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Barbara Drašler,1 Damjana Drobne,1–3 Sara Novak,1 Janez Valant,1 Sabina Boljte,1,4 Lado Otrin,1 Michael Rappolt,5,6 Barbara Sartori,5 Aleš Iglic,7 Veronika Kralj-Iglic,8 Vid Šuštar,9 Darko Makovec,3,10 Sašo Gyergyek,10 Matej Hocevar,11 Matjaž Godec,11 Jernej Zupanc11University of Ljubljana, Biotechnical Faculty, Department of Biology, Ljubljana, 2Centre of Excellence in Advanced Materials and Technologies for the Future, Ljubljana, 3Centre of Excellence in Nanoscience and Nanotechnology, Ljubljana, 4Institute of Microbial Sciences and Technologies, Ljubljana, Slovenia; 5Institute of Inorganic Chemistry, Graz University of Technology, Basovizza, Italy; 6School of Food Science and Nutrition, University of Leeds, Leeds, UK; 7Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, 8Faculty of Health Sciences, Laboratory of Clinical Biophysics, University of Ljubljana, Ljubljana 9Laboratory of Clinical Biophysics, Chair of Orthopaedics, Faculty of Medicine, University of Ljubljana, Ljubljana, 10Institute Jožef Stefan, Ljubljana, 11Institute of Metals and Technology, Ljubljana, SloveniaBackground: The purpose of this work is to provide experimental evidence on the interactions of suspended nanoparticles with artificial or biological membranes and to assess the possibility of suspended nanoparticles interacting with the lipid component of biological membranes.Methods: 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid vesicles and human red blood cells were incubated in suspensions of magnetic bare cobalt ferrite (CoFe2O4) or citric acid (CA)-adsorbed CoFe2O4 nanoparticles dispersed in phosphate-buffered saline and glucose solution. The stability of POPC giant unilamellar vesicles after incubation in the tested nanoparticle suspensions was assessed by phase-contrast light microscopy and analyzed with computer-aided imaging. Structural changes in the POPC multilamellar vesicles were assessed by small angle X-ray scattering, and the shape transformation of red blood cells after incubation in tested suspensions of nanoparticles was observed using scanning electron microscopy and sedimentation, agglutination, and hemolysis assays.Results: Artificial lipid membranes were disturbed more by CA-adsorbed CoFe2O4 nanoparticle suspensions than by bare CoFe2O4 nanoparticle suspensions. CA-adsorbed CoFe2O4-CA nanoparticles caused more significant shape transformation in red blood cells than bare CoFe2O4 nanoparticles.Conclusion: Consistent with their smaller sized agglomerates, CA-adsorbed CoFe2O4 nanoparticles demonstrate more pronounced effects on artificial and biological membranes. Larger agglomerates of nanoparticles were confirmed to be reactive against lipid membranes and thus not acceptable for use with red blood cells. This finding is significant with respect to the efficient and safe application of nanoparticles as medicinal agents.Keywords: CoFe2O4, nanoparticles, agglomerates, human red blood cells, lipid vesicles

Due to their augmented properties, biomimetic polymer/lipid hybrid compartments are a promising substitute for natural liposomes in multiple applications, but the protein-free fusion of those semisynthetic membranes is unexplored to date. Here, we study the charge-mediated fusion of hybrid vesicles composed of poly(dimethylsiloxane)-graft-poly(ethylene oxide) and different lipids and analyze the process by size distribution and the mixing of membrane species at μm and nano scales. Remarkably, the membrane mixing of oppositely charged hybrids surpasses by far the degree in liposomes, which we correlate with properties like membrane disorder, rigidity, and ability of amphiphiles for flip-flop. Furthermore, we employ the integration of two respiratory proteins as a functional content mixing assay for different membrane compositions. This reveals that fusion is also attainable with neutral and cationic hybrids and that the charge is not the sole determinant of the final adenosine triphosphate synthesis rate, substantiating the importance of reconstitution environment. Finally, we employ this fusion strategy for the delivery of membrane proteins to giant unilamellar vesicles as a way to automate the assembly of synthetic cells.

Employing concepts from physics, chemistry and bioengineering, 'learning-by-building' approaches are becoming increasingly popular in the life sciences, especially with researchers who are attempting to engineer cellular life from scratch. The SynCell2020/21 conference brought together researchers from different disciplines to highlight progress in this field, including areas where synthetic cells are having socioeconomic and technological impact. Conference participants also identified the challenges involved in designing, manipulating and creating synthetic cells with hierarchical organization and function. A key conclusion is the need to build an international and interdisciplinary research community through enhanced communication, resource-sharing, and educational initiatives., Competing Interests: OS, JD, EB, AB, AB, KJ, ZM, LO, TD, JS, JS, KA, BJ, MD, KG, DS, SA, MG, ML, AS, JS, GL No competing interests declared, (© 2021, Staufer et al.)

A variety of artificial cells springs from the functionalization of liposomes with proteins. However, these models suffer from low durability without repair and replenishment mechanisms, which can be partly addressed by replacing the lipids with polymers. Yet natural membranes are also dynamically remodeled in multiple cellular processes. Here, we show that synthetic amphiphile membranes also undergo fusion, mediated by the protein machinery for synaptic secretion. We integrated fusogenic SNAREs in polymer and hybrid vesicles and observed efficient membrane and content mixing. We determined bending rigidity and pore edge tension as key parameters for fusion and described its plausible progression through cryo-EM snapshots. These findings demonstrate that dynamic membrane phenomena can be reconstituted in synthetic materials, thereby providing new tools for the assembly of synthetic protocells., (© 2021. The Author(s).)

The bottom-up approach in synthetic biology aims to create molecular ensembles that reproduce the organization and functions of living organisms and strives to integrate them in a modular and hierarchical fashion toward the basic unit of life-the cell-and beyond. This young field stands on the shoulders of fundamental research in molecular biology and biochemistry, next to synthetic chemistry, and, augmented by an engineering framework, has seen tremendous progress in recent years thanks to multiple technological and scientific advancements. In this timely review of the research over the past decade, we focus on three essential features of living cells: the ability to self-reproduce via recursive cycles of growth and division, the harnessing of energy to drive cellular processes, and the assembly of metabolic pathways. In addition, we cover the increasing efforts to establish multicellular systems via different communication strategies and critically evaluate the potential applications.

A recombinant fragment of human κ-Casein, termed RL2, induces cell death of breast cancer cells; however, molecular mechanisms of RL2-mediated cell death have remained largely unknown. In the current study, we have decoded the molecular mechanism of the RL2-mediated cell death and found that RL2 acts via the induction of mitophagy. This was monitored by the loss of adenosine triphosphate production, LC3B-II generation, and upregulation of BNIP3 and BNIP3L/NIX, as well as phosphatase and tensin homolog-induced kinase 1. Moreover, we have analyzed the cross talk of this pathway with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis upon combinatorial treatment with RL2 and TRAIL. Strikingly, we found two opposite effects of this co-treatment. RL2 had inhibitory effects on TRAIL-induced cell death upon short-term co-stimulation. In particular, RL2 treatment blocked TRAIL-mediated caspase activation, cell viability loss, and apoptosis, which was mediated via the downregulation of the core proapoptotic regulators. Contrary to short-term co-treatment, upon long-term co-stimulation, RL2 sensitized the cells toward TRAIL-induced cell death; the latter observation provides the basis for the development of therapeutic approaches in breast cancer cells. Collectively, our findings have important implications for cancer therapy and reveal the molecular switches of the cross talk between RL2-induced mitophagy and TRAIL-mediated apoptosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wohlfromm, Richter, Otrin, Seyrek, Vidaković-Koch, Kuligina, Richter, Koval and Lavrik.)

Cytochrome bo 3 ubiquinol oxidase is a transmembrane protein, which oxidizes ubiquinone and reduces oxygen, while pumping protons. Apart from its combination with F 1 F o -ATPase to assemble a minimal ATP regeneration module, the utility of the proton pump can be extended to other applications in the context of synthetic cells such as transport, signaling, and control of enzymatic reactions. In parallel, polymers have been speculated to be phospholipid mimics with respect to their ability to self-assemble in compartments with increased stability. However, their usability as interfaces for complex membrane proteins has remained questionable. In the present work, we optimized a fusion/electroformation approach to reconstitute bo 3 oxidase in giant unilamellar vesicles made of PDMS- g -PEO and/or phosphatidylcholine (PC). This enabled optical access, while microfluidic trapping allowed for online analysis of individual vesicles. The tight polymer membranes and the inward oriented enzyme caused 1 pH unit difference in 30 min, with an initial rate of 0.35 pH·min -1 To understand the interplay in these composite systems, we studied the relevant mechanical and rheological membrane properties. Remarkably, the proton permeability of polymer/lipid hybrids decreased after protein insertion, while the latter also led to a 20% increase of the polymer diffusion coefficient in polymersomes. In addition, PDMS- g -PEO increased the activity lifetime and the resistance to free radicals. These advantageous properties may open diverse applications, ranging from cell-free biotechnology to biomedicine. Furthermore, the presented study serves as a comprehensive road map for studying the interactions between membrane proteins and synthetic membranes, which will be fundamental for the successful engineering of such hybrid systems., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)