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Specific control of cellular processes

It would be extremely useful to have a remote control system which would enable processes inside cells to be switched on and off at will externally. A group of students supervised by junior professor Dr. Kristian Müller and Dr. Katja Arndt from the University of Freiburg, has succeeded in constructing just such a system. As part of the “2008 international Genetically Engineered Machine” (iGEM) competition, the young biologists constructed a modular building set of artificial receptors and ligands which enabled them to induce certain reactions in the cytosol. The jury from the Massachusetts Institute of Technology (MIT) awarded the Freiburg students second place in the competition. The construction kit might in future enable specific cell interventions.

The engineer’s art is to manipulate nature and use it for specific purposes. The question is, is this also possible on the microscopic and nanoscopic level of living organisms? At the end of 2007, nine biology students at the University of Freiburg participated in a seminar on biological engineering and, excited by the potential of this field, they decided to set up a joint project. The result, in November 2008, was second place in the “international Genetically Engineered Machine” (iGEM) competition organised by the Massachusetts Institute of Technology (MIT) in Boston. The group of 9 were up against 83 teams from 21 countries. In the final, the University of Freiburg beat teams from the American elite universities of Berkeley, Caltech and Harvard. Their molecular building set of artificial nanostructures convinced the jury. “This building set can be used to establish a ‘cell control system’,” said Dr. Kristian Müller from the Institute of Biology III at the University of Freiburg, and supervisor and initiator of the project.
The Freiburg team in front of the MIT's auditorium prior to the prize award: Moritz Busacker, Robert Gawlik, Philipp Mappes, Daniel Hautzinger, Normann Kilb, Kathrin Pieper, Prof. Dr. Kristian Müller; in the front row: Simone Weber, Michael Kneib, Sabine Jägle (Photo: Prof. Dr. Kristian Müller)

Perfect fit

This ’cell control system’, developed in Freiburg with the infrastructural support of numerous laboratories and major financial support through the excellence cluster “Centre for Biological Signalling Studies” (Bioss), consists of four elements. One can imagine the system as a switch on the cell membrane that can only be manipulated with a specific key and which is transduced to the cytosol where a specific mechanism is executed. In biological terms, the system is a synthetic transmembrane receptor system comprising extracellular input devices, the extracellular receiver domain, the transducer to the cytosol and the reporter in the cytosol. The biology students led by Dr. Müller and Dr. Arndt from the “Freiburg Institute for Advanced Studies” (FRIAS) have synthesised the components so that they interacted perfectly with each other. If a cell is genetically engineered to integrate this system into its cell membrane, the mechanism can be induced in the cytosol. Theoretically, different wheels in the molecular gearbox of the cell can be manipulated. The principle represents a potential quantum leap in the control of signalling pathways in cancer cells.

But what do the individual elements look like and how do they interact? “The key comprises a DNA-origami to which hapten molecules are coupled, for example,” explained Normann Kilb, one of the students involved in the project. “This construct interacts with a hapten antibody which can specifically bind haptens.” DNA-origamis are nanoscopic structures of individual DNA building blocks that can be folded into figures of an exactly defined size and shape. Haptens are small organic molecules that can be attached anywhere on the origami scaffold. This enables the researchers to develop a key with exactly defined zigzag patterns. Antibodies or antibody-like proteins constitute the lock for this key. Two haptens, located at a specific distance from each other, interact with one of the antibodies. Each antibody is coupled to a protein that spans the cell membrane and extends into the cytosol. If the key fits, then both antibodies bind and approach each other, thereby bringing the two transmembrane proteins closer together.

A fluorescent reunion

Model of how the receptor system functions: in the inactive state, the two parts are spatially separated from each other (left). A modified DNA-origami piece (top right) brings the two halves together and the protein in the cytosol dimerises. (Photo: Prof. Dr. Kristian Müller)
The dimerisation is transduced into the cytosol and induces the decisive signalling mechanism. Each of the two complementary halves of a protein is located at the cytosolic terminus of the two transmembrane proteins. The protein halves move towards each other and dimerise. “This will then lead to further actions. Instead of coupling so-called split proteins it is also possible to couple different molecular interaction partners which will find each other and interact when the switch system is manipulated,” said Michael Kneib. This interaction can then potentially affect the signalling networks in the cytosol. The students used two halves of blue fluorescent protein (CFP). As the CFP halves only fluoresce under the fluorescence microscope upon dimerisation, the biologists clearly see when their switch system induces the signalling mechanism.

The Freiburg students have deposited the genes of the individual building blocks of the switch system in the “Registry of Standard Biological Parts” where they will be made available to future iGEM participants. The 2008 team has based its work on that of the 2007 Freiburg team. The students spent about six months on their project. How have the students maintained their motivation? “If you see that a sub-step works, you want to continue,” said Kathrin Pieper, another student member of the team. “It is still not clear what will become of the project, but we hope to publish the results in a major scientific journal,” said Müller.

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