact upon applications such as energy generation and nanofabrication. Current designs for constructing organisms from component parts are typically limited in scope, as they utilize a cut-and-paste ideology to create simple stepwise engineered protein-signalling pathways. We propose the addition of a new design element that segregates components into lipid-bound 'proto-organelles', which are interfaced with response elements and housed within a synthetic protocell. This design is inspired by living cells, which utilize multiple types of signalling molecules to facilitate communication between isolated compartments. This paper presents our design and validation of the components required for a simple multi-compartment protocell machine, for coupling a light transducer to a gene expression system. This represents a general design concept for the compartmentalization of different types of artificial cellular machinery and the utilization of non-protein signal molecules for signal transduction.

Interfacial assembly of protein-polymer nano-conjugates into stimulus-responsive biomimetic protocells.

The mechanism of spontaneous assembly of microscale compartments is a central question for the origin of life, and has technological repercussions in diverse areas such as materials science, catalysis, biotechnology and biomedicine. Such compartments need to be semi-permeable, structurally robust and capable of housing assemblages of functional components for internalized chemical transformations. In principle, proteins should be ideal building blocks for the construction of membrane-bound compartments but protein vesicles with cell-like properties are extremely rare. Here we present an approach to the interfacial assembly of protein-based micro-compartments (proteinosomes) that are delineated by a semi-permeable, stimulus-responsive, enzymatically active, elastic membrane consisting of a closely packed monolayer of conjugated protein-polymer building blocks. The proteinosomes can be dispersed in oil or water, thermally cycled to temperatures of 70 °C, and partially dried and re-inflated without loss of structural integrity. As a consequence, they exhibit protocellular properties such as guest molecule encapsulation, selective permeability, gene-directed protein synthesis and membrane-gated internalized enzyme catalysis.

Jeewanu, or the 'particles of life'. The approach of Krishna Bahadur in 20th century origin of life research.

Starting in the 1960s, the Indian chemist Krishna Bahadur, from the University of Allahabad, published on organic and inorganic particles that he had synthesized and baptized 'Jeewanu', or 'particle of life'. Bahadur conceived of the Jeewanu as a simple form of the living. These studies are presented in a historical perspective and positioned within mid-20th century research on the origin of life, notably the so-called 'coacervate theory' of the Soviet biochemist Aleksandr I Oparin. The concepts of life proposed by Bahadur, Oparin and others are discussed from a historical standpoint.

Novel platelet substitutes: disk-shaped biodegradable nanosheets and their enhanced effects on platelet aggregation.

We have studied biocompatible spherical carriers carrying a dodecapeptide, HHLGGAKQAGDV (H12), on their surface as platelet substitutes. This peptide is a fibrinogen γ-chain carboxy-terminal sequence (γ400-411) and specifically recognizes the active form of glycoprotein IIb/IIIa on activated platelets. Our purpose is to assess the possibility of making a novel platelet substitute consisting of disk-shaped nanosheets having a large contact area for the targeting site, rather than conventional small contact area spherical carriers. The H12 peptide was conjugated to the surface of the free-standing nanosheets made of biodegradable poly(d,l-lactide-co-glycolide) (PLGA). These H12-PLGA nanosheets were fabricated onto 3 µm disk-shaped patterned hydrophobic octadecyl regions on a SiO(2) substrate. By way of comparison, spherical H12-PLGA microparticles with the same surface area and conjugation number of H12 were also prepared. The resulting H12-PLGA nanosheets specifically interacted with the activated platelets adhered on the collagen surface at twice the rate of the H12-PLGA microparticles under flow conditions, and showed platelet thrombus formation in a two-dimensional spreading manner. Thus, H12-PLGA nanosheets might be a suitable candidate novel platelet alternative substitute for infused human platelet concentrates for the treatment of bleeding in patients with severe thrombocytopenia.