Susanne F. Fenz

Membrane protein synthesis in cell-free systems: From bio-mimetic systems to bio-membranes

When taking up the gauntlet of studying membrane protein functionality, scientists are provided with a plethora of advantages, which can be exploited for the synthesis of these difficult‐to‐express proteins by utilizing cell‐free protein synthesis systems. Due to their hydrophobicity, membrane proteins have exceptional demands regarding their environment to ensure correct functionality. Thus, the challenge is to find the appropriate hydrophobic support that facilitates proper membrane protein folding. So far, various modes of membrane protein synthesis have been presented. Here, we summarize current state‐of‐the‐art methodologies of membrane protein synthesis in biomimetic‐supported systems. The correct folding and functionality of membrane proteins depend in many cases on their integration into a lipid bilayer and subsequent posttranslational modification. We highlight cell‐free systems utilizing the advantages of biological membranes.

Diffusion and intermembrane distance: case study of avidin and E-cadherin mediated adhesion.

We present a biomimetic model system for cell-cell adhesion consisting of a giant unilamellar vesicle (GUV) adhering via specific ligand-receptor interactions to a supported lipid bilayer (SLB). The modification of in-plane diffusion of tracer lipids and receptors in the SLB membrane due to adhesion to the GUV is reported. Adhesion was mediated by either biotin-neutravidin (an avidin analogue) or the extracellular domains of the cell adhesion molecule E-cadherin (Ecad). In the strong interaction (biotin-avidin) case, binding of soluble receptors to the SLB alone led to reduced diffusion of tracer lipids. From theoretical considerations, this could be attributed partially to introduction of obstacles and partially to viscous effects. Further specific binding of a GUV membrane caused additional slowing down of tracers (up to 15%) and immobilization of receptors, and led to accumulation of receptors in the adhesion zone until full coverage was achieved. The intermembrane distance was measured to be 7 nm from microinterferometry (RICM). We show that a crowding effect due to the accumulated receptors alone is not sufficient to account for the slowing downan additional friction from the membrane also plays a role. In the weak binding case (Ecad), the intermembrane distance was about 50 nm, corresponding to partial overlap of the Ecad domains. No significant change in diffusion of tracer lipids was observed upon either protein binding or subsequent vesicle binding. The former was probably due to very small effective size of the obstacles introduced into the bilayer by Ecad binding, whereas the latter was due to the fact that, with such high intermembrane distance, the resulting friction is negligible. We conclude that the effect of intermembrane adhesion on diffusion depends strongly on the choice of the receptors.

Quantitation of glucocorticoid receptor DNA-binding dynamics by single-molecule microscopy and FRAP

Recent advances in live cell imaging have provided a wealth of data on the dynamics of transcription factors. However, a consistent quantitative description of these dynamics, explaining how transcription factors find their target sequences in the vast amount of DNA inside the nucleus, is still lacking. In the present study, we have combined two quantitative imaging methods, single-molecule microscopy and fluorescence recovery after photobleaching, to determine the mobility pattern of the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR), two ligand-activated transcription factors. For dexamethasone-activated GR, both techniques showed that approximately half of the population is freely diffusing, while the remaining population is bound to DNA. Of this DNA-bound population about half the GRs appeared to be bound for short periods of time (∼0.7 s) and the other half for longer time periods (∼2.3 s). A similar pattern of mobility was seen for the MR activated by aldosterone. Inactive receptors (mutant or antagonist-bound receptors) show a decreased DNA binding frequency and duration, but also a higher mobility for the diffusing population. Likely, very brief (≤1 ms) interactions with DNA induced by the agonists underlie this difference in diffusion behavior. Surprisingly, different agonists also induce different mobilities of both receptors, presumably due to differences in ligand-induced conformational changes and receptor complex formation. In summary, our data provide a consistent quantitative model of the dynamics of GR and MR, indicating three types of interactions with DNA, which fit into a model in which frequent low-affinity DNA binding facilitates the search for high-affinity target sequences.

Probing Biomembrane Dynamics by Dual‐Wavelength Reflection Interference Contrast Microscopy

We present an improved analysis of reflection interference contrast microscopy (RICM) images, recorded to investigate model membrane systems that mimic cell adhesion. The model systems were giant unilamellar vesicles (GUV) adhering via specific ligand-receptor interactions to supported lipid bilayers (SLB) or to patterns of receptors. Conventional RICM and dual-wavelength RICM (DW-RICM) were applied to measure absolute optical distances between the biomembranes and planar substrates. We developed algorithms for a straightforward implementation of an automated, time-resolved reconstruction of the membrane conformations from RICM/DW-RICM images, taking into account all the interfaces in the system and blurring of the data due to camera noise. Finally, we demonstrate the validity and usefulness of this new approach by analyzing the topography and fluctuations of a bound membrane in the steady state and its dynamic adaptation to osmotic pressure changes. These measurements clearly show that macroscopic membrane flow through tightly adhered area is possible in our system.

Cell-free synthesis of membrane proteins: Tailored cell models out of microsomes ☆

Incorporation of proteins in biomimetic giant unilamellar vesicles (GUVs) is one of the hallmarks towards cell models in which we strive to obtain a better mechanistic understanding of the manifold cellular processes. The reconstruction of transmembrane proteins, like receptors or channels, into GUVs is a special challenge. This procedure is essential to make these proteins accessible to further functional investigation. Here we describe a strategy combining two approaches: cell-free eukaryotic protein expression for protein integration and GUV formation to prepare biomimetic cell models. The cell-free protein expression system in this study is based on insect lysates, which provide endoplasmic reticulum derived vesicles named microsomes. It enables signal-induced translocation and posttranslational modification of de novo synthesized membrane proteins. Combining these microsomes with synthetic lipids within the electroswelling process allowed for the rapid generation of giant proteo-liposomes of up to 50 μm in diameter. We incorporated various fluorescent protein-labeled membrane proteins into GUVs (the prenylated membrane anchor CAAX, the heparin-binding epithelial growth factor like factor Hb-EGF, the endothelin receptor ETB, the chemokine receptor CXCR4) and thus presented insect microsomes as functional modules for proteo-GUV formation. Single-molecule fluorescence microscopy was applied to detect and further characterize the proteins in the GUV membrane. To extend the options in the tailoring cell models toolbox, we synthesized two different membrane proteins sequentially in the same microsome. Additionally, we introduced biotinylated lipids to specifically immobilize proteo-GUVs on streptavidin-coated surfaces. We envision this achievement as an important first step toward systematic protein studies on technical surfaces.

Inter-membrane adhesion mediated by mobile linkers: Effect of receptor shortage

Giant unilamellar vesicles (GUVs) adhering to supported bilayers were used as a model system to mimic ligand–receptor mediated cell-cell adhesion. We present the effect of varying the concentration of receptors (neutravidin on the bilayer) and ligands (biotin on the vesicle) on GUV adhesion and the organization of receptors in the adhesion zone. At high concentrations of both ligands and receptors, the adhesion is strong, all the available membrane is adhered and receptors are accumulated under the adhered membrane up to the geometrical limit of close packing. At low concentrations of receptors (<0.5%), and an arbitrary concentration of ligands (≥0.1%), adhesion does not proceed to completion: the membrane is only partially bound and parts of it still fluctuate. The receptors tend to accumulate under the adhered membrane but the filling is partial. Receptors get jammed and form clusters with fractal like shapes along the rim of the adhered vesicle in such a way that the annular cluster prevents further filling of the adhesion disc. We characterize the filling in terms of a compaction factor and the final concentration. Interestingly, the closing of the ring of jammed clusters switches the interior of the adhesion disc from one thermodynamic ensemble to another. In the new ensemble the receptors sealed within the adhesion disc are mobile but their number is fixed. Under such conditions, the usually strong neutravidin/biotin bond is weak. The incomplete adhesion state can be attributed to a combination of the effects of diffusion, jamming and the competition of enthalpy and entropy on bond formation. The formation of jammed receptor clusters reported here represents a new mechanism that influences membrane adhesion.

Switching from Ultraweak to Strong Adhesion

Nature switches from weak to strong adhesion at the cellular level to spectacular effect – for example, for incredibly sensitive recognition and decisive action during immune response. [ 1 ] If realized in an artifi cial system, such a switching could one day be harnessed as a powerful tool to manipulate weakly interacting objects. The fi rst step towards realizing such a system involves understanding how to create and detect ultraweak adhesion and how to then switch-on a strong interaction. So far, in the context of model membranes, weak adhesion has been achieved only with a ligand-receptor of intrinsically low binding affi nity. [ 2 ] Whatever, the intrinsic strength of the bonds, so far they were usually found to be arranged in compact stable domains. [ 3 ] Here, we present experiments and simulations that indicate how to create and detect ultraweak adhesion in the context of fl uid two dimensional membranes interacting via specifi c ligand/receptor bonds. Thus, specifi c adhesion is mediated by transient domains consisting of sparsely distributed bonds. Amazingly, we demonstrate that the avidin/biotin pair – famous for forming the strongest receptor/ligand bond known in nature, mediates ultraweak adhesion under suitable circumstances. This choice of binders allows us to switch on strong binding once sensitive detection is achieved – without resorting to a second binding pair – something not possible with intrinsically weak binders. However, this goal necessitates an appropriate design strategy elaborated below. The in vitro system consists of two membranes: a solid supported lipid bilayer (SLB) and the freely fl uctuating membrane of a giant unillamelar vesicle (GUV). A GUV is a two

Inferring spatial organization of bonds within adhesion clusters by exploiting fluctuations of soft interfaces

Detecting the organization of bonds within adhesion domains connecting two interacting membranes is, at present, extremely challenging. Herein we present a technique, based on Reflection Interference Contrast Microscopy, which uses spontaneous thermal fluctuations of a soft interface as a tool to identify the organization of specific ligand-receptor bonds. The key is a time-resolved analysis of micro-interferometric data that systematically quantifies fluctuations and enables the detection of their suppression due to the formation of bonds, which, in turn, allows the identification of the bond organization without the use of fluorescent labelling. The identification of a new type of bond organization characterized by sparsely distributed bonds, as well as detection of pinning centres of nanometric size is presented.

The molecular size of the extra-membrane domain influences the diffusion of the GPI-anchored VSG on the trypanosome plasma membrane

A plethora of proteins undergo random and passive diffusion in biological membranes. While the contribution of the membrane-embedded domain to diffusion is well established, the potential impact of the extra-membrane protein part has been largely neglected. Here, we show that the molecular length influences the diffusion coefficient of GPI-anchored proteins: smaller proteins diffuse faster than larger ones. The distinct diffusion properties of differently sized membrane proteins are biologically relevant. The variant surface glycoprotein (VSG) of African trypanosomes, for example, is sized for an effective diffusion-driven randomization on the cell surface, a process that is essential for parasite virulence. We propose that the molecular sizes of proteins dominating the cell surfaces of other eukaryotic pathogens may also be related to diffusion-limited functions.

Association rates of membrane-coupled cell adhesion molecules.

Thus far, understanding how the confined cellular environment affects the lifetime of bonds, as well as the extraction of complexation rates, has been a major challenge in studies of cell adhesion. Based on a theoretical description of the growth curves of adhesion domains, we present a new (to our knowledge) method to measure the association rate k(on) of ligand-receptor pairs incorporated into lipid membranes. As a proof of principle, we apply this method to several systems. We find that the k(on) for the interaction of biotin with neutravidin is larger than that for integrin binding to RGD or sialyl Lewis(x) to E-selectin. Furthermore, we find k(on) to be enhanced by membrane fluctuations that increase the probability for encounters between the binders. The opposite effect on k(on) could be attributed to the presence of repulsive polymers that mimic the glycocalyx, which points to two potential mechanisms for controlling the speed of protein complexation during the cell recognition process.