Scientific publications

The immune system distinguishes between self and foreign antigens. The kinetic proofreading (KPR) model proposes that T cells discriminate self from foreign ligands by the different ligand binding half-lives to the T cell receptor (TCR). It is challenging to test KPR as the available experimental systems fall short of only altering the binding half-lives and keeping other parameters of the interaction unchanged. We engineered an optogenetic system using the plant photoreceptor phytochrome B (PhyB) as a ligand to selectively control the dynamics of ligand binding to the TCR by light. This opto-ligand-TCR system was combined with the unique property of PhyB to continuously cycle between the binding and non-binding states under red light, with the light intensity determining the cycling rate and thus the binding duration. Mathematical modeling of our experimental datasets showed that indeed the ligand-TCR interaction half-life is the decisive factor for activating downstream TCR signaling, substantiating KPR. © Yousefi et al.

Franziska Lautenschläger studied physics at the University of Leipzig, Germany and Université Paul Sabatier Toulouse, France, before graduating with a physics diploma from Leipzig. For her PhD on mechanical changes during stem cell differentiation, she joined the laboratory of Jochen Guck at the University of Cambridge, UK. In 2011, Franziska moved to Paris, France, for her postdoctoral work on the migration of immune cells under confinement at the Institut Curie with supervisor Matthieu Piel. Since 2013, she has been an independent group leader and holds a junior professorship in biophysics from Saarland University, Saarbrücken, Germany. In addition, the Leibniz Institute for New Materials, Saarbrücken, appointed her as junior group leader in 2017. Franziska's research focuses on cell migration and polarity, and the links to cell mechanics and the different cytoskeletal networks in cells.

Cryopreservation is an essential tool to meet the increasing demand for stem cells in medical applications. To ensure maintenance of cell function upon thawing, the preservation of the actin cytoskeleton is crucial, but so far there is little quantitative data on the influence of cryopreservation on cytoskeletal structures. For this reason, our study aims to quantitatively describe cryopreservation induced alterations to F-actin in adherent human mesenchymal stem cells, as a basic model for biomedical applications. Here we have characterised the actin cytoskeleton on single-cell level by calculating the circular standard deviation of filament orientation, F-actin content, and average filament length. Cryo-induced alterations of these parameters in identical cells pre and post cryopreservation provide the basis of our investigation. Differences between the impact of slow-freezing and vitrification are qualitatively analyzed and highlighted. Our analysis is supported by live cryo imaging of the actin cytoskeleton via two photon microscopy. We found similar actin alterations in slow-frozen and vitrified cells including buckling of actin filaments, reduction of F-actin content and filament shortening. These alterations indicate limited functionality of the respective cells. However, there are substantial differences in the frequency and time dependence of F-actin disruptions among the applied cryopreservation strategies; immediately after thawing, cytoskeletal structures show least disruption after slow freezing at a rate of 1°C/min. As post-thaw recovery progresses, the ratio of cells with actin disruptions increases, particularly in slow frozen cells. After 120 min of recovery the proportion of cells with an intact actin cytoskeleton is higher in vitrified than in slow frozen cells. Freezing at 10°C/min is associated with a high ratio of impaired cells throughout the post-thawing culture.

During cell spreading, cells undergo many changes to their architecture and their mechanical properties. Vimentin, as an integral part of the cell architecture, and its mechanical stability must adapt to the new state of the cell. This study focuses on the structures formed by vimentin during the first steps of cell adhesion. Very early, ball-like structures, or ‘knots’, are seen and often vimentin filaments emerge in the shape of rings around the nucleus. Although intermediate filaments are not known to be associated to motor proteins to form contractile systems, these rings can nonetheless strongly deform the cell nucleus. In the first 6 h to 12 h of adhesion, these vimentin knots and rings disappear, and the intermediate filament network returns to the state seen before detachment of the cells. As these vimentin structures are very transient in the early steps of cell spreading, they have rarely been described in the literature. However, they can also be seen during mitosis, which is an event that involves partial detachment and re-spreading of the cells. Interestingly, the turnover dynamics of vimentin are reduced in both the knots and rings, compared to vimentin in the lamelipodia. It remains to de defined how the force is transmitted from the ball-like structures to the rings, and to measure the impact of such strong nuclear deformation on gene expression during cell re-spreading and the rearrangement of the vimentin network.

Extraordinarily small (2.4 nm) cobalt ferrite nanoparticles (ESCIoNs) were synthesized by a one-pot thermal decomposition approach to study their potential as magnetic resonance imaging (MRI) contrast agents. Fine size control was achieved using oleylamine alone, and annular dark-field scanning transmission electron microscopy revealed highly crystalline cubic spinel particles with atomic resolution. Ligand exchange with dimercaptosuccinic acid rendered the particles stable in physiological conditions with a hydrodynamic diameter of 12 nm. The particles displayed superparamagnetic properties and a low r2/r1 ratio suitable for a T1 contrast agent. The particles were functionalized with bile acid, which improved biocompatibility by significant reduction of reactive oxygen species generation and is a first step toward liver-targeted T1 MRI. Our study demonstrates the potential of ESCIoNs as T1 MRI contrast agents.

Abstract A strategy for spatial and temporal regulation of ligand presentation within a biomaterial, and the consequent site- and time-specific cellular responses in 4D cell cultures are presented. The key molecular component is a light-activatable adhesive peptidomimetic (cyclo Arg-Gly-Asp-phe-Cys, RGDfC) modified with the two-photon photocleavable group (p-methoxynitrobiphenyl, PMNB) used to functionalize a hydrogel. A scanning laser at 740 nm defines the 4D presentation of active RGD ligands within the gel, and directs basic cellular processes of embedded cells in situ. The excellent photochemical properties of the PMNB photoremovable group allows direct photomanipulation of the cellular environment without appreciable damage of the embedded cells. Light-directed migration of fibroblasts within a crosslinked poly(ethylene glycol) (PEG) hydrogel, and sequential, light-regulated angiogenesis with human umbilical vein endothelial cells (HUVECs) in 4D constructs is demonstrated. The materials presented here represent unique microenvironments to reconstruct dynamic changes in the composition of the extracellular space of cells that occur in in vivo tissues.

Design, optimization and integration of biosensors hold a great potential for the development of cost-effective screening and point-of-care technologies. However, significant progress in this field can still be obtained on condition that sufficiently accurate mathematical models will be developed. Herein, we present a novel approach for the improvement of mechanistic models which do not only combine the fundamental principles but readily incorporate the results of electrochemical and morphological studies. The first generation glucose biosensors were chosen as a case study for model development and to perform cyclic voltammetry (CV) measurements. As initial step in the model development we proposed the interpretation of experimental voltammograms obtained in the absence of substrate (glucose). The model equations describe dynamic diffusion and reaction of the involved species (oxygen, oxidized/reduced forms of the mediator – Prussian Blue/Prussian White). Furthermore, the developed model was applied under various operating conditions as a crucial tool for biosensor design optimization. The obtained qualitative and quantitative dependencies towards amperometric biosensors design optimization were independently supported by results of cyclic voltammetry and multi-analytical studies, such as scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX) and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Remarkably, a linear response of the optimized biosensors tested at the applied voltage (−0.14 V) in the presence of the glucose was obtained from 10−3 to 10−5 M (relative standard deviation (RSD) <7% per electrode). We believe that the presented model can be used to determine the exact mechanism driving the electrochemical reactions and to identify critical system parameters affecting the biosensor response that would significantly contribute to the knowledge on biosensing, device’s design and bioengineering strategies in the future.

In this paper, the removal of noble and non-noble metals from aqueous solutions on multi-dimensional hydroxyapatite microspheres (MD-HAp-Ms) in column experiments was studied. The efficiency of non-noble metal removal, viz. Co(II) and Ni(II) commonly presented in the environment, and the attachment mechanism onto MD-HAp-Ms readily depended on the concentration of H+ in the influent solution. In contrast, ICP-MS, SEM, XRD, TEM/EDX and RAMAN investigations independently revealed that the adsorption of Pb(II) onto the minicolumns was complete over the entire pH range and did not significantly depend on the medium acidity/basicity. The formation of a hydroxylpyromorphite phase with a general formula of Pb5-x/Cax(PO4)3OH onto the calcium MD-HAp-Ms minicolumns during Pb(II) uptake regardless from the used pH range was detected. Compared to non-noble metals, the noble ions Ag+, Pd2+, Pt2+, Au3+ formed nanoparticles with an average size of 10–50 nm during adsorption onto the MD-HAp-Ms in ammoniacal medium. The efficiency of noble ions removal was in accordance with their standard electrode potential (E0).