44 research outputs found

    Linker Engineering of Ligand‐Decorated DNA Origami Nanostructures Affects Biological Activity

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    News from an old acquaintance: The streptavidin (STV)-biotin binding system is frequently used for the decoration of DNA origami nanostructures (DON) to study biological systems. Here, a surprisingly high dynamic of the STV/DON interaction is reported, which is affected by the structure of the DNA linker system. Analysis of different mono- or bi-dentate linker architectures on DON with a novel high-speed atomic force microscope (HS-AFM) enabling acquisition times as short as 50 ms per frame gave detailed insights into the dynamics of the DON/STV interaction, revealing dwell times in the sub-100 millisecond range. The linker systems are also used to present biotinylated epidermal growth factor on DON to study the activation of the epidermal growth factor receptor signaling cascade in HeLa cells. The studies confirm that cellular activation correlated with the binding properties of linker-specific STV/DON interactions observed by HS-AFM. This work sheds more light on the commonly used STV/DON system and will help to further standardize the use of DNA nanostructures for the study of biological processes

    Highly Modular Protein Micropatterning Sheds Light on the Role of Clathrin-Mediated Endocytosis for the Quantitative Analysis of Protein-Protein Interactions in Live Cells

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    Protein micropatterning is a powerful tool for spatial arrangement of transmembrane and intracellular proteins in living cells. The restriction of one interaction partner (the bait, e.g., the receptor) in regular micropatterns within the plasma membrane and the monitoring of the lateral distribution of the bait’s interaction partner (the prey, e.g., the cytosolic downstream molecule) enables the in-depth examination of protein-protein interactions in a live cell context. This study reports on potential pitfalls and difficulties in data interpretation based on the enrichment of clathrin, which is a protein essential for clathrin-mediated receptor endocytosis. Using a highly modular micropatterning approach based on large-area micro-contact printing and streptavidin-biotin-mediated surface functionalization, clathrin was found to form internalization hotspots within the patterned areas, which, potentially, leads to unspecific bait/prey protein co-recruitment. We discuss the consequences of clathrin-coated pit formation on the quantitative analysis of relevant protein-protein interactions, describe controls and strategies to prevent the misinterpretation of data, and show that the use of DNA-based linker systems can lead to the improvement of the technical platform

    Total internal reflection fluorescence microscopy (TIRFM) guided analysis of receptor tyrosine kinase (RTK) signaling

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    Tyrosine kinase receptors (RTKs) are transmembrane proteins which are activated following binding with peptide growth factors or hormones and play key roles in processes such as cellular growth, metabolism, motility and differentiation. Recent evidence suggests that these receptors are involved in the pathogenesis and progression of a variety of cancers. In addition, dysfunction in IR and IGF-IR signaling is strongly linked to obesity and type 2 diabetes mellitus. Due to the fact that these receptors have major influences on human health, they were intensively investigated and became major drug targets for the above mentioned diseases. Aim of this thesis was to set up a novel and workable live cell analysis platform based on micropatterned surfaces for studying the influence of tyrosine kinase receptor signaling modulating substances on the one hand, and on the other hand a screening platform for the identification and characterization of insulin mimetic substances. The respective chapters were published in different scientific journals. (1) Quantification and Kinetic Analysis of Grb2-EGFR Interaction on Micro-Patterned Surfaces for the Characterization of EGFR-Modulating Substances. PLOS ONE (2014). In this chapter we report on a technique which combines micro-patterned surfaces and total internal reflection fluorescence (TIRF) microscopy (-patterning assay) for the quantitative analysis of EGFR activity. Here we quantified the interaction of the key signal transmitting protein Grb2 (growth factor receptor-bound protein 2) with the EGFR in a live cell context. It was possible to demonstrate an EGF dependent recruitment of Grb2 to the EGFR, which was significantly inhibited in the presence of clinically tested EGFR inhibitors. Importantly, in addition to its potential use as a screening tool, our experimental setup offers the possibility to provide insight into the molecular mechanisms of bait-prey interaction. Application of bleaching experiments enabled calculation of the Grb2 exchange rate, which significantly changed upon stimulation or the presence of EGFR activity inhibiting drugs. (2) Analysis of insulin receptor substrate (IRS) signaling dynamics on micro- structured surfaces. FEBS Journal (2015). Here we provide additional insight into IR/IGF-IR downstream signaling via insulin receptor substrate (IRS) proteins. Interaction of IR/IGF-IR with IRS proteins is an initial key event for downstream signaling and bioactivities. Despite the structural similarities, increasing evidences show that IRS family proteins have non-redundant functions. Although the specificity of insulin/IGF signaling and biological responses in part reflects which IRS proteins are dominantly phosphorylated by the receptors, the precise properties of respective IRS interaction with the receptors remain elusive. Again we used the -patterning assay for the quantitative analysis of the interaction between IRS proteins and IR/IGF-IR in living cells. Our experimental setup enabled the measurement of equilibrium associations and interaction dynamics of these molecules with high specificity. We revealed that several domains of IRS including pleckstrin homology and phosphotyrosine binding domains critically determine the turnover rate of the receptors. Furthermore, we found significant differences among IRS proteins in the strength and kinetic stability of the interaction with the receptors, suggesting that these interaction properties could account for the diverse IRS functions. In addition, our analyses using fluorescent recovery after photobleaching revealed that kinases such as c-Jun N-terminal kinase and IkB kinase beta, which phosphorylate serine/threonine residues of IRS and contribute to insulin resistance, altered the interaction kinetics of IRS with insulin receptor. (3) Identification of Novel Insulin Mimetic Drugs by Quantitative Total Internal Reflection Fluorescence (TIRF) Microscopy (2014). Finally, we used quantitative TIRFM to investigate the glucose transporter 4 (GLUT4) translocation modulatory properties of selected phytochemicals (chapter 5). For this purpose, TIRFM was applied to quantify GLUT4 translocation in highly insulin-sensitive CHO-K1 cells expressing a GLUT4-myc-GFP fusion protein. Using our approach, we demonstrated the influence of selected phytamines on GLUT4 translocation and identified novel potential insulin mimetics. An increase in the TIRF signal was found to correlate with an elevated glucose uptake. Variations in the expression level of the human insulin receptor (hInsR) showed that the insulin mimetics identified stimulate GLUT4 translocation by a mechanism that is independent of the presence of the hInsR. Taken together, the results indicate that TIRF microscopy is an excellent tool for the quantification of GLUT4 translocation and for identifying insulin mimetic drugs.Die Familie der Tyrosin-Kinasen-Rezeptoren (TKR) beinhaltet essenzielle Membranprotein die fĂŒr die Zell-Zell-Kommunikation von wesentlicher Bedeutung sind. Hierunter befinden sich auch der Epidermale Wachstumsfaktor Rezeptor (EGFR), der Insulin Rezeptor (IR) und der Insulin-Ă€hnliche Wachstumsfaktor Rezeptor (IGF-IR). Diese Rezeptoren werden durch Wachstumsfaktoren (Insulin, EGF, IGF,...) aktiviert und haben eine SchlĂŒsselrolle bei der Regulierung von Zellprozessen wie Zellwachstum, Metabolismus und Zelldifferenzierung. Neueste Studien belegen, dass diese Rezeptoren aufgrund ihrer zellulĂ€ren Funktion bei der Entstehung einer Vielzahl von unterschiedlichen Krebsarten eine entscheidende Rolle spielen. Des Weiteren konnte gezeigt werden, dass Fehlfunktionen des IR und IGF-IR mit Störungen im Fett- (Fettleibigkeit) und Kohlenhydratstoffwechsel (Diabetes) einhergehen. Durch diese klaren ZusammenhĂ€nge sind diese Rezeptoren in den letzten Jahren zu Hauptangriffspunkten bei der Behandlung der bereits erwĂ€hnten Krankheiten geworden. Um die Wirksamkeit von Rezeptor-modulierenden Substanzen testen und charakterisieren zu können wurde eine Vielzahl an chemischen, biologischen und biophysikalischen Testmethoden entwickelt. Die immer komplexer werdende Welt der Wirkstoffsuche- und Analyse macht es jedoch notwendig, einfache und kostengĂŒnstige Analysenplattformen zu entwickeln. Ziel dieser Studie war einerseits die Etablierung einer in-vitro Analysenmethode basierend auf einem -Biochip zur Erforschung von TKR-modulierenden Substanzen, andererseits die Setup Entwicklung fĂŒr quantitative Totale-Interne-Reflektions-Fluoreszenz-Mikroskopie (TIRFM) zum Screening von Insulin-mimetischen Substanzen. Die in der Dissertation vorliegenden Arbeiten wurden als wissenschaftliche Publikationen in Fachjournalen veröffentlicht. (1) Quantification and Kinetic Analysis of Grb2-EGFR Interaction on Micro-Patterned Surfaces for the Characterization of EGFR-Modulating Substances. PLOS ONE (2014) In dieser Arbeit beschreiben wir die Verwendung von -Biochips in Kombination mit TIRFM zur quantitativen Analyse der in-vitro EGFR AktivitĂ€t durch Charakterisierung der Interaktion mit dem SchlĂŒsselprotein Grb2 (Growth factor binding protein 2). Grb2 ist der erste intrazellulĂ€re Signaltransmitter nach EGFR Stimulation und es konnte gezeigt werden, dass dieses Protein in einem Agonist-abhĂ€ngigen Zusammenhang zum Rezeptor rekrutiert wird. Diese Rekrutierung wurde signifikant inhibiert durch die Gegenwart von klinischen EGFR Inhibitoren. Neben der möglichen Anwendung als Screeningtool ermöglicht dieses Setup auch die Erforschung molekularer Signalisierungsmechanismen, wie mittels FRAP (Fluorescence recovery after photobleaching) Experimenten gezeigt wurde. (2) Analysis of insulin receptor substrate (IRS) signaling dynamics on micro- structured surfaces. FEBS Journal (2015) Mit Hilfe des -patterning Assays wurde in dieser Publikation die Interaktion zwischen IR/IGF-IR und dem DownstreammolekĂŒl IRS (Insulin receptor substrate) charakterisiert. Unterschiedliche IRS-Proteine (IRS1-4) haben schlussendlich auch unterschiedlichste biologische Outputs zur Folge, wobei die genauen Mechanismen noch nicht bekannt sind. Hier zeigen wir eine quantitative Analyse der Bindungskinetiken und Turnoverraten zwischen IR/IGF-IR und IRS und berichten ĂŒber den Einfluss wichtiger Bindungs- und Phosphorylierungsdomainen in diesem Zusammenhang. Es konnte ein signifikanter Unterschied zwischen den IRS-Proteinen hinsichtlich InteraktionsstĂ€rke und BindungsstabilitĂ€t am Rezeptor festgestellt werden. Des Weiteren konnte die Rolle spezifischer Phosphorylierungsmuster an IRS-Proteinen (Serin/Threonin) bezĂŒglich subzellulĂ€rer Lokalisierung weiter vorangetrieben werden. (3) Identification of Novel Insulin Mimetic Drugs by Quantitative Total Internal Reflection Fluorescence (TIRF) Microscopy (2014) Die dritte Publikation behandelt die Etablierung eines quantitativen TIRM-Setups zur Erforschung von sogenannten Phytaminen welche modulierende Wirkungen auf den Glukose Transporter 4 (GLUT4) besitzen. Zu diesem Zweck wurden insulin-sensitive CHO-K1 Zellen mit ĂŒberexprimiertem GLUT4-GFP verwendet. Durch diese prĂ€zise TIRM-Methode konnte der Einfluss unterschiedlicher Phytamine auf die GLUT4-Translokation gezeigt bzw. neuartige insulin-mimetische Substanzen identifiziert werden.eingereicht von: Peter LanzerstorferZsfassung in dt. SpracheLinz, Univ., Diss., 2015OeBB(VLID)39269

    Super-Resolution Live Cell Microscopy of Membrane-Proximal Fluorophores

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    Here, we present a simple and robust experimental setup for the super-resolution live cell microscopy of membrane-proximal fluorophores, which is comparably easy to perform and to implement. The method is based on Structured Illumination Microscopy (SIM) with a switchable spatial light modulator (SLM) and exchangeable objective lenses for epi-illumination and total internal reflection fluorescence (TIRF) microscopy. While, in the case of SIM (upon epi-illumination), cell layers of about 1–2 µm in close proximity to the plasma membrane can be selected by software, layers in the 100 nm range are assessed experimentally by TIRF-SIM. To show the applicability of this approach, both methods are used to measure the translocation of the glucose transporter 4 (GLUT4) from intracellular vesicles to the plasma membrane upon stimulation by insulin or insulin-mimetic compounds, with a lateral resolution of around 100 nm and an axial resolution of around 200 nm. While SIM is an appropriate method to visualize the intracellular localization of GLUT4 fused with a green fluorescent protein, TIRF-SIM permits the quantitative evaluation of its fluorescence in the plasma membrane. These imaging methods are discussed in the context of fluorescence lifetime kinetics, providing additional data for the molecular microenvironment

    Probing Small Distances in Live Cell Imaging

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    For probing small distances in living cells, methods of super-resolution microscopy and molecular sensing are reported. A main requirement is low light exposure to maintain cell viability and to avoid photobleaching of relevant fluorophores. From this point of view, Structured Illumination Microscopy (SIM), Axial Tomography, Total Internal Reflection Fluorescence Microscopy (TIRFM) and often a combination of these methods are used. To show the high potential of these techniques, measurements on cell-substrate topology as well as on intracellular translocation of the glucose transporter GLUT4 are described. In addition, molecular parameters can be deduced from spectral data, fluorescence lifetimes or non-radiative energy transfer (FRET) between a donor and an acceptor molecule. As an example, FRET between the epidermal growth factor receptor (EGFR) and the growth factor receptor-bound protein 2 (Grb2) is described. Since this interaction, as well as further processes of cellular signaling (e.g., translocation of GLUT4) are sensitive to stimulation by pharmaceutical agents, methods (e.g., TIRFM) are transferred from a fluorescence microscope to a multi-well reader system for simultaneous detection of large cell populations

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

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    Depositing biomolecule micropatterns on solid substrates via microcontact printing (”CP) 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 ”CP 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 ”CP procedures and further increases the accessibility of protein micropatterning for cell biological research questions