Cells have their own language that they use to communicate with each other. They need this language to be able to form intact tissues and fulfil their specific functions in the body. If these signalling pathways are disrupted, metabolic processes will suffer and result in diseases. We know many “words” of the cellular language, i.e. signalling molecules that bind to specific surface receptors and thereby trigger chemical reactions inside the cell. But we do not know how these “words” are combined in “sentences” nor do we understand the underlying “grammar”. Researchers at the Karlsruhe Institute of Technology (KIT) have developed a method to decode the grammar of cell signals.
In multicellular organisms, cells already communicate with each other during embryonic development. This is the only way that the two original germ cells can form tissues and organs in an orderly fashion. In adult organisms, cells must continue to exchange information with each other in order to be able to exert intact body functions (e.g. immune defence). Signalling molecules (ligands) represent the “words” of the cell language. Such ligands bind to transmembrane receptors, thereby triggering a cascade of chemical reactions inside a cell. This cell can then transfer its response to other cells in a similar way.
Some of the “words” of some signalling pathways have already been studied in some detail, but little is yet known about how these “words” are combined in “sentences” or about the grammar rules that they follow to mediate a special cellular reaction. The ability to interpret the language of cells is of crucial importance for science, as autoimmune diseases and diseases such as cancer have been shown to be the result of defective receptors and cell signals. A detailed understanding of signalling pathways is therefore key to developing therapies and drugs for treating disease.
Researchers at the Institute for Biological Interfaces (IBG 1) at the Karlsruhe Institute of Technology (KIT) have been studying cell signals for quite some time. “This requires extremely small systems,” says the institute’s director, Prof. Dr. Christof Niemeyer. “We want to study the binding of molecules in the lower nanoscale. The binding of a ligand to a receptor takes place on an area a few nanometres in size. It is truly impressive that such small events can affect a cell around 10 micrometres in size. It is already known that different ligands that bind to the surface of cells represent the letters and words of the cell language. However, we do not yet know how these are used by the cells. Are the signals simply expelled as a “stream of words”, or is there a “grammatical context”, and does the letter depend on the spatial arrangement of the receptors in the cell membrane?”
In order to find answers to these questions, the nanobiotechnology specialists from Karlsruhe have developed MOSAIC (multiscale origami structures as interfaces for cells), a DNA-based platform that they use to position signalling molecules on a specific spot on a nanometre-sized area. “Such a method has not previously been available. This is down to the fact that structures of this size have been difficult to build,” says Niemeyer. “Nowadays, miniaturised systems, i.e. microarrays, are usually created using top-down methods like inkjet printing; these methods are also used for making computer chips. In our case, however, this would mean that we would have to immobilise a large number of different proteins in spots of around 5 nanometres in diameter. As we cannot create such small arrays with existing top-down methods, we therefore combine the advantages of top-down micropatterning of solid surfaces with the advantages of bottom-up approaches which take advantage of the nucleic acids ability to self-assemble."
The so-called DNA origami technique, which was developed in the USA around ten years ago, exploits DNA's ability to fold into arbitrary two- and three-dimensional shapes on the nanoscale. “The idea of using DNA as construction material was already established in the 1980s and has since been developed further,” says the professor. The MOSAIC technology also involves miniature DNA pegboards consisting of short single-strand DNA molecules. The DNA molecules folds in a self-organised manner to form a pegboard 100 nm long and 50 nm wide on which chemically synthesised protein molecules can be positioned at defined places. An atomic force microscope is used to check whether the molecules are correctly arranged. “After the pegboards have been characterised, dozens of them are applied to a glass carrier with a special printer and the cell line to be investigated is subsequently added. We then use a microscope to find out whether the cells respond to certain nanostructures differently to others,” says Niemeyer explaining the test principle.
To provide proof of principle, the scientists used origami constructs using EGF (epidermal growth factor) as ligand to activate the EGF receptor. EGFR signalling plays a key role in mitosis and is already relatively well studied. “It is already known how EGFR-mediated signalling works and that the EGF ligand addresses four to five different signalling pathways inside cells. However, it is not yet clear how the cell knows which of the different signalling pathways to chose,” says Niemeyer. “Currently, there is no method to assess individual signals. Our test helped us to find out which receptor molecule clusters are responsible for signal integration. It is assumed that such molecule clusters are involved in many signalling pathways. Now we have a method that we can use to examine this with relevant cells.”
Now that the researchers know that their method works, they will apply it to investigating other pathways. The researchers’ next project will be carried out in cooperation with research groups in Heidelberg and Dortmund. It will focus on signalling pathways that play a key role in immune defence (immunological synapses). “Once we understand the processes, we will be able – at least in principle – to develop drugs for restoring defective signalling pathways. But this is still a pipe dream,” says Niemeyer. “However, with the MOSAIC method, I am confident that we will achieve this goal a lot more quickly.”