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.

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.

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.

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.

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.

BLNCR is a long non-coding RNA adjacent to integrin beta-1 that is rapidly lost during epidermal progenitor cell differentiation.

As our understanding of transcriptional regulation improves so does our appreciation of its complexity. Both coding and (long) non-coding RNAs provide cells with multiple levels of control and thereby flexibility to adapt gene expression to the environment. However, few long non-coding RNAs (lncRNAs) have been studied in human epidermal stem cells. Here, we characterized the expression of 26 lncRNAs in human epidermal keratinocytes, 7 of which we found to be dynamically expressed during differentiation. We performed in depth analysis of a lncRNA located proximal to the epidermal stem cell marker integrin beta-1 (ITGB1) and transcribed in the opposite direction. We dubbed this gene Beta1-adjacent long non-coding RNA, or BLNCR, and found that its expression is regulated by p63 and AP1 transcription factors. Furthermore, BLNCR expression is regulated downstream the integrin and EGF signaling pathways that are key to epidermal stem cell maintenance. Finally, we found that BLNCR expression is rapidly reduced upon induction of differentiation, preceding the down regulation of integrin beta-1 expression. These dynamics closely mirror the loss of proliferative and adhesion capacity of epidermal stem cells in colony formation assays. Together, these results suggest that loss of BLNCR expression marks the switch from a proliferative state towards terminal differentiation in human epidermal stem cells.