Institute of Applied Physics - Biophysics
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Lipid interaction of the T cell receptor complex

V 538 Richter Programme; Eva Sevcsik , IAP, TU Wien

Every cell of our body is surrounded by a plasma membrane that separates the inside of a cell from the outside. This is not just a static barrier but the stage of an intricate interplay of invisibly small structures, proteins and even smaller lipids that move and assemble themselves in complex ways to mediate membrane function. A special type of cells, T-cells, is major players in our immune system. One protein in their plasma membrane, the T-cell receptor (TCR), recognizes a part of a pathogen (the antigen), which is presented by another cell, that has previously found this intruder in our body. Upon this recognition process, the T-cell becomes activated and a series of events is initiated ultimately leading to an immune response. We have already a good understanding of the protein players involved in this process, but the role of lipids is largely unclear. This is because lipids are extremely hard to study, particularly in living cells. Lipids and proteins are much too small to be directly imaged using optical microscopic methods, and they are constantly moving around. However, by labelling only a tiny subset of these molecules with fluorescent dyes they can be detected as individual diffraction limited spots and we can follow their movement over time and watch, how they interact with each other. Still, this is not good enough for lipids, because their interactions with proteins are often too short. Further, they cannot only influence proteins by direct, one-on-one interaction, but they also make up the 2-dimensional matrix of the plasma membrane itself. There are thousands of different lipid species with very different properties and their organization and dynamics shape the properties of the membrane. Here, a novel experimental approach will be employed and will allow, for the first time, to interrogate the interaction of lipids with the TCR. T-cells will be interfaced with microstructured substrates which mimic antigen presenting cells; this allows to immobilize and enrich the TCR specifically within certain areas of the T-cell plasma membrane in live cells. Thus, it becomes possible to extract quantitative parameters on the interaction of the immobile interaction partner (the TCR) with a mobile interaction partner (the lipid). Several powerful single molecule fluorescence techniques will be used to for the first time observe the behavior of the lipids with respect to the TCR to elucidate their role in the process of T-cell activation.

Nana-analytics of cellular systems (NanoCell)

FWF graduate college; coordinator Peter Hinterdorfer,Johannes Kepler University Linz (JKU)

A highly interdisciplinary graduate college funded by the Austrian Science Fund (FWF), titled Nano-Analytics of Cellular Systems (NanoCell), has the major goal to train next generation multidisciplinary graduates for frontier research in life- and cellular nano-science. Its scientific aim is to span the gap between the processes of molecular recognition and structural rearrangements on the one side, and membrane transport and cell motility on the other. The educational and research program integrates a broad basis of scientific and technological fields related to biophysics, cell biology, nanotechnology, applied physics, theoretical physics, bioorganic and inorganic chemistry, structural and molecular biology, mathematical modelling, and scientific computing. Participating institutions are: Johannes Kepler University Linz (JKU), Johann Radon Institute for Computational and Applied Mathematics (RICAM), Institute of Science and Technology (IST), and Vienna University of Technology (TUW). Selected PhD students will be employed for at least 3 years with a contract that includes social benefits. They will be encouraged to spend one semester abroad joining other collaborating laboratories. All selection procedures, training activities, and communication will be in English.

Effect of palmitoylation in early T cell signaling

FWF Stand-alone project, Florian Baumgart, Vienna University of Technology

T-lymphocyte activation relies on the specific molecular recognition of peptide-loaded MHC (pMHC) on an antigen-presenting cell (APC) by the T-cell receptor (TCR). TCR-pMHC binding causes the spatial rearrangement of components of the TCR signaling machinery and the phosphorylation of multiple tyrosines on TCR-associated CD3 by the non-receptor tyrosine kinase Lck. These initial phosphorylation events result in the binding of downstream molecules and ultimately in T-cell activation. Lck is therefore key for TCR triggering and its absence abolishes T-cell activation. Although the sequence of events that leads to T-cell activation has been extensively studied, a detailed mechanistic understanding of how the spatial organization of different signaling components controls their function is still lacking. Notably, Lck contains an N-terminal membrane anchor, which is necessary and sufficient for its membrane targeting as well as a prerequisite for its function. The membrane anchor consists of three lipid modifications: an irreversibly linked myristic acid and two reversibly attached palmitic acid chains. The presence of three lipid modifications appears interesting, since, in principal, myristoylation plus one palmitate chain is sufficient for tight membrane anchorage. Even more so, protein-protein interactions like the association of Lck with the transmembrane co-receptors CD4 or CD8 would actually make acylation dispensable for membrane targeting. Strikingly, CD4 and CD8, as well as other molecules that modulate Lck activity are also palmitoylated. Some of them, like CD4 and CD8, are transmembrane proteins, where the function of palmitoylation is not obvious. At the same time, studies addressing the function of CD4 and CD8 palmitoylation yielded contradictory results. The present project is designed to gain a fundamental understanding of how protein palmitoylation modulates the spatial organization and thereby the functionality of key signaling molecules during early T-cell activation. In the past, the effect of protein palmitoylation has been mostly addressed in biochemical ensemble experiments or classical diffraction-limited light microscopy. In the present proposal, single molecule microscopy and murine primary T-cell model systems are used to get unprecedented molecular insights into the regulation of early T-cell signaling by protein palmitoylation.

Transmembrane Transporters in Health and Disease

Subunit stoichiometry and supermolecular organization of transmembrane transporters FWF-SFB; coordinator Harald Sitte, Medical University of Vienna

We employ state-of-the-art single molecule fluorescence microscopy for obtaining insights into the oligomeric state, the interaction kinetics, the mobility, and the nanoscopic organization of transmembrane transporters. The single molecule approach allows us to go beyond conventional ensemble measurements: with brightness analysis and two-color colocalization studies, we precisely quantify molecular associations; the high temporal resolution of one millisecond is employed to capture transient phenomena such as weak interactions with immobile sites in the membrane; finally, we exploit the high localization precision of ~20nm for unraveling structures influencing the diffusional paths and for obtaining insights into the nanoscopic arrangements of the molecules. In particular, we address the organization of the monoamine transporters SERT and DAT in live cells; this study is performed in close collaboration with Harald Sitte and Michael Freissmuth, who provide and test GFP fusion constructs, fluorescent ligands, cell lines, and ex vivo cultured neurons. The developed techniques shall be further applied for studying the organization of ABC and PDR transporters. We coordinate all experiments with Peter Hinterdorfer, who performs complimentary force spectroscopy measurements to determine the interaction energies between transmembrane transporter

In Vivo Nanopatterning of Membrane Proteins

FWF Stand-alone project; collaboration with Iris Bergmair, Profactor GmbH Steyr, Austria

Specific activation of T cells is initiated in the contact zone with an antigen presenting cell, the so-called immunological synapse. The T cell receptor recognizes its cognate antigen presented by MHCII molecules on the antigen-presenting cell, which initiates a plethora of highly regulated protein interactions. Early signaling is accompanied by spatial reorganization of proteins within the synapse, leading to a large-scale segregation into microclusters and supramolecular activation clusters. Currently, interactions are inferred from the colocalization of proteins within the developing synapse via two-color fluorescence microscopy. That approach, however, does not measure the interaction per se, and thus is highly prone to false positive results.
We propose here to measure protein-protein interactions during early T cell signaling directly by slightly retarding one of the interaction partners (“bait”), and recording the co-retardation of a second fluorescently tagged protein (”prey”). The idea is based on a method previously introduced by us to study protein-protein interactions in the live cell plasma membrane using protein micropatterning (Schwarzenbacher et al., Nat. Methods 5:1053 (2008)). For this, a bait-specific ligand is immobilized on a glass surface in a characteristic micropattern. When cells are grown on such substrates, the bait located in the plasma membrane follows these patterns. Interaction with a fluorescent prey leads to the rearrangement of the prey to form the same patterns, which can be easily detected via total internal reflection fluorescence microscopy.
Up to now, the method was predominantly used to obtain a static and averaged picture of a particular interaction, i.e. no spatial or temporal variations were addressed. However, as our approach allows for live cell analysis, spatiotemporal changes of protein interactions can be directly recorded, what shall be the subject of this proposal. We will study interactions between the T cell receptor and a selected choice of potential interaction partners during the formation of the immunological synapse between the T cell and a functionalized surface. Such mimicries of an antigen-presenting cell are frequently used in the community, and have been successfully established also in our lab (Huppa et. al, Nature, in press). Our idea is to provide the cell with a micro- or nanostructured substrate, which allows on the one hand for the formation of spatiotemporal heterogeneities, and on the other hand for sufficient retardation of the bait molecules so that interaction with fluorescent prey can be analyzed by its co-patterning. To achieve this, it will be necessary to attach the bait to the surface via a reversible linkage with an adjustable off-rate. The NTA-Ni2+-oligohistidine complex will be used for reversible ligation, and its affinity will be down-modulated to the value required for proper functioning by applying free histidine.
Analysis of spatial heterogeneities in the protein-protein interaction requires the interrogation of contrast values at a spatial frequency which is higher than the desired resolution of the map. Our method was established using a feature size of 3µm, which is insufficient to resolve details within spatial features of the immunological synapse. To improve the resolution down to length-scales characteristic of the synapse (~1µm), feature sizes of 250nm or smaller have to be employed. We propose here to use nanocontact printing for this purpose, which shall be set up as the basis for our patterning technology. The miniaturization strategies will be implemented in two steps: i) Features will be generated, which can be imaged by classical diffraction-limited optics (~250nm). ii) Features below ~250nm, referred to as nanopatterns, cannot be imaged by classical optics devices. Nanopatterns will be read out by photoactivation localization microscopy (PALM), a new technology developed to obtain subdiffraction resolution in light microscopy by photoactivation and localization of single fluorophores. The resolution is then only limited by the localization precision of single molecules, which is ~50nm.

Single Molecule Platform for Protein Interaction Analysis

FWF Stand-alone project, collaboration with Stefan Howorka, CBL GmbH Linz, Austria

The plasma membrane of T-cells contains a variety of protein complexes, which are important regulators of T-cell function. During activation, some complexes change their composition by fusing, segregating, or recruiting additional proteins. One prominent example is the T-cell receptor (TCR) complex, in which the T-cell receptor α- and β-chain are stably linked to CD3γ, CD3δ, CD3ε, CD3ζ, but also transiently associated with e.g. Lck, CD2, LAT, or ZAP-70. A second example is the coreceptor CD4, which recruits the kinase Lck to the TCR for tyrosine phosphorylation. In both examples, the stoichiometric composition of the complexes and the variability are only vaguely known.
With current technologies it has been difficult to obtain quantitative information on the hetero-oligomeric nature of such complexes. In this project, we will directly approach this need by developing a microscopy-based platform, which allows for quantitative measurements of the composition of individual protein complexes in the cellular plasma membrane. The new method will combine state-of-the-art strategies to nanostructure surfaces and to image single molecules in cells. It is based on a bait-prey technique previously introduced by us for studying protein-protein interactions in the live cell plasma membrane via protein micropatterning. For this, a bait-specific ligand is immobilized on a glass surface in a characteristic micropattern. When cells are grown on such substrates, the bait located in the plasma membrane follows these patterns. Interaction with a fluorescent prey leads to the rearrangement of the prey within the same patterns.
We propose now to extend such micropatterning platforms to the nanoscopic regime, and to combine them with single molecule microscopy. Our idea is to produce combined micro- and nanostructured surfaces of capture antibodies to the bait protein which is part of the complex to be investigated. Upon growing cells on such surfaces, the bait proteins and thereby the complexes will be immobilized along the nanopatterns. Combined micro- and nanopatterns will be designed to generate an analysis area, where the protein complexes can be immobilized to 50nm spots at mutual distances of 1µm, which provides sufficient separation for single molecule imaging. Additional microscale bulk areas of high capture antibody density will be used to remove the majority of the bait excess from the analysis area. This will allow us to use single molecule microscopy in the analysis area to count the number of different protein molecules contained in each complex. One part of the project aims at the development of the new platforms. In addition, single molecule tools will provide the readout for the second part of the project, the application of the new platform to T-cell biology.

StarPATT – Micropatterning-based protein-protein-interaction detection platform

FFG Bridge; collaboration with Julian Weghuber, Fachhochschule Wels, Austria, and with EVG

Mechanical Forces in T-Cell Antigen Recognition

WWTF Life Science Call 2013; collaboration with Johannes Huppa, Medical University of Vienna

T-cells readily detect the presence of even a single antigenic peptide/MHC complex (pMHC) among thousands of endogenous pMHCs via clonotypic T-cell receptors (TCRs) on the surface of antigen presenting cells (APCs). The mechanisms underlying this phenomenal sensitivity have remained elusive, but more recent studies suggest mechanical forces to be instrumental. To address their role most directly we will introduce calibrated force sensors into the immunological synapse to allow for quantitative visualization of forces acting between TCR and pMHC on opposing cell surfaces. Sensors will link the pMHC directly to the APC surface and function as a short spring, with fluorescent dyes site-specifically coupled to both ends: the spring collapses in case of zero force, giving rise to high Förster resonance energy transfer (FRET) between donor and acceptor dyes; FRET decreases when the spring is stretched. Forces exerted on the TCR will be measured in synapses of T-cells contacting functionalized planar lipid bilayers and also physiological synapses between T-cells and APCs. To evaluate their function in ligand discrimination and T-cell triggering, forces will be correlated to the stimulatory potency of chosen pMHCs, to TCR-pMHC bond rupture or TCR-proximal signaling, to be monitored simultaneously. We expect to reveal new principles underlying T-cell sensitivity, a true hallmark of adaptive immunity, and to break new ground in studying cell-cell contacts.

From organismic to biomolecular interactions: Visualizing signaling complexes in the fungal membrane during phytopathogenic attack

WWTF Life Science Call 2013; collaboration with Susanne Zeilinger, Vienna University of Technology

Mycoparasitic Trichoderma fungi are among the most successful biofungicides in today’s agriculture although our understanding of the exact molecular mechanisms of their activity still is fragmentary. The attack of phytopathogens by the mycoparasite is preceded by chemotropic growth towards the prey and pre-contact induction of its “molecular weapons”, consequently the receptors and signaling pathways involved in sensing and responding to the prey are of special interest. Cell surface receptors for host recognition, their ligands, interactors and localization in the membrane as well as membrane architecture itself are largely unexplored in pathogenic filamentous fungi. The objective of the proposed project is to gain detailed insights into the signaling dynamics and hence function of the virulence-associated Gpr1 receptor during chemotropism and interaction of Trichoderma with a prey. By combining fungal genetics, optical microscopy and biophysics, receptor localization and movement such as polarization towards the prey and association with its interactors shall be monitored at the single molecule level in the native cell membrane by recent super-resolution microscopy of living fungal hyphae. Imaging of Gpr1 signaling complexes at the single-molecule level in the mycoparasite during sensing and directed growth to the prey will reveal unprecedented insights into fundamental biological processes underlying fungal pathogenicity and will open up new avenues in super-resolution microscopy of fungal organisms.

MEIBio: Molecular and Elemental Imaging in Biosciences

TU Vienna; coordinator Martina Marchetti-Deschmann Link


FFG Nano Initiative; coordinator: Profactor GmbH Steyr, Austria Link