Engineering Mesoscale T Cell Receptor Clustering by Plug-and-Play Nanotools.

T cell receptor (TCR) clustering and formation of an immune synapse are crucial for TCR signaling. However, limited information is available about these dynamic assemblies and their connection to transmembrane signaling. Here, we controlled TCR clustering via plug-and-play nanotools based on an engineered irreversible conjugation pair and a peptide-loaded major histocompatibility complex (pMHC) molecule to compare receptor assembly in a ligand (pMHC)-induced or ligand-independent manner. A streptavidin-binding peptide displayed in both tools enabled their anchoring in streptavidin-pre-structured matrices. Strikingly, pMHC-induced clustering in the confined regions exhibited higher density and dynamics than the ligand-free approach, indicating that the size and architecture of the pMHC ligand influences TCR assembly. Our approach enables the control of membrane receptor clustering with high specificity and provide the possibility to explore different modalities of receptor activation. This article is protected by copyright. All rights reserved.

Multiphoton lithography with protein photoresists.

Recently, 2D/3D direct laser writing has attracted increased attention due to its broad applications ranging from biomedical engineering to aerospace. 3D nanolithography of water-soluble protein-based scaffolds have been envisioned to provide a variety of tunable properties. In this paper, we present a functional protein-based photoresist with tunable mechanical properties that is suitable for multiphoton lithography (MPL). Through the use of methacrylated streptavidin or methacrylated bovine serum albumin in combination with polyethylene glycol diacrylate or methacrylated hyaluronic acid as crosslinkers and a vitamin-based photoinitiator, we were able to write two- and three-dimensional structures as small as 200 nm/600 nm lateral/axial features, respectively. We also demonstrated that Young's modulus can be tuned by the photoresist composition, and we were able to achieve values as low as 40 kPa. Furthermore, we showed that Young's modulus can be recovered after drying and rehydration (i.e. shelf time determination). The retained biological functionality of the streptavidin scaffolds was demonstrated using fluorescently labelled biotins. Using single-molecule fluorescence microscopy, we estimated the density of streptavidin in the written features (1.8 ± 0.2 × 105 streptavidins per 1.00 ± 0.05 µm³ of feature volume). Finally, we showed applicability of our 2D scaffold as a support for a fluorescence absorbance immuno-assay (FLISA), and as a delivery platform of extracellular vesicles to HeLa cells.

Surface-Patterned DNA Origami Rulers Reveal Nanoscale Distance Dependency of the Epidermal Growth Factor Receptor Activation.

The nanoscale arrangement of ligands can have a major effect on the activation of membrane receptor proteins and thus cellular communication mechanisms. Here we report on the technological development and use of tailored DNA origami-based molecular rulers to fabricate "Multiscale Origami Structures As Interface for Cells" (MOSAIC), to enable the systematic investigation of the effect of the nanoscale spacing of epidermal growth factor (EGF) ligands on the activation of the EGF receptor (EGFR). MOSAIC-based analyses revealed that EGF distances of about 30-40 nm led to the highest response in EGFR activation of adherent MCF7 and Hela cells. Our study emphasizes the significance of DNA-based platforms for the detailed investigation of the molecular mechanisms of cellular signaling cascades.

Differential effects of glucose-dependent insulinotropic polypeptide receptor/glucagon-like peptide-1 receptor heteromerization on cell signaling when expressed in HEK-293 cells.

The incretin hormones: glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are important regulators of many aspects of metabolism including insulin secretion. Their receptors (GIPR and GLP-1R) are closely related members of the secretin class of G-protein-coupled receptors. As both receptors are expressed on pancreatic ß-cells there is at least the hypothetical possibility that they may form heteromers. In the present study, we investigated GIPR/GLP-1R heteromerization and the impact of GIPR on GLP-1R-mediated signaling and vice versa in HEK-293 cells. Real-time fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) saturation experiments confirm that GLP-1R and GIPR form heteromers. Stimulation with 1 µM GLP-1 caused an increase in both FRET and BRET ratio, whereas stimulation with 1 µM GIP caused a decrease. The only other ligand tested to cause a significant change in BRET signal was the GLP-1 metabolite, GLP-1 (9-36). GIPR expression had no significant effect on mini-Gs recruitment to GLP-1R but significantly inhibited GLP-1 stimulated mini-Gq and arrestin recruitment. In contrast, the presence of GLP-1R improved GIP stimulated mini-Gs and mini-Gq recruitment to GIPR. These data support the hypothesis that GIPR and GLP-1R form heteromers with differential consequences on cell signaling.

A Simplified and Robust Activation Procedure of Glass Surfaces for Printing Proteins and Subcellular Micropatterning Experiments.

Depositing biomolecule micropatterns on solid substrates via microcontact printing (µCP) usually requires complex chemical substrate modifications to initially create reactive surface groups. Here, we present a simplified activation procedure for untreated solid substrates based on a commercial polymer metal ion coating (AnteoBindTM Biosensor reagent) that allows for direct µCP and the strong attachment of proteins via avidity binding. In proof-of-concept experiments, we identified the optimum working concentrations of the surface coating, characterized the specificity of protein binding and demonstrated the suitability of this approach by subcellular micropatterning experiments in living cells. Altogether, this method represents a significant enhancement and simplification of existing µCP procedures and further increases the accessibility of protein micropatterning for cell biological research questions.