Structural basis for the shielding function of the dynamic trypanosome variant surface glycoprotein coat.

The most prominent defence of the unicellular parasite Trypanosoma brucei against the host immune system is a dense coat that comprises a variant surface glycoprotein (VSG). Despite the importance of the VSG family, no complete structure of a VSG has been reported. Making use of high-resolution structures of individual VSG domains, we employed small-angle X-ray scattering to elucidate the first two complete VSG structures. The resulting models imply that the linker regions confer great flexibility between domains, which suggests that VSGs can adopt two main conformations to respond to obstacles and changes of protein density, while maintaining a protective barrier at all times. Single-molecule diffusion measurements of VSG in supported lipid bilayers substantiate this possibility, as two freely diffusing populations could be detected. This translates into a highly flexible overall topology of the surface VSG coat, which displays both lateral movement in the plane of the membrane and variation in the overall thickness of the coat.

Facilitating trypanosome imaging.

Research on trypanosomes as a model organism has provided a substantial contribution to a detailed understanding of basic cellular processes within the last few years. At the same time, major advances in super-resolution microscopy have been achieved, facilitating the resolution of biological structures in living cells at a scale of a few nm. However, the motility of trypanosomes has prevented access to high resolution microscopy of live cells. Here, we present a hydrogel based on poly(ethylene glycol) functionalized with either norbornene or thiol moieties for UV induced thiol-ene crosslinking for the embedding and imaging of live trypanosomes. The resulting gel exhibits low autofluorescence properties, immobilizes the cells efficiently on the nanometer scale and is compatible with cell viability for up to one hour at 24 °C. We applied super-resolution imaging to the inner plasma membrane leaflet using lipid-anchored eYFP as a probe. We find specific domains within the membrane where the fluorescence either accumulates or appears diluted rather than being homogenously distributed. Based on a Ripley's analysis, the size of the domains was determined to be raccumulated=170±5 nm and rdilute>115±15 nm. We hypothesize that this structuring of the membrane is associated with the underlying cytoskeleton.

N-glycosylation enables high lateral mobility of GPI-anchored proteins at a molecular crowding threshold.

The protein density in biological membranes can be extraordinarily high, but the impact of molecular crowding on the diffusion of membrane proteins has not been studied systematically in a natural system. The diversity of the membrane proteome of most cells may preclude systematic studies. African trypanosomes, however, feature a uniform surface coat that is dominated by a single type of variant surface glycoprotein (VSG). Here we study the density-dependence of the diffusion of different glycosylphosphatidylinositol-anchored VSG-types on living cells and in artificial membranes. Our results suggest that a specific molecular crowding threshold (MCT) limits diffusion and hence affects protein function. Obstacles in the form of heterologous proteins compromise the diffusion coefficient and the MCT. The trypanosome VSG-coat operates very close to its MCT. Importantly, our experiments show that N-linked glycans act as molecular insulators that reduce retarding intermolecular interactions allowing membrane proteins to function correctly even when densely packed.

CXCR4 signaling is controlled by immobilization at the plasma membrane.

Understanding of the regulation mechanisms of CXCR4 signaling is essential for revealing its role in physiological and pathological processes. Though biochemical pathways following CXCR4 activation by its ligand CXCL12 are well established, knowledge about the receptor dynamics on the plasma membrane remains limited. Here we used Ewing sarcoma-derived cells to unravel the processes that are involved in regulating CXCR4 dynamics on the plasma membrane during receptor signaling. Single-molecule epi-fluorescence microscopy showed that CXCR4 was present in monomeric state on the plasma membrane independent of receptor stimulation. However, upon activation freely diffusing receptors were immobilized in a ligand concentration-dependent manner. CXCR4 immobilization was strongly correlated with the ability for G-protein signaling and was a precursor of subsequent endocytotic events. Our data suggest that, a balanced regulation of G-protein dependent and independent pathways is required for controlling CXCR4 receptor mobility, and potentially subsequent controlled signal transduction.

Crowding of receptors induces ring-like adhesions in model membranes.

The dynamics of formation of macromolecular structures in adherent membranes is a key to a number of cellular processes. However, the interplay between protein reaction kinetics, diffusion and the morphology of the growing domains, governed by membrane mediated interactions, is still poorly understood. Here we show, experimentally and in simulations, that a rich phase diagram emerges from the competition between binding, cooperativity, molecular crowding and membrane spreading. In the cellular context, the spontaneously-occurring organization of adhesion domains in ring-like morphologies is particularly interesting. These are stabilized by the crowding of bulky proteins, and the membrane-transmitted correlations between bonds. Depending on the density of the receptors, this phase may be circumvented, and instead, the adhesions may grow homogeneously in the contact zone between two membranes. If the development of adhesion occurs simultaneously with membrane spreading, much higher accumulation of binders can be achieved depending on the velocity of spreading. The mechanisms identified here, in the context of our mimetic model, may shed light on the structuring of adhesions in the contact zones between two living cells. This article is part of a Special Issue entitled: Mechanobiology.

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.

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.

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.

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.

Giant vesicles as cell models.

Tremendous progress has been made in recent years in understanding the working of the living cell, including its micro-anatomy, signalling networks, and regulation of genes. However, an understanding of cellular phenomena using fundamental laws starting from first principles is still very far away. Part of the reason is that a cell is an active and exquisitely complex system where every part is linked to the other. Thus, it is difficult or even impossible to design experiments that selectively and exclusively probe a chosen aspect of the cell. Various kinds of idealised systems and cell models have been used to circumvent this problem. An important example is a giant unilamellar vesicle (GUV, also called giant liposome), which provides a cell-sized confined volume to study biochemical reactions as well as self-assembly processes that occur on the membrane. The GUV membrane can be designed suitably to present selected, correctly-oriented cell-membrane proteins, whose mobility is confined to two dimensions. Here, we present recent advances in GUV design and the use of GUVs as cell models that enable quantitative testing leading to insight into the working of real cells. We briefly recapitulate important classical concepts in membrane biophysics emphasising the advantages and limitations of GUVs. We then present results obtained over the last decades using GUVs, choosing the formation of membrane domains and cell adhesion as examples for in-depth treatment. Insight into cell adhesion obtained using micro-interferometry is treated in detail. We conclude by summarising the open questions and possible future directions.

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.

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.