Publikationen

Molecular signals are sensed by their respective receptors and information is transmitted and processed by a sophisticated intracellular network controlling various biological functions. Optogenetic tools allow the targeting of specific signaling nodes for a precise spatiotemporal control of downstream effects. These tools are based on photoreceptors such as phytochrome B (PhyB), cryptochrome 2, or light-oxygen-voltage-sensing domains that reversibly bind to specific interaction partners in a light-dependent manner. Fusions of a protein of interest to the photoreceptor or their interaction partners may enable the control of the protein function by light-mediated dimerization, a change of subcellular localization, or due to photocaging/-uncaging of effectors. In this review, we summarize the photoreceptors and the light-based mechanisms utilized for the modulation of signaling events in mammalian cells focusing on non-neuronal applications. We discuss in detail optogenetic tools and approaches applied to control signaling events mediated by second messengers, Rho GTPases and growth factor-triggered signaling cascades namely the RAS/RAF and phosphatidylinositol-3-kinase pathways. Applying the latest generation of optogenetic tools allows to control cell fate decisions such as proliferation and differentiation or to deliver therapeutic substances in a spatiotemporally controlled manner. Optogenetic tools enable the spatiotemporal control of the function of genetically encoded proteins by light. The authors discuss optogenetic tools and approaches applied to control signaling events mediated by second messengers, Rho GTPases and signaling cascades namely the RAS/RAF and PI3K/AKT pathways. Applying the latest generation of optogenetic tools allows to control cell fate decisions such as proliferation and differentiation or to deliver therapeutic substances in a spatiotemporally-controlled manner. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fluorogenic RNAs that are based on the complex formed by 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) derivatives and the RNA aptamer named Spinach were used to engineer a new generation of in vitro and in vivo sensors for bioanalytics. With the resolved crystal structure of the RNA/small molecule complex, the engineering map becomes available, but comprehensive information regarding the thermodynamic profile of the molecule is missing. Here, we reconstructed the full thermodynamic binding and stability landscapes between DFHBI and a truncated sequence of first-generation Spinach. For this purpose, we established a systematic screening procedure for single- and double-point mutations on a microfluidic large-scale integrated chip platform for 87-nt long RNAs. The thermodynamic profile with single base resolution was used to engineer an improved fluorogenic spinach generation via a directed rather than evolutional approach. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Background: Multicellular organisms depend on the exchange of information between specialized cells. This communication is often difficult to decipher in its native context, but synthetic biology provides tools to engineer well-defined systems that allow the convenient study and manipulation of intercellular communication networks. Results: Here, we present the first mammalian synthetic network for reciprocal cell-cell communication to compute the border between a sender/receiver and a processing cell population. The two populations communicate via Ltryptophan and interleukin-4 to highlight the population border by the production of a fluorescent protein. The sharpness of that visualized edge can be adjusted by modulating key parameters of the network. Conclusions: We anticipate that this network will on the one hand be a useful tool to gain deeper insights into the mechanisms of tissue formation in nature and will on the other hand contribute to our ability to engineer artificial tissues. © 2015 Kolar et al.

The perivascular niche is a complex microenvironment containing mesenchymal stem cells (MSCs), among other perivascular cells, as well as temporally organized biochemical and biophysical gradients. Due to a lack of conclusive phenotypic markers, MSCs' identity, heterogeneity and function within their native niche remain poorly understood. The in vitro reconstruction of an artificial three-dimensional (3D) perivascular niche would offer a powerful alternative to study MSC behavior under more defined conditions. To this end, we here present a poly(ethylene glycol)-based in vitro model that begins to mimic the spatiotemporally controlled presentation of biological cues within the in vivo perivascular niche, namely a stably localized platelet-derived growth factor B (PDGF-BB) gradient. We show that 3D-encapsulated MSCs respond to soluble PDGF-BB by proliferation, spreading, and migration in a dose-dependent manner. In contrast, the exposure of MSCs to 3D matrix-tethered PDGF-BB gradients resulted in locally restricted morphogenetic responses, much as would be expected in a native perivascular niche. Thus, the herein presented artificial perivascular niche model provides an important first step towards modeling the role of MSCs during tissue homeostasis and regeneration. © 2015 The Royal Society of Chemistry.

The in vitro formation of physiologically relevant engineered tissues is still limited by the availability of adequate growth-factor-presenting cell-instructive biomaterials, allowing simultaneous and three-dimensionally localized differentiation of multiple tissue progenitor cells. Together with ever improving technologies such as microfluidics, printing, or lithography, these biomaterials could provide the basis for generating provisional cellular constructs, which can differentiate to form tissue mimetics. Although state-of-the-art biomaterials are endowed with sophisticated modules for time- and space-controlled positioning and release of bioactive molecules, reports on 3D arrangements of differentiation-inducing growth factors are scarce. This paper describes the stable and localized immobilization of biotinylated bioactive molecules to a modular, Factor XIII-cross-linked poly(ethylene glycol) hydrogel platform using a genetically engineered streptavidin linker. Linker incorporation is demonstrated by Western blot, and streptavidin functionality is confirmed by capturing biotinylated alkaline phosphatase (ALP). After optimizing bone morphogenetic protein 2 (BMP-2) biotinylation, streptavidin-modified hydrogels are able to bind and present bioactive BMP-2-biotin. Finally, with this immobilization scheme for BMP-2, the specific osteogenic differentiation of mesenchymal stem cells is demonstrated by inducing ALP expression in confined 3D areas. In future, this platform together with other affinity-based strategies will be useful for the local incorporation of various growth factors for engineering cell-responsive constructs. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular switches that are controlled by chemicals have evolved as central research instruments in mammalian cell biology. However, these tools are limited in terms of their spatiotemporal resolution due to freely diffusing inducers. These limitations have recently been addressed by the development of optogenetic, genetically encoded, and light-responsive tools that can be controlled with the unprecedented spatiotemporal precision of light. In this article, we first provide a brief overview of currently available optogenetic tools that have been designed to control diverse cellular processes. Then, we focus on recent developments in light-controlled gene expression technologies and provide the reader with a guideline for choosing the most suitable gene expression system. © 2015 by De Gruyter.

The rapid development of mammalian optogenetics has produced an expanding number of gene switches that can be controlled with the unprecedented spatiotemporal resolution of light. However, in the "pre-optogenetic" era many networks, cell lines and transgenic organisms have been engineered that rely on chemically-inducible transgene expression systems but would benefit from the advantages of the traceless inducer light. To open the possibility for the effortless upgrade of such systems from chemical inducers to light, we capitalized on the specific Med25VBD inhibitor of the VP16/VP64 transactivation domain. In a first step, we demonstrated the efficiency and selectivity of Med25VBD in the inhibition of VP16/VP64-based transgene expression systems. Then, we fused the inhibitor to the blue light-responsive B-LID degron and optimized the performance of this construct with regard to the number of Med25VBD repeats. This approach resulted in an optogenetic upgrade of the popular Tet-OFF (TetR-VP64, tetO7-PhCMVmin) system that allows tunable, blue light-inducible transgene expression in HEK-293T cells. © 2015 Wiley Periodicals, Inc..

Purpose The aim of this study was to compare the proliferation and attachment behavior of fibroblasts and epithelial cells on differently structured abutment materials. Materials and Methods Three different surface topographies were prepared on zirconia and titanium alloy specimens and defined as follows: machined (as delivered without further surface modification), smooth (polished), and rough (sandblasted). Energy-dispersive X-ray spectroscopy, topographical analysis, and water contact angle measurements were used to analyze the surface properties. Fibroblasts (HGF1) and epithelial cells (HNEpC) grown on the specimens were investigated 24 hours and 72 hours after seeding and counted using fluorescence imaging. To investigate adhesion, the abundance and arrangement of the focal adhesion protein vinculin were evaluated by immunocytochemistry. Results Similar surface topographies were created on both materials. Fibroblasts exhibited significant higher proliferation rates on comparable surface topographies of zirconia compared with the titanium alloy. The proliferation of fibroblasts and epithelial cells was optimal on different substrate/topography combinations. Cell spreading was generally higher on polished and machined surfaces than on sandblasted surfaces. Rough surfaces provided favorable properties in terms of cellular adhesion of fibroblasts but not of epithelial cells. Conclusions Our data support complex soft tissue cell-substrate interactions: the fibroblast and epithelial cell response is influenced by both the material and surface topography.

The surface of the medical grade titanium alloy (Ti–6Al–4V) was modified using a millisecond laser to alter the topography and wetting behavior. We combined anisotropic surface patterning with a nitriding process which is essential for the protection of dental implants against corrosion and wear. While a homogenous nitride layer was achieved, the laser induced a surface topography composed of both micro- and nano-structures. The surface topography leads to anisotropy in wetting which is desired in next generation implant applications such as nature-analogue anisotropic dental restoration.