Simon Schulz

A red/far-red light-responsive bi-stable toggle switch to control gene expression in mammalian cells

Growth and differentiation of multicellular systems is orchestrated by spatially restricted gene expression programs in specialized subpopulations. The targeted manipulation of such processes by synthetic tools with high-spatiotemporal resolution could, therefore, enable a deepened understanding of developmental processes and open new opportunities in tissue engineering. Here, we describe the first red/far-red light-triggered gene switch for mammalian cells for achieving gene expression control in time and space. We show that the system can reversibly be toggled between stable on- and off-states using short light pulses at 660 or 740 nm. Red light-induced gene expression was shown to correlate with the applied photon number and was compatible with different mammalian cell lines, including human primary cells. The light-induced expression kinetics were quantitatively analyzed by a mathematical model. We apply the system for the spatially controlled engineering of angiogenesis in chicken embryos. The system’s performance combined with cell- and tissue-compatible regulating red light will enable unprecedented spatiotemporally controlled molecular interventions in mammalian cells, tissues and organisms.

Multi-chromatic control of mammalian gene expression and signaling

The emergence and future of mammalian synthetic biology depends on technologies for orchestrating and custom tailoring complementary gene expression and signaling processes in a predictable manner. Here, we demonstrate for the first time multi-chromatic expression control in mammalian cells by differentially inducing up to three genes in a single cell culture in response to light of different wavelengths. To this end, we developed an ultraviolet B (UVB)-inducible expression system by designing a UVB-responsive split transcription factor based on the Arabidopsis thaliana UVB receptor UVR8 and the WD40 domain of COP1. The system allowed high (up to 800-fold) UVB-induced gene expression in human, monkey, hamster and mouse cells. Based on a quantitative model, we determined critical system parameters. By combining this UVB-responsive system with blue and red light-inducible gene control technology, we demonstrate multi-chromatic multi-gene control by differentially expressing three genes in a single cell culture in mammalian cells, and we apply this system for the multi-chromatic control of angiogenic signaling processes. This portfolio of optogenetic tools enables the design and implementation of synthetic biological networks showing unmatched spatiotemporal precision for future research and biomedical applications.

Mechano-transduction in periodontal ligament cells identifies activated states of MAP-kinases p42/44 and p38-stress kinase as a mechanism for MMP-13 expression

BackgroundMechano-transduction in periodontal ligament (PDL) cells is crucial for physiological and orthodontic tooth movement-associated periodontal remodelling. On the mechanistic level, molecules involved in this mechano-transduction process in PDL cells are not yet completely elucidated.ResultsIn the present study we show by western blot (WB) analysis and/or indirect immunofluorescence (IIF) that mechanical strain modulates the amount of the matrix metalloproteinase MMP-13, and induces non-coherent modulation in the amount and activity of signal transducing molecules, such as FAK, MAP-kinases p42/44, and p38 stress kinase, suggesting their mechanistic role in mechano-transduction. Increase in the amount of FAK occurs concomitant with increased levels of the focal contact integrin subunits β3 and β1, as indicated by WB or optionally by IIF. By employing specific inhibitors, we further identified p42/44 and p38 in their activated, i.e. phosphorylated state responsible for the expression of MMP-13. This finding may point to the obedience in the expression of this MMP as extracellular matrix (ECM) remodelling executioner from the activation state of mechano-transducing molecules. mRNA analysis by pathway-specific RT-profiler arrays revealed up- and/or down-regulation of genes assigning to MAP-kinase signalling and cell cycle, ECM and integrins and growth factors. Up-regulated genes include for example focal contact integrin subunit α3, MMP-12, MAP-kinases and associated kinases, and the transcription factor c-fos, the latter as constituent of the AP1-complex addressing the MMP-13 promotor. Among others, genes down-regulated are those of COL-1 and COL-14, suggesting that strain-dependent mechano-transduction may transiently perturbate ECM homeostasis.ConclusionsStrain-dependent mechano-/signal-transduction in PDL cells involves abundance and activity of FAK, MAP-kinases p42/44, and p38 stress kinase in conjunction with the amount of MMP-13, and integrin subunits β1 and β3. Identifying the activated state of p42/44 and p38 as critical for MMP-13 expression may indicate the mechanistic contribution of mechano-transducing molecules on executioners of ECM homeostasis.

Environmental constraints guide migration of malaria parasites during transmission.

Migrating cells are guided in complex environments mainly by chemotaxis or structural cues presented by the surrounding tissue. During transmission of malaria, parasite motility in the skin is important for Plasmodium sporozoites to reach the blood circulation. Here we show that sporozoite migration varies in different skin environments the parasite encounters at the arbitrary sites of the mosquito bite. In order to systematically examine how sporozoite migration depends on the structure of the environment, we studied it in micro-fabricated obstacle arrays. The trajectories observed in vivo and in vitro closely resemble each other suggesting that structural constraints can be sufficient to guide Plasmodium sporozoites in complex environments. Sporozoite speed in different environments is optimized for migration and correlates with persistence length and dispersal. However, this correlation breaks down in mutant sporozoites that show adhesion impairment due to the lack of TRAP-like protein (TLP) on their surfaces. This may explain their delay in infecting the host. The flexibility of sporozoite adaption to different environments and a favorable speed for optimal dispersal ensures efficient host switching during malaria transmission.

Focal adhesion kinase (FAK) perspectives in mechanobiology: implications for cell behaviour.

Mechanobiology is a scientific interface discipline emerging from engineering and biology. With regard to tissue-regenerative cell-based strategies, mechanobiological concepts, including biomechanics as a target for cell and human mesenchymal stem cell behaviour, are on the march. Based on the periodontium as a paradigm, this mini-review discusses the key role of focal-adhesion kinase (FAK) in mechanobiology, since it is involved in mediating the transformation of environmental biomechanical signals into cell behavioural responses via mechanotransducing signalling cascades. These processes enable cells to adjust quickly to environmental cues, whereas adjustment itself relies on the specific intramolecular phosphorylation of FAK tyrosine residues and the multiple interactions of FAK with distinct partners. Furthermore, interaction-triggered mechanotransducing pathways govern the dynamics of focal adhesion sites and cell behaviour. Facets of behaviour not only include cell spreading and motility, but also proliferation, differentiation and apoptosis. In translational terms, identified and characterized biomechanical parameters can be incorporated into innovative concepts of cell- and tissue-tailored clinically applied biomaterials controlling cell behaviour as desired.

Modulation of focal adhesion constituents and their down-stream events by EGF: On the cross-talk of integrins and growth factor receptors.

Within the concept of integrin growth factor receptor (GFR) cross-talk, little is known about the effects of GFRs on focal adhesions (FAs). Therefore, we tested the hypothesis whether EGF can modulate constituents of FAs and subsequent down-stream events. To this end, EGF-treated keratinocytes were subjected to combined fluorescence imaging and western blotting, to quantify expression and/or activation of molecules, involved in integrin GFR cross-talk, and receptor proximal and distal signaling events. Generally, EGF response revealed an amplified redistribution or activation of molecules under study, which will be explained in detail from the plasma membrane to the cell interior. In addition to significant activation of EGF receptor (EGFR) at tyrosine Tyr845, a remarkable redistribution was detectable for the focal adhesion constituents, integrin ß1 and ß3, and zyxin. Increased activation also applied to focal adhesion kinase (FAK) by phosphorylation at Tyr397, Tyr576, and Src at Tyr418, while total FAK remained unchanged. Risen activity was seen as well for the analyzed distal down-stream events, p190RhoGAP and MAP kinases p42/44. Intriguingly, Src-specific inhibitor Herbimycin A abrogated the entire EGF response except FAK Tyr397 phosphorylation, independent of EGF presence. Mechanistically, our results show that EGF modulates adhesion in a dual fashion, by firstly redistributing focal adhesion constituents to adhesion sites, but also by amplifying levels of activated RhoA antagonist p190RhoGAP, important for cell motility. Further, the findings suggest that the observed EGF response underlies an EGFR integrin cross-talk under recruitment of receptor proximal FAK and Src, and MAP kinase and p190RhoGAP as receptor distal events.

Expression of keratinocyte biomarkers is governed by environmental biomechanics.

In solid body tissues, environmental biomechanics is indispensable for tissue homeostasis. While characteristics of homeostasis include morphogenesis, proliferation and differentiation, the influences through biomechanics in corneal keratinocytes are poorly understood. Here we show for the first time that corneal keratinocytes, established in a defined biomechanical microenvironment of micropatterned soft pillars, exhibit favoritism of late and terminal differentiation at large pillar patterns of 11 μm with matched small 5 μm arrays. At 11 μm, epithelial cells expressed decreased levels of early differentiation marker cytokeratin 19 (KRT19), which was antagonized by an increase in biomarkers of late and terminal differentiation, i.e. cytokeratin 12 (KRT12), involucrin and filaggrin. Keratinocytes showed proper morphogenesis on 5 μm arrays, whereas 11 μm yielded in morphological disorders. While the propensity of keratinocyte proliferation appeared attenuated at large pillar patterns, stem cell marker ABCG2 was weak though homogeneous at 5 μm, but strong at 11 μm. Thus, corneal keratinocytes reveal interference of biomarker expression, morphogenesis and proliferation, which are at least in part characteristics of tissue homeostasis by mechanisms, depending on environmental biomechanics of micropattern-allocated cell adhesion points in vitro.

Soft micropillar interfaces of distinct biomechanics govern behaviour of periodontal cells.

A soft micropillar extracellular environment of distinct biomechanics is established by fabricating polydimethylsiloxane (PDMS) interfaces with pillar distances of 5, 7, 9 and 11 microm and elasticity moduli of 0.6, 1.0 and 3.5 Mega Pascal. To allow for cell adhesion, the biomimetic concept of pillar head fibronectin (FN) biofunctionalisation is applied. This environmental set-up aims at the analysis of favourable conditions for cell behaviour of three periodontal cell-types, here reflected by the establishment of regular cell morphology and optimal collagen gene expression. Biomechanics of these predefined functionalized model surfaces reveal progressive deterioration of regular cell morphology with increasing pillar distance, independent from pillar elasticity and cell type. Analysis of collagen gene expression demonstrates interdependency to the elasticity and the micropattern of the extracellular environment in all cell types under study. The results suggest that biomechanics of the extracellular environment govern tissue-specific cell behaviour in different periodontal cell types. Moreover, they form the basis for the creation of new biomaterials which address distinct cell functions by specific biomechanical properties.

Elution of Monomers from Provisional Composite Materials

The aim of this study was to evaluate the elution of substances from different materials used for the manufacturing of temporary indirect restorations, after storage in saliva and ethanol 75%. 10 samples of three chemically cured materials (Protemp 3 Garant, Systemp.c&b, and Trim) and one light-cured material (Clip F) were stored in saliva and ethanol 75% for 24 h, 7, and days 28 days. From the storage media at each time period, samples were prepared and analysed by LC-MS/MS, in order to access the elution of monomers. The results differed among the materials ( ≤ 0.05). No monomers were detected in the samples of Protemp 3 Garant and Clip F. Substances were detected only in ethanol samples of Systemp.c&b and Trim. The amount of BisGMA, TEGDMA, and UDMA 2 released from Systemp.c&b was higher compared to Trim. Storage time affected the release of substances ( ≤ 0.05). The highest release was observed within the first 24 h. It can be concluded that provisional resin composite materials do not show high release of monomers and this release is material dependent. However, the detection of additional peaks during the analysis, suggesting the formation of by-products of the eluted substances, may not be in favour of these materials with respect to their toxicity.

Environmental Biomechanics Substantiated by Defined Pillar Micropatterns Govern Behavior of Human Mesenchymal Stem Cells

While evidence on the impact of the biomechanical environment elasticity on human mesenchymal stem cell (hMSC) behavior is growing, the aspect of micropatterning is still poorly understood. Thus, the present study aimed at investigating the influence of defined environmental micropatterning on hMSC behavior. Following characterization, hMSCs were grown on defined pillar micropatterns of 5, 7, 9, and 11 μm. With respect to cell behavior, primary hMSC adhesion was detected by indirect immunofluorescence (iIF) for paxillin, vinculin, integrin αV, and actin, while proliferation was visualized by histone H3. Morphogenesis was monitored by scanning electron microscopy and the expression of stem cell-specific biomarkers by real-time PCR. Favoritism of primary adhesion of hMSCs on pillar tops occurred at smaller pillar micropatterns, concomitant with cell flattening. While vinculin, integrin αV, and paxillin appeared initially more cytoplasmic, high pillar micropatterns favored a progressive redistribution with polarization to cell tension sites and at cell borders. Accomplishment of morphogenesis at day 3 revealed establishment of fully rotund cell somata at 5 μm, while hMSCs appeared progressively elongated at rising micropatterns. The hMSC proliferation capacity was influenced by pillar micropatterns and gene expression analysis of stem cell- and differentiation-associated biomarkers disclosed clear modulation by distinct pillar micropatterns. In response to environmental biomechanics, our results show that hMSC behavior is governed by pillar micropatterning. In turn, these findings may form the basis to prospectively direct lineage specificity of hMSCs in a customized fashion.