Biology subjects

Steinkühler, J.

Publications and source records attributed to Steinkühler, J..

5 recordsLinked to original sources

bioRxiv2023

Remodeling of lipid-foam prototissues by network-wide tension fluctuations induced by active particles

Recent advances in the field of bottom-up synthetic biology have led to the development of synthetic cells that mimic some features of real cells, such as division, protein synthesis, or DNA replication. Larger assemblies of synthetic cells may be used to form prototissues. However, existing prototissues are limited by their relatively small lateral dimensions or their lack of remodeling ability. In this study, we introduce a lipid-based tissue mimetic that can be easily prepared and functionalized, consisting of a millimeter-sized "lipid-foam" with individual micrometer-sized compartments bound by lipid bilayers. We characterize the structural and mechanical properties of the lipid-foam tissue mimetic. We demonstrate self-healing capabilities enabled by the fluidity of the lipid bilayers. Upon inclusion of bacteria in the tissue compartments, we observe that the tissue mimetic exhibits network-wide tension fluctuations driven by membrane tension generation by the swimming bacteria. Active tension fluctuations facilitate the fluidization and reorganization of the lipid-foam tissue mimetic, providing a versatile platform for understanding and mimicking biological tissues.

synthetic biology↗

bioRxiv2023

Improving cell-free expression of membrane proteins by tuning ribosome co-translational membrane association and nascent chain aggregation

Cell-free gene expression (CFE) systems are powerful tools for transcribing and translating genes outside of a living cell. Given their diverse roles in nature, synthesis of membrane proteins is of particular interest, but their yield in CFE is substantially lower than for soluble protein. In this paper, we study factors that affect the cell-free synthesis of membrane proteins and develop a quantitative kinetic model of their production. We identify that stalling of membrane protein translation on the ribosome is a strong predictor of membrane protein synthesis and creates a negative feedback loop in which stalled peptide sequences quench ribosome activity through aggregation between the ribosome nascent chains. Synthesis can be improved by the addition of lipid membranes which incorporate protein nascent chains and, therefore, kinetically competes with aggregation. Using both quantitative modeling and experiment, we show that the balance between peptide-membrane association and peptide aggregation rates determines the total yield of synthesized membrane protein. We then demonstrate that this balance can be shifted by altering membrane composition or the protein N-terminal domain sequence. Based on these findings, we define a membrane protein expression score that can be used to rationalize the engineering of N-terminal domain sequences both of a native and computationally designed membrane proteins produced through CFE.

synthetic biology↗

bioRxiv2022

Probing the force-from-lipid mechanism with synthetic polymers

A central feature of mechanotransduction is the ability of mechanosensitive channels to respond to mechanical stimuli from the surrounding lipid bilayer. Accordingly, the mechanical properties of membranes should play an important role in modulating force transmission to embedded channels, yet the nature of this relationship remains unclear for a wide class of mechanosensitive channels across prokaryotic and eukaryotic systems. Here, we use a synthetic amphiphile to modulate the membrane mechanical properties of cell-derived vesicles and probe channel activation. Using precise membrane mechanical characterization approaches that have rarely been used in conjunction with electrophysiology techniques, we directly characterize three membrane properties and the activation threshold of the E. coli mechanosensitive channel of large conductance (MscL). Our study reveals that decreases in the membrane area expansion modulus, KA, and bending rigidity, kc, correlate with increases in the pressure required to activate MscL and that this effect is reproducible with the mammalian channel, TREK-1. MD simulations demonstrate that polymer-mediated changes in interfacial tension is the best mechanism to describe these experimental results. Together, our results bolster the force-from-lipids mechanism by demonstrating the generality of the relationship between changes in specific membrane mechanical properties and the gating pressure of MscL and TREK-1. In addition, our results reveal the mechanical mechanism by which membrane amphiphiles alter the activity and sensitivity of mechanosensitive channels through changes in long-range force transmission.

biophysics↗

bioRxiv2022

Enzymatically-active bacterial microcompartments follow substrate gradients and are protected from aggregation in a cell-free system

The ability to dynamically control organelle movement and position is essential for cellular function. Yet the underlying mechanisms driving this organization have not been fully resolved. Here, we draw from recent experimental observations and theoretical models of enzyme chemotaxis to demonstrate the chemotaxis of a bacterial organelle, the 1,2 propanediol (1,2-PD) utilization bacterial microcompartment (MCP) from Salmonella enterica. Upon encapsulating MCPs in a cell-like, biomimetic compartment, we observed the directed movement of MCPs along an external gradient of substrate. Our analysis shows that MCPs not only chemotax towards their substrate but also that enzymatic activity and substrate turnover protect them against large-scale aggregation. Our results provide a first experimental demonstration of organelle chemotaxis in a synthetic cellular system and support a recent theoretical model of chemotaxis. Together this work reveals a potentially significant driver of organelle organization while contributing to the construction of synthetic cell-like materials.

synthetic biology↗

bioRxiv2020

Super-elasticity of plasma- and synthetic membranes by coupling of membrane asymmetry and liquid-liquid phase separation

Biological cells are contained by a fluid lipid bilayer (plasma membrane, PM) that allows for large deformations, often exceeding 50% of the initial (or projected) PM area. Biochemically isolated lipids self-organize into membranes, but the extraordinary deformability of the plasma membrane is lost. Pure lipid bilayers are prone to rupture at small (<2-4%) area strains and this limits progress for synthetic reconstitution of cellular features such as migration, phagocytosis and division. Here, we show that by preserving PM structure and composition during isolation from cells, vesicles with cell-like elasticity are obtained. We found that these plasma membrane vesicles store significant area in the form of nanotubes in their lumen. These are recruited by mechanical tension applied to the outer vesicle membrane showing an apparent elastic response. This "super-elastic" response emerges from the interplay of lipid liquid-liquid phase separation and membrane asymmetry. This finding allows for bottom-up engineering of synthetic vesicles that appear over one magnitude softer and with three fold larger deformability than conventional lipid vesicles.

biophysics↗