1: DWI Leibniz Institute for Interactive Material, Germany; 2: Roy & Diana Vagelos Laboratories, UPEN, United States of America; 3: Institute of Computational Molecular Science, Temple University, United States of America; 4: Institute for Bioengineering of Catalonia (IBEC), Spain; 5: Institució Catalana de Reserca I Estudis Acançats (ICREA), Spain
In this talk, I will address the following question: How can artificial superselectivity be achieved at synthetic cell membrane mimics? This is important not only to expand the understanding of biological systems, but also to develop synthetic protocells with life-like functions. I will introduce our concept for superselectivity in synthetic cell membranes, which requires the integration of (i) specificity, (ii) multivalency (to enhance binding but maintain reversibility), (iii) 2D organization of receptors, and (iv) concepts of cooperativity in binding. To tackle this question, we have designed and synthesized new families of amphiphilic molecules, Janus dendrimers and comb polymers, that self-assemble into cell-mimetic vesicles. Although these molecules do not occur in nature, they closely mimic the thickness, flexibility, and lateral 2D organization of cell membranes. These properties are precisely encoded in the chemical structure, architecture, and topology of the membrane´s macromolecular building blocks. As an example, I will present our recent work where we discovered that the reactivity of sugar receptors towards lectins is enhanced by the 2D organization of sugars into nanoarrays (clustering) and raft-mimics (cooperativity) at the periphery of protocells. In addition, this talk will show how life-like functions such as endocytosis of live bacteria and viruses can be introduced and how this concept can be used to develop phagocytic synthetic cells.
1: Max Planck Institute for Medical Research, Germany; 2: Saarland University, Germany; 3: Biochemistry Center, Heidelberg University, Germany
Proteins represent biological macromolecules of extremely high interest, performing a huge array of functions. They can regulate processes inside the cells as catalyzing metabolic reactions, DNA replication and providing structure to cells. Very intriguing are specific peripheral proteins, namely fibroblast growth factors (FGFs) which participate in multiple signaling pathways. FGF2 is the prototype member of this family. It follows an unconventional secretory pathway while traversing the cell membrane and, beyond the functions of FGFs in normal cell growth and differentiation, plays critical roles under pathophysiological conditions1. In particular, it inhibits the apoptosis of tumoral cells by an autocrine secretion-signaling loop causing tumor cell resistance against conventional anti-cancer therapies. Therefore, understanding its translocation path is crucial to develop further research against tumor cell resistance. Here, we employ Atomic Force Microscopy (AFM) together with current measurement in microfluidic devices (e-µflu) to deepen the functional dynamic of pore formation. In particular, AFM is performed on supported lipid bilayers after FGF2 injection and is here used to visualize the oligomeric assembly in different shapes. Current measurements are instead performed on membranes spanning on µflu devices and reveal current traces which show transient pores and suggest, in conjunction with the AFM results, a trimodal assembly of the oligomers that explain the estimated different pore sizes. We further demonstrated, carrying experiments with mutated FGF2, how the protein binding and insertion depend on the interaction between certain amino acids and lipids as cholesterol and phosphoinositide.
1: Max Planck Institute for Medical Research, Germany; 2: Karlsruhe Institute of Technology, Germany
Toward the ambitious goal of manufacturing synthetic cells from the bottom up, various cellular components have already been reconstituted inside lipid vesicles. However, the deterministic positioning of these components inside the compartment has remained elusive. Here, by using two-photon 3D laser printing, 2D and 3D hydrogel architectures are manufactured inside preformed giant unilamellar lipid vesicles (GUVs) with high precision and nearly arbitrary shape (T. Abele, T. Messer, K. Jahnke, M. Hippler, M. Bastmeyer, M. Wegener, K. Göpfrich, Advanced Materials, 2021). The required water-soluble photoresist is brought into the GUVs by diffusion in a single mixing step. Crucially, femtosecond two-photon printing inside the compartment does not destroy the GUVs. Beyond this proof-of-principle demonstration, early functional architectures are realized. In particular, a transmembrane structure acting as a pore is 3D printed, thereby allowing for the transport of biological cargo, including DNA, into the synthetic compartment. We further developed DNA-based photoresists enabling printing and erasing of hydrogel structures inside GUVs (T. Walther, K. Jahnke, T. Abele, K. Göpfrich, Advanced Functional Materials, 2022). These experiments show that two-photon 3D laser microprinting can be an important addition to the existing toolbox of synthetic biology.
Max Planck Institute for Medical Research, Germany
In the pursuit to produce functioning synthetic cells from the bottom up, DNA nanotechnology has proven to be a powerful tool.
However, the crowded yet highly organized arrangement in living cells, bridging from the nano- to the micron-scale, remains challenging to recreate with DNA-based architectures. Here, laser microprinting is established to print and erase shape-controlled DNA hydrogels inside the confinement of water-in-oil droplets and giant unilamellar lipid vesicles (GUVs). The DNA-based photoresist consists of a photocleavable inactive DNA linker which interconnects Y-shaped DNA motifs when activated by local irradiation with a 405 nm laser. An alternative linker design allows to erase custom features from a preformed DNA hydrogel with feature sizes down to 1.38 µm.
The present work demonstrates that the DNA hydrogels can serve as an internal support to stabilize non-spherical GUV shapes. Overall, DNA-based photoresists for laser printing in confinement allow to build up architectures on the interior of synthetic cells with light, which diversifies the toolbox of bottom-up synthetic biology.
University of Groningen, the Netherlands
Living cells constantly consume energy to perform their dynamic processes and functions. To this end, intricate metabolic reaction networks orchestrate the supply, regeneration, and dissipation of energy. Ultimately, this energy homeostasis is responsible for the flawless exertion of all cellular functions over time, and thus, keeping the cell alive. However, mimicking energy homeostasis in a synthetic cell from scratch is intrinsically difficult. The prolonged performance of man-made non-equilibrium systems is often constricted by the finite availability of fuel molecules and the accumulation of waste products. Here, we present a versatile platform approach that allows for a sustained feed of adenosine triphosphate across synthetic vesicle systems. We demonstrate both energy-providing and energy-consuming organelles. We foresee generic applications both inside and outside the synthetic cell context e.g., for the sustained fueling of biochemical non-equilibrium reactions, as compartmentalized nanoreactors and other life-like material systems.
1: Center of Food and Fermentation Tech (TFTAK), Estonia; 2: Tallinn University of Technology (TTU), Estonia
In developing the Single Cell Modelling Framework (SCM), we joined the Cooper-Helmstetter-Donachie cell cycle model with the constraint-based approach in the analysis and design of the metabolism of growing bacterial cells. We assumed that ribosomes are the main actors in the self-reproduction processes in the composition of the cellular chassis responsible for the doubling of cells. This made possible to calculate all the growing cell parameters – cell size, growth rate, cell composition taking into account the geometry of cells, metabolic flux patterns, etc. in the growth space, and determine the growth space limits based on the knowledge of the structure of metabolic reactions network and molecular characteristics of enzymes and other cellular components. We developed models of cells of different complexity, including simple cells allowing us to present and explain the peculiarities of the SCM framework, but also Genome-Wide Models that made possible modelling and analysis of real cells. We also developed a software environment making it possible development of models of different complexity containing tools for dynamic curation of the metabolic networks (Escher tool etc.) and semiautomatic transformation of Cobra models into SCM models. The software environment developed could be found and tested at www.singlecellmodel.com. Finally, we validated the approach described with the data of E. coli, Str. thermophilus and other bacteria.