Mikrofluidische Systemtechnikentwicklung für das Studium von regulativen Protein Interaktionen im Zell-Metabolismus
Final Report Abstract
This study aimed to develop and use microfluidic chip technologies for the investigation of protein interaction networks that regulate the human cell metabolism. Microfluidics has been termed the science of controlling fluidic properties and their contents within structures of micrometer dimensions. While electrical computer chips perform mathematical operations, microfluidic chips perform chemical operations. The microfluidic chips and corresponding manufacturing technologies developed in this work can be applied to (i) engineer biophysical biosensors, (ii) culture and control the chemical microenvironments of human 2- and 3-dimensional stem cell cultures, and/or (iii) automate complex quantitative protein assays with minimal volume consumption rates. In the first project part of the project we pursued the idea of engineering biosensors for metabolites based on naturally occurring proteins and aptamers with affinities to sugars and amino acids. Massive parallel on-chip screening and the biophysical characterization of large-molecule libraries were implemented on microfluidic chip platforms. One major achievement was the construction of 12 equally strongly responding glycine aptamers with binding affinities and rate constants spanning two orders of magnitude. Our developed microfluidic chip technology and established general concepts for the design of metabolite biosensors pave the ground for using biosensors in online measurements of metabolite fluxes in living organisms. In the second part of the project we developed microfluidic chip technologies to control the chemical microenvironment of 2D and 3D cell culture systems. Upon designing fluid networks using miniaturized valves it was possible to control hundreds of separated cell cultures on a single chip with dimensions of less than 10 cm. With the chip technology in hand we investigated the details of signal transduction through the metabolic-responsive mTOR kinase pathway on timescales from seconds to weeks. To quantitate signal transduction events after changing the cellular chemical microenvironment on the chip, a multiplexed proximity assay was developed. The assay exploits DNA barcoded antibodies and allows the enumeration of proteins, and their interactions and/or phosphorylation events at the single-cell level. Using this integrated technology, it was possible to reveal that the nutrient and growth signals processed via the mTOR pathway differ in their kinetic profiles. Although this has been widely assumed, only with the developed technology could this be proven. Subsequently, the technology was applied to record the protein interactions and spatial position profiles of the mTOR kinase during the differentiation of stem cells into mature white fat cells. It was found that not only quantitative changes in the abundance of mTOR kinase but also shifts in its subcellular position regulates stem cell maturation, which represents a breakthrough in our understanding of kinase signaling. To complement our understanding of protein changes in response to metabolites, we developed a temporal protein labeling method by combining genetic code expansion and genome engineering. This method allows us to attach labels to a target protein upon the addition of UAAs and is tailored for chip integration. In combination with the previously developed quantitative analytical assay technology it is possible to determine protein lifetimes at the single-cell level. Knowing that the subcellular position of kinases is important for signal transduction, it was of high interest to zoom our focus out and investigate, whether the position of the cell in the context of the tissue also influences mTOR signaling. For this purpose, we engineered microfluidic chip technologies for the formation and long-term culture of 3D cell culture models. Upon the integration of a tissue clearing method, a full analytical workflow for the high-throughput imaging of proteins in deep tissue structures could be established. All the central milestones of the project were achieved. Our interdisciplinary work was scientifically recognized and the achievements of the Emmy-Nöther project led to the award of an ERC Consolidator grant.
Publications
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Proximity Ligation Assay for High-content Profiling of Cell Signaling Pathways on a Microfluidic Chip. Molecular & Cellular Proteomics 12, 3898–3907 (2013)
Blazek, M. et al.
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Design and validation of DNA libraries for multiplexing proximity ligation assays. PLoS ONE 9, e112629 (2014)
Gobet, N., Ketterer, S. & Meier, M.
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Through-holes, cavities and perforations in polydimethylsiloxane (PDMS) chips. RSC Adv. 4, 48012–48016 (2014)
Santisteban, T. S., Zengerle, R. & Meier, M.
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Transcription factor sensor system for parallel quantification of metabolites on-chip. Anal Chem 86, 12152–12158 (2014)
Ketterer, S. et al.
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Analysis of fast protein phosphorylation kinetics in single cells on a microfluidic chip. Lab Chip 15, 726–734 (2015)
Blazek, M., Santisteban, T. S., Zengerle, R. & Meier, M.
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Systematic reconstruction of binding and stability landscapes of the fluorogenic aptamer spinach. Nucleic Acids Res. 43, 9564-72 (2015)
Ketterer, S., Fuchs, D., Weber, W. & Meier, M.
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Engineering and characterization of fluorogenic glycine riboswitches. Nucleic Acids Res. 44, 5983-5992 (2016)
Ketterer, S., Gladis G., Kozica, A., Meier, M.
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In situ characterization of the mTORC1 during adipogenesis of human adult stem cells on chip, PNAS, 29, E41143-E4150 (2016)
Wu, X. et al.
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Efficient in situ detection of mRNAs using the Chlorella virus DNA ligase for padlock probe ligation, RNA, 23, 250-256 (2017)
Schneider, N, Meier, M.
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Rapid spheroid clearing on a microfluidic chip, LabChip, 18, 153-161 (2018)
Silva, T. Rabajania, O., Kalinina, I., Robinson, S., Meier, M.
