Ilia Louban

Impact of local versus global ligand density on cellular adhesion

α(v)β(3) integrin-mediated cell adhesion is crucially influenced by how far ligands are spaced apart. To evaluate the impact of local ligand density versus global ligand density of a given surface, we used synthetic micronanostructured cell environments with user-defined ligand spacing and patterns to investigate cellular adhesion. The development of stable focal adhesions, their number, and size as well as the cellular adhesion strength proved to be influenced by local more than global ligand density.

Nanopatterning by block copolymer micelle nanolithography and bioinspired applications

This comprehensive overview of block copolymer micelle nanolithography (BCMN) will discuss the synthesis of inorganic nanoparticle arrays by means of micellar diblock copolymer approach and the resulting experimental control of individual structural parameters of the nanopattern, e.g., particle density and particle size. Furthermore, the authors will present a combinational approach of BCMN with conventional fabrication methods, namely, photolithography and electron beam lithography, which combines the advantages of high-resolution micronanopatterning with fast sample processing rates. In addition, the authors will demonstrate how these nanoparticle assemblies can be transferred to polymer substrates with a wide range of elasticity. In the second part of this report the authors will introduce some of the most intriguing applications of BCMN in biology and materials science: The authors will demonstrate how nanoparticle arrays may be used as anchor points to pattern functional proteins with single molecule resolution for studying cellular adhesion and present a technological roadmap to high-performance nanomaterials by highlighting recent applications for biomimetic optics and nanowires.

Immune synapse formation determines interaction forces between T cells and antigen-presenting cells measured by atomic force microscopy

During adaptive immune responses, T lymphocytes recognize antigenic peptides presented by MHC molecules on antigen-presenting cells (APCs). This recognition results in the formation of a so-called immune synapse (IS) at the T-cell/APC interface, which is crucial for T-cell activation. The molecular composition of the IS has been extensively studied, but little is known about the biophysics and interaction forces between T cells and APCs. Here, we report the measurement of interaction forces between T cells and APCs employing atomic force microscopy (AFM). For these investigations, specific T cells were selected that recognize an antigenic peptide presented by MHC-class II molecules on APCs. Dynamic analysis of T-cell/APC interaction by AFM revealed that in the presence of antigen interaction forces increased from 1 to 2 nN at early time-points to a maximum of ≈14 nN after 30 min and decreased again after 60 min. These data correlate with the kinetics of synapse formation that also reached a maximum after 30 min, as determined by high-throughput multispectral imaging flow cytometry. Because the integrin lymphocyte function antigen-1 (LFA-1) and its counterpart intercellular adhesion molecule-1 (ICAM-1) are prominent members of a mature IS, the effect of a small molecular inhibitor for LFA-1, BIRT377, was investigated. BIRT377 almost completely abolish the interaction forces, emphasizing the importance of LFA-1/ICAM-1-interactions for firm T-cell/APC adhesion. In conclusion, using biophysical measurements, this study provides precise values for the interaction forces between T cells and APCs and demonstrates that these forces develop over time and are highest when synapse formation is maximal.

Polymeric substrates with tunable elasticity and nanoscopically controlled biomolecule presentation

Despite tremendous progress in recent years, nanopatterning of hydrated polymeric systems such as hydrogels still represents a major challenge. Here, we employ block copolymer nanolithography to arrange gold nanoparticles on a solid template, followed by the transfer of the pattern to a polymeric hydrogel. In the next step, these nanoparticles serve as specific anchor points for active biomolecules. We demonstrate the engineering of poly(ethylene glycol) hydrogel surfaces with respect to elasticity, nanopatterning, and functionalization with biomolecules. For the first time, biomolecule arrangement on the nanometer scale and substrate stiffness can be varied independently from each other. Youngs moduli, a measure of the compliance of the substrates, can be tuned over 4 orders of magnitude, including the values for all of the different tissues found in the human body. Structured hydrogels can be used to pattern any histidine-tagged protein as exemplified for his-protein A as an acceptor for immunoglobulin. When cell-adhesion-promoting peptide cRGDfK is selectively coupled to gold nanoparticles, the surfaces provide cues for cell-surface interaction and allow for the study of the modulation of cellular adhesion by the mechanical properties of the environment. Therefore, these substrates represent a unique multipurpose platform for studying receptor/ligand interactions with adhering cells, mechanotransduction, and cell-adhesion-dependent signaling.

Single cell force spectroscopy of T cells recognizing a myelin-derived peptide on antigen presenting cells

T-cell recognition of peptide-MHC complexes on APCs requires cell-cell interactions. The molecular events leading to T-cell activation have been extensively investigated, but the underlying physical binding forces between T-cells and APCs are largely unknown. We used single cell force spectroscopy for quantitation of interaction forces between T-cells and APCs presenting a tolerogenic peptide derived from myelin basic protein. When T-cells were brought into contact with peptide-loaded APCs, interaction forces increased with time from about 0.5nN after 10s interaction to about 15nN after 30min. In the absence of antigen, or when ICAM-1-negative APC was used, no increase in binding forces was observed. The temporal development of interaction forces correlated with the kinetics of immune synapse formation, as determined by LFA-1 and TCR enrichment at the interface of T-cell/APC conjugates using high throughput multispectral imaging flow cytometry. Together, these results suggest that ICAM-1/LFA-1 redistribution to the contact area is mainly responsible for development of strong interaction forces. High forces will keep T-cells and APCs in tight contact, thereby providing a platform for optimal interaction between TCRs and peptide-MHC complexes.

Intelligent induction of active biosystem responses at interfaces

Abstract Intelligent interfaces make use of a fundamental molecular understanding of biosystems for the induction of their specific responses. Biological cells especially have an enormous spatial resolution for organizing their transmembrane receptor molecules, which is translated into specific cell functions. In turn, interfaces which provide a counter organization of molecules to required transmembrane receptor organizations are able to induce specific cell responses accordingly. This mission requires a patterning technology at interfaces which operates at the resolution of single molecules. Here, we report on self-assembly technologies for providing such patterns and their subsequent functionalization with cell receptor binding molecules. Cells explore such surfaces and show a very distinct cell response. In the future, such interfaces may “learn” how to induce cell responses properly by analyzing cell responses and providing dynamically the adequate interface pattern which will allow cells to act more...