Covalent and density-controlled surface immobilization of E-cadherin for adhesion force spectroscopy.

E-cadherin is a key cell-cell adhesion molecule but the impact of receptor density and the precise contribution of individual cadherin ectodomains in promoting cell adhesion are only incompletely understood. Investigating these mechanisms would benefit from artificial adhesion substrates carrying different cadherin ectodomains at defined surface density. We therefore developed a quantitative E-cadherin surface immobilization protocol based on the SNAP-tag technique. Extracellular (EC) fragments of E-cadherin fused to the SNAP-tag were covalently bound to self-assembled monolayers (SAM) of thiols carrying benzylguanine (BG) head groups. The adhesive functionality of the different E-cadherin surfaces was then assessed using cell spreading assays and single-cell (SCSF) and single-molecule (SMSF) force spectroscopy. We demonstrate that an E-cadherin construct containing only the first and second outmost EC domain (E1-2) is not sufficient for mediating cell adhesion and yields only low single cadherin-cadherin adhesion forces. In contrast, a construct containing all five EC domains (E1-5) efficiently promotes cell spreading and generates strong single cadherin and cell adhesion forces. By varying the concentration of BG head groups within the SAM we determined a lateral distance of 5-11 nm for optimal E-cadherin functionality. Integrating the results from SCMS and SMSF experiments furthermore demonstrated that the dissolution of E-cadherin adhesion contacts involves a sequential unbinding of individual cadherin receptors rather than the sudden rupture of larger cadherin receptor clusters. Our method of covalent, oriented and density-controlled E-cadherin immobilization thus provides a novel and versatile platform to study molecular mechanisms underlying cadherin-mediated cell adhesion under defined experimental conditions.

Tension monitoring during epithelial-to-mesenchymal transition links the switch of phenotype to expression of moesin and cadherins in NMuMG cells.

Structural alterations during epithelial-to-mesenchymal transition (EMT) pose a substantial challenge to the mechanical response of cells and are supposed to be key parameters for an increased malignancy during metastasis. Herein, we report that during EMT, apical tension of the epithelial cell line NMuMG is controlled by cell-cell contacts and the architecture of the underlying actin structures reflecting the mechanistic interplay between cellular structure and mechanics. Using force spectroscopy we find that tension in NMuMG cells slightly increases 24 h after EMT induction, whereas upon reaching the final mesenchymal-like state characterized by a complete loss of intercellular junctions and a concerted down-regulation of the adherens junction protein E-cadherin, the overall tension becomes similar to that of solitary adherent cells and fibroblasts. Interestingly, the contribution of the actin cytoskeleton on apical tension increases significantly upon EMT induction, most likely due to the formation of stable and highly contractile stress fibers which dominate the elastic properties of the cells after the transition. The structural alterations lead to the formation of single, highly motile cells rendering apical tension a good indicator for the cellular state during phenotype switching. In summary, our study paves the way towards a more profound understanding of cellular mechanics governing fundamental morphological programs such as the EMT.

Three-dimensional microscaffolds exhibiting spatially resolved surface chemistry.

Spatial control over the surface chemistry of 3D organic-inorganic hybrid microscaffolds is achieved by a two-photon-triggered cycloaddition. Following a coating step with photoactivatable dienes via silanization, surface irradiation with a femtosecond-pulsed laser in the presence of functional dienophiles enables a site-selective alteration of the surface chemistry. Bioconjugation with fluorescent protein tags is employed to reveal the 3D molecular patterns.

SNAP-tag as a tool for surface immobilization.

SNAP-tag technology has been an important tool for protein study for more than a decade and in the meanwhile has found a number of applications beyond the field of molecular biology and protein purification. Based on covalent interaction of SNAP-tag, 20 kDA mutant of DNA repair protein and benzylguanine, it enables irreversible and controllable protein modification. In this mini review, recent developments in the use of SNAP-tag for the design of protein arrays and nanoparticle biofunctionalization are presented and discussed. A short overview of other applications that paved the way to surface modifications is also given with emphasis on fluorescent labeling through the use of SNAP-tag fusion proteins. Finally, the future of the SNAP-tag methodology for surface patterning and 3D structural scaffolding is addressed.

Design of chemically activated polymer microwells by one-step UV-lithography for stem cell adhesion.

A novel method to produce sub-microwalled chemically activated polymer microwells by one-step UV-lithography under ambient conditions which are selectively coated with gelatin is introduced. The dimensions as well as the shape of the resulting polystyrene structures are both tunable merely by the irradiation time through one and the same mask. It is shown that the UV-irradiation initiates three effects at those surface areas which are not covered by the mask: (i) oxidation, (ii) cross-linking, and (iii) degradation of polystyrene. The superposition of those effects results in the formation of microscaled, oxidized polymer wells separated by polymer walls, whereas the polymer walls are formed below the mask structures. Topographical changes induced by the UV-irradiation are investigated by atomic force microscopy after different irradiation times. It is shown by X-ray photoelectron spectroscopy and ellipsometric investigations that the chemical composition of the irradiated areas and the degradation of polystyrene reach an equilibrium state after an irradiation time of 10 min. The lateral distribution of the cross-linked and oxidized and of the nonmodified polystyrene after irradiation was determined by fluorescence microscopy and time-of-flight secondary ion mass spectrometry. After the irradiated samples were treated with gelatin solution, it was found that stem cells selectively attach to the irradiated areas. This is due to the selective immobilization of the gelatin on the irradiated polymer areas, which was proved by X-ray photoelectron spectroscopy experiments.

Cold-inducible RNA binding protein (CIRP), a novel XTcf-3 specific target gene regulates neural development in Xenopus.

BACKGROUND: As nuclear mediators of wnt/beta-catenin signaling, Lef/Tcf transcription factors play important roles in development and disease. Although it is well established, that the four vertebrate Lef/Tcfs have unique functional properties, most studies unite Lef-1, Tcf-1, Tcf-3 and Tcf-4 and reduce their function to uniformly transduce wnt/beta-catenin signaling for activating wnt target genes. In order to discriminate target genes regulated by XTcf-3 from those regulated by XTcf-4 or Lef/Tcfs in general, we performed a subtractive screen, using neuralized Xenopus animal cap explants. RESULTS: We identified cold-inducible RNA binding protein (CIRP) as novel XTcf-3 specific target gene. Furthermore, we show that knockdown of XTcf-3 by injection of an antisense morpholino oligonucleotide results in a general broadening of the anterior neural tissue. Depletion of XCIRP by antisense morpholino oligonucleotide injection leads to a reduced stability of mRNA and an enlargement of the anterior neural plate similar to the depletion of XTcf-3. CONCLUSION: Distinct steps in neural development are differentially regulated by individual Lef/Tcfs. For proper development of the anterior brain XTcf-3 and the Tcf-subtype specific target XCIRP appear indispensable. Thus, regulation of anterior neural development, at least in part, depends on mRNA stabilization by the novel XTcf-3 target gene XCIRP.