Colony-like Protocell Superstructures.

We report the formation, growth, and dynamics of model protocell superstructures on solid surfaces, resembling single cell colonies. These structures, consisting of several layers of lipidic compartments enveloped in a dome-shaped outer lipid bilayer, emerged as a result of spontaneous shape transformation of lipid agglomerates deposited on thin film aluminum surfaces. Collective protocell structures were observed to be mechanically more stable compared to isolated spherical compartments. We show that the model colonies encapsulate DNA and accommodate nonenzymatic, strand displacement DNA reactions. The membrane envelope is able to disassemble and expose individual daughter protocells, which can migrate and attach via nanotethers to distant surface locations, while maintaining their encapsulated contents. Some colonies feature "exocompartments", which spontaneously extend out of the enveloping bilayer, internalize DNA, and merge again with the superstructure. A continuum elastohydrodynamic theory that we developed suggests that a plausible driving force behind subcompartment formation is attractive van der Waals (vdW) interactions between the membrane and surface. The balance between membrane bending and vdW interactions yields a critical length scale of 236 nm, above which the membrane invaginations can form subcompartments. The findings support our hypotheses that in extension of the "lipid world hypothesis", protocells may have existed in the form of colonies, potentially benefiting from the increased mechanical stability provided by a superstructure.

Transport among protocells via tunneling nanotubes.

We employ model protocell networks for evaluation of molecular transport through lipid nanotubes as potential means of communication among primitive cells on the early Earth. Network formation is initiated by deposition of lipid reservoirs onto a SiO2 surface in an aqueous environment. These reservoirs autonomously develop into surface-adhered protocells interconnected via lipid nanotubes while encapsulating solutes from the ambient buffer. We observe the uptake of DNA and RNA, and their diffusive transport between the lipid compartments via the interconnecting nanotubes. By means of an analytical model we determine key physical parameters affecting the transport, such as nanotube diameter and compartment size. We conclude that nanotube-mediated transport could have been a possible pathway of communication between primitive cells on the early Earth, circumventing the necessity for crossing the membrane barrier. We suggest this transport as a feasible means of RNA and DNA exchange under primitive prebiotic conditions, possibly facilitating early replication.

Protocells: Milestones and Recent Advances.

The origin of life is still one of humankind's great mysteries. At the transition between nonliving and living matter, protocells, initially featureless aggregates of abiotic matter, gain the structure and functions necessary to fulfill the criteria of life. Research addressing protocells as a central element in this transition is diverse and increasingly interdisciplinary. The authors review current protocell concepts and research directions, address milestones, challenges and existing hypotheses in the context of conditions on the early Earth, and provide a concise overview of current protocell research methods.

Manipulation of Lipid Membranes with Thermal Stimuli.

We describe a protocol for the assembly and application of infrared (IR-B) laser-based set-ups to be used for localized heating of solid-supported planar and vesicular lipid membrane assemblies.

Mixed fatty acid-phospholipid protocell networks.

Self-assembled membranes composed of both fatty acids and phospholipids are permeable for solutes and structurally stable, which was likely an advantageous combination for the development of primitive cells on the early Earth. Here we report on the solid surface-assisted formation of primitive mixed-surfactant membrane compartments, i.e. model protocells, from multilamellar lipid reservoirs composed of different ratios of fatty acids and phospholipids. Similar to the previously discovered enhancement of model protocell formation on solid substrates, we achieve spontaneous multi-step self-transformation of mixed surfactant reservoirs into closed surfactant containers, interconnected via nanotube networks. Some of the fatty acid-containing compartments in the networks exhibit colony-like growth. We demonstrate that the compartments generated from fatty acid-containing phospholipid membranes feature increased permeability coefficients for molecules in the ambient solution, for fluorescein up to 7 × 10-6 cm s-1 and for RNA up to 3.5 × 10-6 cm s-1. Our findings indicate that surface-assisted autonomous protocell formation and development, starting from mixed amphiphiles, is a plausible scenario for the early stages of the emergence of primitive cells.

Did Solid Surfaces Enable the Origin of Life?

In this perspective article, I discuss whether and how solid surfaces could have played a key role in the formation of membranous primitive cells on the early Earth. I argue why surface energy could have been used by prebiotic amphiphile assemblies for unique morphological transformations, and present recent experimental findings showing the surface-dependent formation and behavior of sophisticated lipid membrane structures. Finally, I discuss the possible unique contributions of such surface-adhered architectures to the transition from prebiotic matter to living systems.

Subcompartmentalization and Pseudo-Division of Model Protocells.

Membrane enclosed intracellular compartments have been exclusively associated with the eukaryotes, represented by the highly compartmentalized last eukaryotic common ancestor. Recent evidence showing the presence of membranous compartments with specific functions in archaea and bacteria makes it conceivable that the last universal common ancestor and its hypothetical precursor, the protocell, may have exhibited compartmentalization. To the authors' knowledge, there are no experimental studies yet that have tested this hypothesis. They report on an autonomous subcompartmentalization mechanism for protocells which results in the transformation of initial subcompartments to daughter protocells. The process is solely determined by the fundamental materials properties and interfacial events, and does not require biological machinery or chemical energy supply. In the light of the authors' findings, it is proposed that similar events may have taken place under early Earth conditions, leading to the development of compartmentalized cells and potentially, primitive division.

Biological lipid nanotubes and their potential role in evolution.

The membrane of cells and organelles are highly deformable fluid interfaces, and can take on a multitude of shapes. One distinctive and particularly interesting property of biological membranes is their ability to from long and uniform nanotubes. These nanoconduits are surprisingly omnipresent in all domains of life, from archaea, bacteria, to plants and mammals. Some of these tubes have been known for a century, while others were only recently discovered. Their designations are different in different branches of biology, e.g. they are called stromule in plants and tunneling nanotubes in mammals. The mechanical transformation of flat membranes to tubes involves typically a combination of membrane anchoring and external forces, leading to a pulling action that results in very rapid membrane nanotube formation - micrometer long tubes can form in a matter of seconds. Their radius is set by a mechanical balance of tension and bending forces. There also exists a large class of membrane nanotubes that form due to curvature inducing molecules. It seems plausible that nanotube formation and functionality in plants and animals may have been inherited from their bacterial ancestors during endosymbiotic evolution. Here we attempt to connect observations of nanotubes in different branches of biology, and outline their similarities and differences with the aim of providing a perspective on their joint functions and evolutionary origin.

Rapid Growth and Fusion of Protocells in Surface-Adhered Membrane Networks.

Elevated temperatures might have promoted the nucleation, growth, and replication of protocells on the early Earth. Recent reports have shown evidence that moderately high temperatures not only permit protocell assembly at the origin of life, but can have actively supported it. Here, the fast nucleation and growth of vesicular compartments from autonomously formed lipid networks on solid surfaces, induced by a moderate increase in temperature, are shown. Branches of the networks, initially consisting of self-assembled interconnected nanotubes, rapidly swell into microcompartments which can spontaneously encapsulate RNA fragments. The increase in temperature further causes fusion of adjacent network-connected compartments, resulting in the redistribution of the RNA. The experimental observations and the mathematical model indicate that the presence of nanotubular interconnections between protocells facilitates the fusion process.

Microfluidic technology for investigation of protein function in single adherent cells.

Instrumental techniques and associated methods for single cell analysis, designed to investigate and measure a broad range of cellular parameters in search of unique features, address key limitations of conventional cell-based assays with their ensemble average response. While many different single cell techniques exist for suspension cultures, which can process and characterize large numbers of individual cells in rapid succession, the access to surface-immobilized cells in typical 2D and 3D culture environments remains challenging. Open space microfluidics has created new possibilities in this area, allowing for exclusive access to single cells in adherent cultures, even at high confluency. In this chapter, we briefly review new microtechnologies for the investigation of protein function in single adherent cells, and present an overview over related recent applications of the multifunctional pipette (Biopen), a microfluidic multi-solution dispensing system that uses hydrodynamic confinement in open volume environments in order to establish a superfusion zone over selected single cells in adherent cultures.

Styrene maleic acid copolymer induces pores in biomembranes.

We investigated the interactions between styrene-maleic acid (SMA) copolymers and phospholipid bilayers, using confocal microscopy and surface acoustic wave resonance (SAR) sensing. For the first time we experimentally observed and followed pore formation by SMA copolymers in intact supported bilayers and unilamellar vesicles, showing that fluorescein, a water-soluble organic compound with a mean diameter of 6.9 Å, can traverse the membrane. Our findings are in agreement with recent theoretical predictions, which suggested that SMA copolymers may insert along the plane of the bilayer to form stable toroidal pores.

A Hypothesis for Protocell Division on the Early Earth.

I hypothesize that the division of the first protocell might have occurred before genetic polymers were synthesized and redistributed. In the light of recent findings, it is conceivable that the first division event of a primitive protocell might have occurred at the same time as its surface-assisted formation.

Molecular Lipid Films on Microengineering Materials.

In this study, we have systematically investigated the formation of molecular phospholipid films on a variety of solid substrates fabricated from typical surface engineering materials and the fluidic properties of the lipid membranes formed on these substrates. The surface materials comprise of borosilicate glass, mica, SiO2, Al (native oxide), Al2O3, TiO2, ITO, SiC, Au, Teflon AF, SU-8, and graphene. We deposited the lipid films from small unilamellar vesicles (SUVs) by means of an open-space microfluidic device, observed the formation and development of the films by laser scanning confocal microscopy, and evaluated the mode and degree of coverage, fluidity, and integrity. In addition to previously established mechanisms of lipid membrane-surface interaction upon bulk addition of SUVs on solid supports, we observed nontrivial lipid adhesion phenomena, including reverse rolling of spreading bilayers, spontaneous nucleation and growth of multilamellar vesicles, and the formation of intact circular patches of double lipid bilayer membranes. Our findings allow for accurate prediction of membrane-surface interactions in microfabricated devices and experimental environments where model membranes are used as functional biomimetic coatings.

Nanotube-Mediated Path to Protocell Formation.

Cellular compartments are membrane-enclosed, spatially distinct microenvironments that confine and protect biochemical reactions in the biological cell. On the early Earth, the autonomous formation of compartments is thought to have led to the encapsulation of nucleotides, thereby satisfying a starting condition for the emergence of life. Recently, surfaces have come into focus as potential platforms for the self-assembly of prebiotic compartments, as significantly enhanced vesicle formation was reported in the presence of solid interfaces. The detailed mechanism of such formation at the mesoscale is still under discussion. We report here on the spontaneous transformation of solid-surface-adhered lipid deposits to unilamellar membrane compartments through a straightforward sequence of topological changes, proceeding via a network of interconnected lipid nanotubes. We show that this transformation is entirely driven by surface-free energy minimization and does not require hydrolysis of organic molecules or external stimuli such as electrical currents or mechanical agitation. The vesicular structures take up and encapsulate their external environment during formation and can subsequently separate and migrate upon exposure to hydrodynamic flow. This may link the self-directed transition from weakly organized bioamphiphile assemblies on solid surfaces to protocells with secluded internal contents.

A cellular automaton for modeling non-trivial biomembrane ruptures.

A novel cellular automaton (CA) for simulating biological membrane rupture is proposed. Constructed via simple rules governing deformation, tension, and fracture, the CA incorporates ideas from standard percolation models and bond-based fracture methods. The model is demonstrated by comparing simulations with experimental results of a double bilayer lipid membrane expanding on a solid substrate. Results indicate that the CA can capture non-trivial rupture morphologies such as floral patterns and the saltatory dynamics of fractal avalanches observed in experiments. Moreover, the CA provides insight into the poorly understood role of inter-layer adhesion, supporting the hypothesis that the density of adhesion sites governs rupture morphology.

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum.

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al2O3 substrate. The adhesion is mediated by Ca2+ in the ambient buffer. Subsequent depletion of Ca2+ by means of BAPTA/EDTA causes retraction of the membrane, resulting in spontaneous lipid nanotube network formation. The method only comprises phospholipids and microfabricated surfaces for simple formation of an ER model and does not require the addition of proteins or chemical energy (e.g., GTP or ATP). In contrast to the 3D morphology of the cellular endoplasmic reticulum, the model is two-dimensional (albeit the nanotube dimensions, geometry, structure, and dynamics are maintained). This unique in vitro ER model consists of only a few components, is easy to construct, and can be observed under a light microscope. The resulting structure can be further decorated for additional functionality, such as the addition of ER-associated proteins or particles to study transport phenomena among the tubes. The artificial networks described here are suitable structural models for the cellular ER, whose unique characteristic morphology has been shown to be related to its biological function, whereas details regarding formation of the tubular domain and rearrangements within are still not completely understood. We note that this method uses Al2O3 thin-film-coated microscopy coverslips, which are commercially available but require special orders. Therefore, it is advisable to have access to a microfabrication facility for preparation.

Formation and dynamics of endoplasmic reticulum-like lipid nanotube networks.

We report on the self-organized formation and dynamics of artificial lipid nanotube networks, which, in terms of morphology and behavior, resemble the endoplasmic reticulum(ER) of biological cells. The networks, initially generated from a solid-supported planar phospholipid membrane, undergo a morphological transformation, triggered by the chelation and removal of Ca2+ from the environment surrounding the membrane. Calcium depletion gradually causes de-pinning, thus de-wetting, at the membrane-substrate interface. We observe dynamic re-arrangements very similar to the ones reported for the cellular ER, such as sliding of tubes and formation of new junctions, and quantify these transformations. We also show occurrences of the dynamic replacement of lipidic particles on nanotubes as indicators for the existence of a tension gradient throughout the network, as well as the spontaneous formation of small vesicles from semi-free floating tubes. We propose that these artificial networks are suitable to serve as a bottom-up-generated structural model for the cellular ER, whose fascinating characteristic morphology is suggested to be tied to its biological function, but with respect to formation, dynamics, and functional details still incompletely understood.

Peridynamic Modeling of Ruptures in Biomembranes.

We simulate the formation of spontaneous ruptures in supported phospholipid double bilayer membranes, using peridynamic modeling. Experiments performed on spreading double bilayers typically show two distinct kinds of ruptures, floral and fractal, which form spontaneously in the distal (upper) bilayer at late stages of double bilayer formation on high energy substrates. It is, however, currently unresolved which factors govern the occurrence of either rupture type. Variations in the distance between the two bilayers, and the occurrence of interconnections ("pinning sites") are suspected of contributing to the process. Our new simulations indicate that the pinned regions which form, presumably due to Ca2+ ions serving as bridging agent between the distal and the proximal bilayer, act as nucleation sites for the ruptures. Moreover, assuming that the pinning sites cause a non-zero shear modulus, our simulations also show that they change the rupture mode from floral to fractal. At zero shear modulus the pores appear to be circular, subsequently evolving into floral pores. With increasing shear modulus the pore edges start to branch, favoring fractal morphologies. We conclude that the pinning sites may indirectly determine the rupture morphology by contributing to shear stress in the distal membrane.

Deformation of a single mouse oocyte in a constricted microfluidic channel.

Single oocyte manipulation in microfluidic channels via precisely controlled flow is critical in microfluidic-based in vitro fertilization. Such systems can potentially minimize the number of transfer steps among containers for rinsing as often performed during conventional in vitro fertilization and can standardize protocols by minimizing manual handling steps. To study shape deformation of oocytes under shear flow and its subsequent impact on their spindle structure is essential for designing microfluidics for in vitro fertilization. Here, we developed a simple yet powerful approach to (i) trap a single oocyte and induce its deformation through a constricted microfluidic channel, (ii) quantify oocyte deformation in real-time using a conventional microscope, and (iii) retrieve the oocyte from the microfluidic device to evaluate changes in their spindle structures. We found that oocytes can be significantly deformed under high flow rates, e.g., 10 µl/min in a constricted channel with a width and height of 50 and 150 µm, respectively. Oocyte spindles can be severely damaged, as shown here by immunocytochemistry staining of the microtubules and chromosomes. The present approach can be useful to investigate underlying mechanisms of oocyte deformation exposed to well-controlled shear stresses in microfluidic channels, which enables a broad range of applications for reproductive medicine.

Bio-inspired cryo-ink preserves red blood cell phenotype and function during nanoliter vitrification.

Current red-blood-cell cryopreservation methods utilize bulk volumes, causing cryo-injury of cells, which results in irreversible disruption of cell morphology, mechanics, and function. An innovative approach to preserve human red-blood-cell morphology, mechanics, and function following vitrification in nanoliter volumes is developed using a novel cryo-ink integrated with a bioprinting approach.