Engineering supported membranes for cell biology

Cell membranes exhibit multiple layers of complexity, ranging from their specific molecular content to their emergent mechanical properties and dynamic spatial organization. Both compositional and geometrical organizations of membrane components are known to play important roles in life processes, including signal transduction. Supported membranes, comprised of a bilayer assembly of phospholipids on the solid substrate, have been productively served as model systems to study wide range problems in cell biology. Because lateral mobility of membrane components is readily preserved, supported lipid membranes with signaling molecules can be utilized to effectively trigger various intercellular reactions. The spatial organization and mechanical deformation of supported membranes can also be manipulated by patterning underlying substrates with modern micro- and nano-fabrication techniques. This article focuses on various applications and methods to spatially patterned biomembranes by means of curvature modulations and spatial reorganizations, and utilizing them to interface with live cells. The integration of biological components into synthetic devices provides a unique approach to investigate molecular mechanisms in cell biology

Spatiomechanical Modulation of EphB4-Ephrin-B2 Signaling in Neural Stem Cell Differentiation.

Interactions between EphB4 receptor tyrosine kinases and their membrane-bound ephrin-B2 ligands on apposed cells play a regulatory role in neural stem cell differentiation. With both receptor and ligand constrained to move within the membranes of their respective cells, this signaling system inevitably experiences spatial confinement and mechanical forces in conjunction with receptor-ligand binding. In this study, we reconstitute the EphB4-ephrin-B2 juxtacrine signaling geometry using a supported-lipid-bilayer system presenting laterally mobile and monomeric ephrin-B2 ligands to live neural stem cells. This experimental platform successfully reconstitutes EphB4-ephrin-B2 binding, lateral clustering, downstream signaling activation, and neuronal differentiation, all in a configuration that preserves the spatiomechanical aspects of the natural juxtacrine signaling geometry. Additionally, the supported bilayer system allows control of lateral movement and clustering of the receptor-ligand complexes through patterns of physical barriers to lateral diffusion fabricated onto the underlying substrate. The results from this study reveal a distinct spatiomechanical effect on the ability of EphB4-ephrin-B2 signaling to induce neuronal differentiation. These observations parallel similar studies of the EphA2-ephrin-A1 system in a very different biological context, suggesting that such spatiomechanical regulation may be a common feature of Eph-ephrin signaling

A Tethered Bilayer Assembled on Top of Immobilized Calmodulin to Mimic Cellular Compartmentalization

International audienceBACKGROUND: Biomimetic membrane models tethered on solid supports are important tools for membrane protein biochemistry and biotechnology. The supported membrane systems described up to now are composed of a lipid bilayer tethered or not to a surface separating two compartments: a "trans" side, one to a few nanometer thick, located between the supporting surface and the membrane; and a "cis" side, above the synthetic membrane, exposed to the bulk medium. We describe here a novel biomimetic design composed of a tethered bilayer membrane that is assembled over a surface derivatized with a specific intracellular protein marker. This multilayered biomimetic assembly exhibits the fundamental characteristics of an authentic biological membrane in creating a continuous yet fluid phospholipidic barrier between two distinct compartments: a "cis" side corresponding to the extracellular milieu and a "trans" side marked by a key cytosolic signaling protein, calmodulin. METHODOLOGY/PRINCIPAL FINDINGS: We established and validated the experimental conditions to construct a multilayered structure consisting in a planar tethered bilayer assembled over a surface derivatized with calmodulin. We demonstrated the following: (i) the grafted calmodulin molecules (in trans side) were fully functional in binding and activating a calmodulin-dependent enzyme, the adenylate cyclase from Bordetella pertussis; and (ii) the assembled bilayer formed a continuous, protein-impermeable boundary that fully separated the underlying calmodulin (trans side) from the above medium (cis side). CONCLUSIONS: The simplicity and robustness of the tethered bilayer structure described here should facilitate the elaboration of biomimetic membrane models incorporating membrane embedded proteins and key cytoplasmic constituents. Such biomimetic structures will also be an attractive tool to study translocation across biological membranes of proteins or other macromolecules

Mechanisms of Focal Adhesions

Focal adhesions are dynamic multiprotein structures connecting cells to their surrounding microenvironment. Cells receive critical mechanical signals from adhesions that control many cellular processes including wound healing, differentiation, development, and cancer. Proteins that form adhesions are called adhesion proteins and some of these proteins can be mechanosensitive, meaning that they respond to mechanical stimuli. During spreading and migration, cells mechanically test extracellular matrix rigidity by contracting matrix to a constant displacement. Transmission and processing of such mechanical signals rely upon the dynamic regulation of the adhesions, which is tightly coordinated with activation of intracellular signaling cascades involving various adhesion molecules. However, the molecular mechanisms of mechanical signals that are transmitted through the adhesions to control cell behavior are poorly understood. In this thesis, we discovered novel phenomenon and mechanisms to elucidate roles of mechanical signals for multiple key aspects of basic cell behavior, especially cell growth. We performed live cell imaging of cells spreading on fibronectin coated micropillars to understand adhesion formation, adhesion regulation, and their impact on cell behavior. One of the earliest molecules to arrive at an adhesion formation site is a mechanosensitive protein called talin which binds to several other entities to form the backbone of focal adhesions. We found a novel role of talin cleavage, which previously was thought to play a role only in focal adhesion turnover. We found that talin cleavage is a force dependent process that regulates proper adhesion formation, thereby governing several critical cellular processes. In the absence of this talin cleavage, cells formed abnormal adhesions and showed inhibited growth. Further, we found that upon inhibition of talin cleavage, one of the key cellular behaviors of increased cellular motility upon stimulation by epidermal growth factor seemed to disappear. Epidermal growth factor receptor is a transmembrane protein and has previously been shown to play important role in various cancers where cells exhibit altered rigidity sensing. Surprisingly, we found that epidermal growth factor receptor was required for cellular rigidity sensing only on rigid substrates, highlighting the importance of the interplay between mechanical and biochemical signals in determining cell behavior

The influence of adhesion molecules on binding and protein organization in cell contacts

Interactions between immune cells such as T cells and antigen-presenting cells (APCs) are integral for mounting an adaptive immune response. The interaction between the T cell receptor (TCR) and the antigen-presenting major histocompatibility complex (pMHC) on a contacting T cell and APC, is widely accepted to be the key interaction. If the interaction is favourable, then T cell activation occurs. A large pool of research has been aimed at characterizing this interaction by measuring the binding kinetics and relating it to the T cell response. A simplified model membrane system called a supported lipid bilayer (SLB) is often used to mimic the membrane of the APC. In many T cell activation studies, the SLB contains the nickel-chelating lipid DGS-NTA(Ni) to functionalize the SLB with histidine-tagged proteins. In the first part of this thesis I show that interactions between DGS-NTA(Ni) and the T cells can lead to, unwanted, T cell signaling. It was found that increasing the concentration of DGS-NTA(Ni) both increased cell adhesion and the fraction of signaling cells. Adding bovine serum albumin (BSA) functioned as a blocking agent, preventing unspecific cell adhesion and decreased the fraction of signaling cells down to a basal level. A low level of signaling was also obtained when functionalizing the blocked SLBs with adhesion molecules binding to receptors on the T cell. In contrast, without blocking these functionalized SLBs again signaled at a similar level to the unblocked, not functionalized DGS-NTA(Ni) SLBs. The DGS-NTA(Ni) signaling was argued to be due to TCR-DGS-NTA(Ni) interactions and stressed the importance of adequately blocking these interactions in T cell activation studies.In the second part of the thesis, a new method to measure the two-dimensional dissociation constant (2D Kd) of ligand-receptor interactions on single cells is presented. This is measured on individual cell-SLB contacts, providing an accurate new means of measuring binding affinity and to study differences in the 2D Kd in the cell population. In the final part of the thesis, the interaction of TCR-pMHC in the presence of adhesion molecules of different length and density is studied. Adhesion molecule pairs of similar height as TCR-pMHC have been argued to facilitate the TCR-pMHC interaction by physically keeping the opposing membranes at an optimal distance for binding. However, adhesion pairs of different height than that of TCR-pMHC are also important for cell-cell contact formation and have been shown to result in an impaired T cell response if removed. To better understand how, and if, adhesion molecules of different lengths influences TCR-pMHC binding the 2D Kd of TCR-pMHC in the presence of differently-sized adhesion molecules was studied. For this purpose, a SLB functionalized with TCR and an adhesion ligand, was allowed to bind cell with pMHC and the corresponding adhesion receptor. It was found that the 2D Kd of the TCR-pMHC interaction could be up to an order of magnitude higher (weaker) than the corresponding value for TCR-pMHC alone when having height-mismatched molecules. In addition, the TCR-pMHC distributed non-homogeneously in the cell-SLB contacts when having height-mismatched adhesion molecules, but homogeneously when having height-matched adhesion molecules. Furthermore, even for height-matched adhesion molecules the 2D Kd of the TCR-pMHC interaction was found to be dependent on the relative density fraction of TCR to adhesion molecules, with low fractions of TCR molecules giving 2-3 times weaker binding. This indicates that TCR-pMHC binding in cell contacts depends significantly on the local environment and not only on the protein-protein interaction per se

Protein segregation and conformation in antigen-receptor triggering: a quantitative fluorescence microscopy study

Antigen receptors play a central role in determining whether an immune response is required. The remarkable diversity of their extracellular domains allows them to detect unfamiliar pathogens. In response, information is conveyed across the plasma membrane, resulting in the phosphorylation of cytoplasmic domains. This process, known as triggering, has been the subject of much controversy. Antigen-independent triggering has been demonstrated for the T-cell receptor, challenging conventional views of signal transduction. The kinetic-segregation model proposes that size-dependent exclusion of phosphatases increases net receptor phosphorylation. Rather than initiating downstream signalling autonomously, ligand binding serves to hold the receptor within phosphatase-depleted regions of the membrane. The strength of this model is that receptor-ligand interactions are considered in the broader context of their physical environment. This thesis asks how intermembrane distance affects antigen-receptor triggering. Total internal reflection fluorescence microscopy is used to establish that phosphatase exclusion decreases when intermembrane distance increases. This inverse relationship, demonstrated for both B cells and T cells, is consistent with the kinetic-segregation model. However, in T cells, increases in intermembrane distance are not found to affect downstream signalling. The experiments described here thus fail to establish a relationship between phosphatase segregation and triggering. Part of this thesis addresses an unanticipated problem with the experimental system, namely that lymphocytes are triggered by nickel-chelating lipids in supported lipid bilayers. An effective and easily implemented solution is identified, which can be used in future work with this popular model surface. A short section focusing on the kinase Lck is also included. The feasibility of using Förster resonance energy transfer to identify spatiotemporal changes in Lck conformation is explored. Understanding antigen-receptor triggering is crucial, as failure to discriminate between self and non-self antigens can have life-threatening consequences. Here, live-cell imaging is used to examine kinase and phosphatase behaviour, in an attempt to shed some light on how decisions are made.Wellcome Trust Cambridge Philosophical Societ

Development of Droplet-Based Microfluidics for Synthetic Biology Applications

Microfluidics combines principles of science and technology, and enables the user to handle, process and manipulate fluids of very small volumes. This technology permits the integration of multiple laboratory applications into one single microfabricated chip, requires minimal manual user intervention and sample consumption, and allows enhanced data analysis speed and precision. Due to these numerous advantages, the potential for this technology to be applied in fundamental biophysical and biomedical research is vast. The major aim of this thesis was to explore the capacities of microfluidics, particularly droplet-based microfluidic technology in the following topics: 1) Mimicry of the immune system cellular environment, with the ultimate goal of programing T cells for adoptive T cell therapy; 2) Bottom-up assembly of minimal synthetic cells. Towards this end, a novel approach to form gold-nanostructured and specifically biofunctionalized water-in-oil droplets was developed. This thesis highlights the advanced properties of nanostructured droplets to serve as 3D antigen presenting cell (APC) surrogates for T-cell stimulation. The combination of flexible biofunctionalization and pliable physical droplet properties work in tandem, providing a flexible and modular system that closely models in situ APC-T cell interactions. The research within this thesis focused also on the dissection of complex cellular sensory machinery implementing an automated droplet-based microfluidic approach. Towards this goal, nanostructured droplets as cell-sized compartments and droplet-based pico-injection technology were used to achieve the bottom-up assembly of the minimal number of proteins required for a “simple synthetic cell.” While the applied methodology has a potential for assembly of a wide range of subcellular functional units, the focus in this thesis was on the reconstitution of the actomyosin cortex. Successful optimization of the biochemical and biophysical conditions within the droplets allowed to achieve precise control over the actin polymerization and actomyosin network organization by their linkage to the droplets periphery. These experimental steps were also necessary to generate signaling events including myosin-driven droplet migration and self-propulsion with reduced molecular complexity compared to living cells

DNA Nanotechnology to Map and Manipulate Adhesion Forces at Fluid Interfaces

Cells transmit piconewton (pN) receptor forces to ligands in the extracellular matrix (ECM) and on the surface of adjacent cells. These forces regulate functions ranging from adhesion to clotting and the immune response. Whereas adhesion mechanics on rigid substrates are well characterized, understanding mechanotransduction at cell-cell junctions remains challenging due to a lack of tools. We develop and apply new classes of DNA-based force probes to map and manipulate receptor forces on supported lipid bilayers (SLBs), planar membranes that mimic an adjacent cell. We use these probes to elucidate force balance in podosomes, which are multipurpose protrusive structures that form at cell-cell and cell-ECM interfaces. Podosomes have a core-ring architecture, and previous works demonstrated that the podosome’s actin core generates nanonewton protrusive forces. However, the podosome’s contractile landscape remained poorly understood. In Aim 1 (Chapter 3), we develop and apply Molecular Tension- Fluorescence Lifetime Imaging Microscopy to map integrin receptor forces and clustering on SLBs. We demonstrate that integrin receptors apply pN tension in podosome rings. We then introduce photocleavable probes to site-specifically perturb adhesion forces and apply rupturable DNA-based force probes to test the role of receptor tension in podosome formation and maintenance. These studies confirm a local mechanical feedback between podosome core protrusion and integrin receptor tension. In Aim 2 (Chapter 4), we evaluate structure and energy transfer across a library of DNA-based tension probes using spectroscopy and microscopy. We then demonstrate the functional implications of probe design on cellular imaging. This work expands our understanding of receptor forces in podosome mechanobiology and contributes new insight and tools for studying juxtacrine receptor interactions.Ph.D

Nanoscale spherical-supported membranes as novel platforms for improving the phage display screening of antibody mimetics against membrane protein targets

Membrane proteins represent the majority of therapeutic targets for the antibody-based drugs available today. These are routinely identified via phage display screening, but traditional antigen presentation methods require membrane protein targets to be detergent-solubilised in order to preserve their native conformations post-purification. Unfortunately, detergent solubilisation can not only lead to gradual target denaturation over time, but the detergent micelles can also occlude important epitopes on the extramembranous loops and thus prevent the discovery of antibody binders. The current thesis aimed to demonstrate that, by reconstituting purified membrane proteins into spherical-supported bilayer lipid membranes (SSBLMs) deposited on nanosized substrates, a versatile platform can be constructed for performing phage display screening against membrane protein targets, while not only presenting these within a native-like lipid environment, but also eliminating detergents from the screening phase altogether. For providing proof-of-concept, 100- and 200 nm silica nanoparticles were covered with POPC SSBLMs embedding the bacterial nucleoside transporter NupC. Full substrate coverage and the correct formation of the lipid bilayer components were established via spectrofluorometry, using fluorescent labelling and small-angle X-ray scattering (SAXS) respectively, while Western blotting and high-affinity antibody binding confirmed the presence of SSBLM-embedded NupC. The platform was then used to screen designed ankyrin repeat proteins (DARPins) against a His6-tagged construct of NupC across different screening formats so as to offer a comparison to the classic 96-well plate antigen presentation method. Following that, the DARPin binders showing the highest potential affinity for NupC were purified and subjected to further binding validation assays against two other constructs – detergent-solubilised double Strep-tagged NupC and SSBLM-embedded untagged/wild-type NupC – in order to identify any binders targeting extramembranous epitopes that would be accessible in vivo as well. Ultimately, the results presented throughout indicated that SSBLMs constitute a promising means of screening antibody binders against membrane protein targets embedded in a close-to-native format

High Resolution Atomic Force Microscopy of Functional Biological Molecules

Nanoscale dynamic biological processes are central to the regulation of cellular pro- cesses within the body. The direct visualisation of these processes represents a challenge because of the intrinsic difficulties of imaging at the nanoscale, well below the diffraction limit of light. Here we use the Atomic Force Microscope to ‘feel’ the structure of single biomolecules adsorbed to a flat substrate at sub-nanometre resolution. We have enhanced the performance and resolution of Atomic Force Microscopy (AFM) for imaging DNA plasmids in solution, resolving its secondary structure in the form of the double helix. We are able to observe local deviations from the average structure, and in particular variations in the depth of the grooves in the double-stranded DNA which may be attributed to supercoiling of the DNA. Such local variations of the DNA double helix structure are important in mediating protein-DNA binding specificity and thus in regulating gene expression. We show preliminary data on DNA minicircles, which can be used as a synthetic system to study how supercoiling affects DNA structure and influences DNA-protein binding interactions with implications for many genetic processes. Going from fundamental science to a biomedical application, we have used AFM to study the functional mechanisms of antimicrobial peptides, which are developed in response to the growing problem of antimicrobial resistance. Antimicrobial peptides disrupt microbial phospholipid membranes but direct observation of the mode of action for the disruption is lacking. Here we visualise the mode of action of syn- thetic antimicrobial cationic alpha-helical peptides. Two of these peptides attack membrane via previously unknown mechanism: Amhelin forms pores which are not limited in size but expand from the nano to micrometre scale; Amhelit also forms pores which penetrate a single layer of the lipid bilayer that forms the membrane. We present the first nanoscale visualisation of membrane disruption by the naturally occurring antimicrobial peptide cecropin B. This is complemented by the visualisa- tion of peptides similar in sequence to cecropin B, but with structural modifications which are used to elucidate the structural origins of cecropin B’s mechanism of ac- tion. Improvements in imaging capabilities of the AFM, as tested on DNA, were shown to benefit imaging of the mode of action for antimicrobial peptides, including time-lapse imaging of a novel expanding monolayer state. We have thus used AFM to elucidate mechanisms of action for antimicrobial pep- tides. Relating these mechanisms to the peptide sequences, we can gain insight into how peptide sequence affects structure and function for these antimicrobial agents. This may aid in the development and improvement of novel peptide antibiotics