The Biophysics Division at the Department of Biology of the University of Osnabrück promotes research and teaching in molecular and cellular biophysics. Our mission is to bridge the gap between the biological and the physicochemical perspective on cellular processes. To this end we have developed a broad spectrum of biophysical tools for quantifying molecular functions in complex environment, often in the context of lipid membranes. We apply these tools for unraveling the mechanistic determinants of signal transduction across the plasma membrane and we share them in numerous collaborations at the Department of Biology and beyond. Within several Bachelor and Master Courses, we introduce the inherently interdisciplinary concepts of biophysics, which we believe are pivotal for fundamental progress in the understanding of life.
Ligand-induced signal activation via specific receptor proteins in the plasma membrane is a key process for the cell in order to fulfill its complex role within multicellular organisms. While signaling pathways have been mapped in detail, molecular and cellular determinants governing signal propagation across the membrane have remained controversially debated. For cytokine receptors, receptor tyrosine kinases and related receptors, an intricate interplay of lateral interactions and conformational changes induced by the interaction of the ligand to multiple receptor subunits is probably responsible for activating cytosolic effector proteins. Interestingly, a critical role of these processes in regulating signaling specificity is emerging. Our research is focused on developing experimental approaches to quantitatively dissect the molecular and cellular processes involved in receptor assembly and signal activation in the context of the membrane. We tackle this challenge by an interdisciplinary approach based on the following strategies:
Reconstitute signaling complexes on surfaces and in polymer-supported membranes for quantitative interaction studies.
Apply surface-sensitive detections techniques for monitoring protein-protein interactions at interfaces and within lipid membranes.
Unravel the spatiotemporal organization of signaling complexes in the plasma membrane of living cells by single molecule localization microscopy.
Exploit unique properties of nanoparticle as versatile reporter and actuator of molecular functions in living cells.
With a main focus on cytokine receptors, these approaches are used to explore receptor assembly and effector recruitment as well as its regulation by negative feedback regulators. Developing these technologies both profit from and feed into the unique research environment at the University of Osnabrück with its Collaborative Research Center "Physiology and Dynamics of Cellular Microcompartments"(DFG, SFB 944) and the interdisciplinary Center of Cellular Nanoanalytics (CellNanOs)
The spatiotemporal organization of cytokine receptor signaling complexes is currently controversially debated. While originally dimerization of two or more receptor subunits by simultaneous interaction with the ligand has been proposed, pre-dimerization and pre-clustering has emerged as an alternative concept. We have devised single molecule fluorescence imaging techniques for quantifying cytokine receptor assembly at the plasma membrane of living cells under close to physiological conditions, i.e. low cell surface receptor expression levels (1).
Binding of fluorescent IFN to endogenous IFNAR in HeLa cells at saturating concentrations.
For this purpose, we have employed posttranslational enzymatic labeling as well as nanobodies for efficient labeling with photostable fluorescence dyes. Using dual-color total internal reflection fluorescence (TIRF) microscopy, direct visualization of diffusion and interaction of the entire population of receptors was achieved in the plasma membrane of living cells (1, 2). Single molecule localization provides unique opportunities for quantitative evaluation of spatial co-organization of receptor subunits.
Single molecule localization-based analyses for quantifying the spatiotemporal receptor organization in the plasma membrane.
Detailed studies on the Type I Interferon (IFN) receptor excluded receptor predimerization or pre-organization in absence of the ligand. However, efficient receptor dimerization was observed upon addition of IFN (1).
Transient dimerization observed in presence IFN furthermore confirmed dynamic equilibrium between binary and ternary complexes.
Transient formation of an individual IFNAR1-IFNAR2 dimer observed by single molecule co-locomotion analysis.
Two-step dimerization of the IFN receptor and formation of a dynamic ternary complex.
Meanwhile, we have confirmed ligand-induced dimerization for several other cytokine receptors including the heterodimeric type II interleukin 4 (IL-4) receptor (2) and the homodimeric erythropoietin (Epo) receptor (3), both members of the class I cytokine receptor family. Surrogate ligands based on receptor-dimerizing diabodies were found to similarly activate signaling (3). These results establish a key role of ligand-induced receptor dimerization for cytokine receptor activation.
|K. Christopher Garcia||Stanford University||US|
|Thomas Müller||University of Würzburg||Germany|
|Christophe Lamaze||Institut Curie, Paris||France|
|Ian Hitchcock||University of York||UK|
Role of USP18 in the dimerization efficiency of IFN variants and mutants with different IFNAR1 binding affinities.
Several cytokine receptors bind different ligands, which can differentially activate cellular responses. The IFN receptor is a paradigm for such receptor plasticity, with 15 different ligands being recognized by a single cell surface receptor. Detailed studies of IFN recognition by its receptor subunits IFNAR1 and IFNAR2 revealed that binding affinity rather than structural differences are responsible for differential IFN activities, suggesting regulation at the level of receptor assembly (1-3). Indeed, systematic tailoring of IFN activities was shown to be possible by engineering its receptor binding affinities (2). Recently, negative feedback regulation by the ubiquitin-specific protease USP18 was found to be a key determinant for differential IFN activity (4). It turned out that USP18 negatively regulates receptor dimerization, probably by binding to the subunit IFNAR2 and thus interfering with possible interactions between the JAKs, as observed by single molecule dimerization experiments (5).
Based on this insight, we have proposed that differential IFN activities are caused by a temporal change in potency, which is determined by a complex interplay of different feedback mechanisms (6, 7).
Feedback mechanisms controlling differential interferon activities, which are characterized by different affinity-potency relationships observed for short-term and long-term cellular responses (6).
|Gideon Schreiber||The Weizmann Institute of Science, Rehovot||Israel|
|Gilles Uzé||CNRS Montpellier||France|
|K. Christopher Garcia||Stanford University||US|
|Christophe Lamaze||Institut Curie, Paris||France|
|Sandra Pellegrini||Institut Pasteur||France|
|Ignacio Moraga||University of Dundee||UK|
Tracking of a labeled IFN bound to transmembrane IFNAR2 reconstituted into micropatterned PSM (boundary indicated by the white dashed line).
Reconstitution into polymer-supported membranes (PSM) is a promising approach for studying membrane protein interaction and conformations in a controlled lipid environment by advanced imaging techniques. Based on a dense PEG polymer brush and hydrophobic anchoring groups for capturing protein liposomes, we have developed a robust approach for PSM assembly on glass-type surfaces, which is compatible with single molecule fluorescence imaging, fluorescence correlation spectroscopy and atomic force microscopy (1). Rapid diffusion of a large variety of transmembrane proteins reconstituted into these PSM was found, as well as retained functionality with respect to protein-protein interactions (1, 2).
As the lipid composition can be controlled in these PSM, separation of ld and lo lipid phases is readily achieved (3). Moreover, micropatterned organization of anchoring groups provides unique possibilities to control PSM formation and lipid phase-separation in a spatially resolved manner (3).
Spatially controlled assembly of phase-separated PSM based on micropatterned palmitic and oleic acid moieties.
As only very minor protein quantities are typically demanded for single molecule applications, production and rapid transfer from mammalian cells is possible (4).
Rapid transfer of transmembrane receptors expressed in HeLa cells into polymer-supported membranes for single molecule fluorescence imaging.
Based on this approach, ligand-induced heterodimerization of the IFN receptor could be quantified in a reconstituted system (4).
Single molecule dimerization of transmembrane IFNAR1 and IFNAR2 in polymer-supported membranes.
Next to transmembrane receptors, reconstitution of integral membrane proteins was achieved including beta-barrel outer membrane proteins, for which spontaneous aggregation into clusters was observed (5).
Micropatterned phase-separated PSMs open exciting perspectives for bioanalytical applications. Based on membrane tethering of His-tagged proteins via tris-NTA lipid, phase partitioning upon dimerization was exploited as a readout for protein-protein interaction (6). Thus, weak binding affinities up to 1 mM could be determined for proteins directly captured to surfaces from mammalian cell lysates (6).
|Heinz-Jürgen Steinhoff||University of Osnabrück||Germany|
|Ünal Coskun||Paul Langerhans Institute Dresden||Germany|
|Jörg Enderlein||University of Göttingen||Germany|
Inorganic nanoparticles provide unique physical properties for visualizing and manipulating cellular processes down to the single molecule level. However, biofunctionalization of these nanoparticles for efficient and stoichiometrically defined conjugation with target proteins without biasing their function remains challenging. We have developed approaches for controlling conjugation stoichiometry by exploiting electrostatic steering of coupling reactions (1, 2). Thus, unbiased diffusion of protein diffusion and interaction at the plasma membrane could be probed using monofunctional quantum dots for cell surface receptor labeling.
Spatiotemporal dynamics of IFN-receptor complexes in living cells visualized by dual color quantum dot tracking.
For efficient nanoparticle targeting inside living cells, we have optimized a substrate for fast, covalent reaction with HaloTag fusion proteins (3). On our quest to apply nanoparticles in the cytosol of living cells, we identified autophagy as a critical mechanism for nanoparticle clearance (4). Using massive PEGylation, monofunctionalized stealth nanoparticles were generated, which were efficiently targeted to a protein in the outer mitochondrial membrane (4).
Magnetogenetic manipulation of Rho GTPase signaling in living cells (5).
Based on these approaches for nanoparticle biofunctionalization, we demonstrated spatially controlled manipulation of active G-protein signaling platforms assembled on the surface of magnetic nanoparticles in living cell (5).
The concept of "magnetogenetic"manipulation opens exciting perspectives as a tool for fundamental cell biology and for medical applications. However, refined surface functionalization and delivery strategies on the basis of sub 50 nm-sized magnetic nanoparticles (6) will be required for practical applications.
|Maxime Dahan||Institut Curie, Paris||France|
|Rolf Heumann||University of Bochum||Germany|
|Alicia El Haj||University of Keele||UK|
|Christine Ménager||CNRS UPMC, Paris||France|
|Markus Haase||University of Osnabrück||Germany|
Protein immobilization on solid support opens versatile possibilities for protein interaction analysis. We have developed surface modification for functional protein immobilization based on site-specific protein capturing. For stable, yet reversible protein immobilization we have developed tris(nitrilotriacetic acid) (tris-NTA), which binds oligohististidine-tagged proteins with nanomolar affinity due to multivalent interactions (1, 2). We have established a large repertoire of molecular tools for surface modification and protein labeling based on tris-NTA (3). For increased specificity and stability of protein capturing, this approach was complemented by enzyme-based coupling techniques including enzymatic phosphopantetheinyl transfer as well as the HaloTag (4). For spatially resolved protein immobilization, we have combined these capturing techniques with soft and photolithographic surface patterning techniques, yielding a versatile toolbox for functional protein organization on surfaces (5, 6).
Microtubule (red) transported by motor proteins (green) immobilized via micropattern tris-NTA (5).
A fundamental objective of these approaches is to develop robust tools for interfacing proteins with surfaces while maintaining the physiological context, e.g. lipid environment of membrane proteins or cellular interaction partners. To this end, we developed micropatterned surface architectures for capturing protein in the plasma membrane of living cells (7, 8).
Surface architecture for capturing HaloTag fusion proteins in the plasma membrane of living cells.
Based on this technique protein-protein interactions at membranes such as receptor hetero-dimerization or effector recruitment can be readily detected and quantified in living cells.
Triggered micropatterning of IFNAR1 (green) in the plasma membrane of living cells followed by ligand-induced association of IFNAR2 (red) (8).
For studying cytosolic protein complexes, we have established surface architectures for pull-down of GFP-tagged proteins from single cells (SiCPull). In combination with single molecule TIRF microscopy, we succeeded to quantify stability and stoichiometry of complexes captured by SiCPull.
Concept of single cell pulldown for quantifying the stability and the stoichiometry of protein complexes (9).
Micropatterned surface capturing of a GFP-tagged cytosolic protein upon cell lysis (9).
By including nanoparticles as additional spectroscopic reporters into micropatterned surface architectures, versatile readouts for biomolecular interactions and conformational changes can be generated. Thus, we implemented label-free detection imaging of protein-protein interactions by localized surface plasmon resonance (LSPR) with micropatterned gold nanoparticles (10).
|Jürgen Klingauf||University of Münster||Germany|
|Thomas Schröder||IHP Frankfurt/Oder||Germany|
|Bernd Witzigmann||University of Kassel||Germany|
Visualization of cellular processes in living cells with highest spatial and temporal resolution plays a key role in modern cell biology. Recent breakthroughs in overcoming the diffraction limitation in far-field fluorescence microscopy have opened exciting perspectives for live cell imaging. We have focused on single molecule localization microscopy (SMLM) techniques such as fluorescence photoactivation localization microscopy (FPALM) and stochastic optical reconstruction microscopy (STORM). By combining FPALM and direct STORM, we could demonstrate triple color superresolution imaging in living cells (1).
Triple-color superresolution imaging in living cells by combining FPALM (PAGFP and PAmCherry) and dSTORM (ATTO655 coupled via the HaloTag) reveals coorganization of receptor subunits (green, red) in the context of the cortical actin cytoskeleton (blue).
While PALM is typically limited to a single read-out cycle, life cell dSTORM opens exciting possibilities for superresolution imaging of cellular nanostructures over prolonged time scales (1).
Time-lapse superresolution imaging of clathrin dynamics at the plasma membrane using dSTORM (ATTO655).
Time-lapse dual-color TALM imaging of IFNAR2 (green) and its effector protein STAT2 (red).
Classic SMLM requires high overexpression of target proteins in order to achieve sufficient density to fulfill the Nyquist criterion, which may affect cellular functions and morphologies. For SMLM at physiological expression levels, we have introduced tracking and localization microscopy (TALM), which is based on a small ensemble of permanently fluorescent species, which successively explore cellular nanostructures by diffusion in the living cell (2, 3). This technique is particularly applicable to membrane proteins as their mobility is compatible with precise localization. Thus, not only morphologies and proteins mobility, but also connectivities or barriers can be identified. Pair correlation applied to TALM imaging thus provides information about spatial and temporal protein organization, which opens exciting possibilities for identifying and quantifying protein clustering and co-clustering (4).
Application of SMLM on biological questions requires access to dedicated image analysis tools not only for robust localization and tracking of individual emitters, but also for versatile quantification of spatial and temporal properties such as (co-)clustering, co-locomotion, complex stoichiometries or morphologies. We are constantly developing an integrated software package providing versatile tools for such analyses. We share these advanced imaging and evaluation techniques within the framework of the DFG-funded Integrated Bioimaging Facility Osnabrück (iBiOs) at the Center of Cellular Nanoanalytics (CellNanOs).
|Eric Betzig||Janelia Farm Research Campus||US|
|Stefan Kunis||University of Osnabrück||Germany|
|Karin Busch||University of Münster||Germany|
Annett Reichel, Imke Peters, Pia Müller, Ramunas Valiokas, Peter Lamken, Suman Lata, Yvonne Becker, Martynas Gavutis, Eva Jaks, Natalie Al-Furoukh, Irina Ohlmer, Jennifer Julia Strunk, Maniraj Bhagawati, Sharon Waichman, Yulia Podoplelova, Dirk Paterok, Friedrich Roder, Markus Staufenbiel, Oliver Beutel, Amine Aladag, Stephan Wilmes, David Richter, Oliver Birkholz
We can offer a spirited and committed scientific atmosphere in an interdisciplinary group jointly working on exciting topics of molecular cell biophysics.
State of the art technology in the field of molecular, structural and cell biology, protein and peptide biochemistry as well as optical spectroscopy and biophysics is available.
Students of different nationalities work in our research department, and thus, the language of all research seminars is English.
The doctoral students in the group have joined us from many different Universities in Europe and have studied Biochemistry, Biology or Chemistry. PhD-students are usually paid according to TVöD E13, or by fellowships from the DFG, which is in the same order of magnitude.
Apply for position
Typically, postdoctoral fellows should be successful in obtaining a fellowship supporting at least part of the time they spent in the group. Not all fellowships are open to all nationalities and therefore strategies have to be specifically devised. We will, however, be committed to help with these applications.
Apply for position
Several departments encourage or even require students to spend some time in a research lab to acquire experimental skills. We regularly host such students, several of whom have returned later for diploma and/or doctoral work. Usually such stays last from 6 weeks to 4 months, and the students must have basic training in experimental biochemistry before coming. They would work under the guidance of an experienced doctoral student in an ongoing project. Please be aware such stays need to be arranged well in advance, and therefore contact us in due course.
Apply for position
Exit A30 at Hellern and follow signs towards the inner city. At the inner city ring, turn left and follow signs "Fachhochschule", "Universität Osnabrück, Standort Westerberg". Building 35-37 is located at the end of the Barbarastasse.
From Münster-Osnabrück airport (FMO) take the shuttle bus to the train station in Osnabrück. (Timetable). To reach us from the train station please refer to the "Travelling by Train" information.
From the train station "Bussteig 1" take bus 21 to the stop "Hochschulen Westerberg" (Timetable). From there you can walk within 5 minutes to our institute, located in building 36. You will find us on the first floor, left corridor.