Single-molecule tracking tools to study plasma membrane receptor dynamics
Applications to EGF receptor
Promotion date: July 3.
Promotor: Prof.dr. Vinod Subramaniam
Assistant Promotor: Dr. Hans Kanger
Tools were developed to advance single-molecule tracking microscopy. In this way the signaling mechanism of receptor signaling can be studied. In this thesis, we applied the tools to the ErbB (HER) receptor family, and in particular to the epidermal growth factor receptor (EGFR). This receptor family plays an important role in the development and progression of various human cancers. They therefore form an attractive target for drug discovery research.
The organization of many receptors in the plasma membrane, shows dynamic clustering and hindered motion on nano- and micro-scale levels. Recording trajectories of EGFR and analyzing these in terms of dimerization and interactions with other cellular structures were realized. For this purpose a microscope was built; a framework to analyze trajectories in terms of different diffusion populations was devised; and an existing protein labeling system – SNAP-tag – was advanced to the single molecule level.
The thesis concludes with a proof of principle demonstration to locally activate receptors using ligand functionalized AFM tips. Locally induced stimulation of receptors by these tips can provide additional insight in molecular interactions and signal propagation, and were combined with single-molecule tracking.
How would you explain your research to a relative layman?
The way proteins move through living cells may provide insight in the mechanisms orchestrating the signaling processes of proteins. Protein signaling provides the communication that cells need to live but, under malignant conditions, can cause human cancers and other diseases to develop.
To understand these signaling processes, and to measure the molecular effects of medication rationally designed to prevent certain signaling processes, tracking receptor proteins in live cells can provide essential new insights to understand the signaling process. Working on the knowledge base of these interactions with cellular structures which can be tracked by microscopic techniques, is an actual topic in science these days.
To improve the ability to record protein trajectories in live cells, I started looking for a promising labeling system. We showed that SNAP-tag, which can be coupled to proteins, can be labeled at the single-molecule level in multiple distinct colors.
Since the SNAP-tag does not influence the function of the protein and is intrinsically monovalent (it does not artificially cluster the proteins it is attached to), this labeling system is very suitable to study receptor signaling. This might be a step towards better fluorescent labels to study the lifetimes of dimers (two receptors combining, this makes the receptor start signaling). When two colors, e.g. red and green excitable dyes, are used to label the receptor, dimer formation can be directly visualized as a green and red labeled receptor being together.
Once the optimal fluorescent dye was selected to label proteins, we could follow proteins on the plasma membrane of live cells for about thirty seconds, and record their individual trajectories. Being able to do so at video rate (24 fps), allowed us to follow the proteins, which are diffusing at relatively high speeds. This required building a custom microscope, as microscopes capable of tracking single molecules are not commercially available yet. To realize such a microscope, we used lasers and high quality optical filters to provide a good image quality to detect signals from weakly emitting fluorophores.
Was your research fundamental in nature or application oriented?
Though the work was quite fundamental, the work was situated at the heart of actual developments in the single-molecule tracking scientific community. Also the applications of my tools on ErbB receptors was very actual in medical oncology research on the molecular function of anti-cancer drugs.
Researchers from the medical oncology group at the UMC in Groningen were very enthusiastic about our ability to track the protein trajectories in such detail. Together we came up with relevant ideas to perform experiments in promising lines of research, and they were of great help in interpreting the experimental data, and to make my fundamental work applicable and meaningful.
Did your work involve other collaborations?
Also collaboration with researchers at the NCMLS Radboud from Nijmegen was very fruitful, as they applied my tools on other receptors related to immunology. They discovered a new signaling mechanism for such receptors, and we are hoping to publish these results very soon. Furthermore, colleagues from Max Planck Institute in Dortmund brought in expertise on molecular cloning of SNAP-tag to the receptors studied. Colleagues from TU Eindhoven helped fabricating SNAP-tag substrates and a cysteine mutated ligand suitable for easy coupling to AFM tips. The coupling I learned at the AFM group of the University of Linz (Austria).
Can you recall some special moments during your PhD research?
After one year into my PhD period I could visualize the first single molecules, and after another year I was able to couple fluorescent molecules to receptors in a live cell. To observe their seemingly random motion was very nice. Also the collaboration started with the UMC Groningen, I recall as inspiring and decisive as their enthusiastic reactions convinced me of the relevance of the fundamental work I was working at.
My initial line of research, concerning ligand functionalized AFM tips, I could return to in the last half year of my PhD period as new mutant ligands became available. To work at the frontline of advancing a microscopy technique, and to apply this technique to a very relevant biological problem (malignant signaling by ErbB proteins) was a very fulfilling process making my PhD period an interesting journey.
Were your articles published in some good journals?
Two papers have been accepted for publication in the Biophysical Journal. I am very happy with that, as this journal is well-read by many renowned colleagues in the field. Another paper for which I contributed is currently in review.
What are your future plans?
I would like to go and work in industrial research, as I find it fulfilling to work on technical innovation while being able to achieve some short time results from time to time. In industry I hope to be able to work in a team pursuing shared goals, which I sometimes missed during my PhD project.
How did you value working within Mesa+?
For this project I have not used equipment available within the specific laboratories of Mesa+. The setup used in my research was established in close collaboration with my group colleagues. For example, technicians built the AFM equipment suitable for use on biological samples enabling me to perform my AFM experiments on cells.
The Mesa+ Days I found inspiring to widen my view. One can meet colleagues from very different research areas. This gives you a good sense of the scientific scope present at Mesa+. I learned many more researchers are active in biophysical related topics.