Super-resolution microscopy that allows researchers to see inside nerve cells, track disease-causing proteins and watch cell division in living organisms earned three scientists the Nobel Prize for chemistry in 2014. Building on that breakthrough, a Marie Curie post-doctoral fellow has developed a pioneering technique to peer even more closely inside living cells at the nano-scale, using fluorescent dyes to label the tiny amino acids that are the building blocks of all proteins.
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“What you cannot label you cannot see,” says Ivana Nikić, who led the ground-breaking research in Edward Lemke’s lab at the European Molecular Biology Laboratory in Heidelberg, Germany. “More than other microscopy techniques, super-resolution microscopy depends on optimal sample preparation. As the precision of the optics, instrumentation and software has improved, new ways to dye and label proteins at an even smaller scale have become necessary to make them visible under the microscope.”
Nikić’s research in the Marie Curie-supported NUPTAG project has led to the smallest protein labelling technique developed to date. This has resulted in two patents being filed, numerous scientific papers published in leading journals, and widespread collaboration with other scientists around the world. More broadly, the work advances the use of super-resolution microscopy in a variety of research areas, not least to improve understanding of cell activity and contribute to the development of new treatments for disease.
Tiny tags for tiny proteins
Small synthetic dyes have long been considered very attractive for use as labelling tags but their direct attachment to proteins remained a challenge. For super-resolution microscopy, tags with specific photo-stable fluorescent properties are required and the tags should be as small as possible to maximise labelling density, ultimately making the targeted protein more visible and traceable under the microscope.
“Larger labelling tags can affect the function of proteins, so small tags are therefore essential for labelling proteins that are not accessible to other methods, such as viruses. Small tags also allow for very high labelling densities because dyes can be put closely together,” Nikić explains.
To solve the problem of attaching small labelling tags to proteins, Nikić used a state-of-the-art technique known as genetic code expansion. By slightly modifying the genetic make-up of a cell, she was able to equip it with specially designed protein building blocks in the form of single ‘unnatural’ amino acids, containing functional groups for the dye to attach to. In a subsequent step, the dye is added using non-toxic click-chemistry, a technique that enables very fast chemical reactions allowing living cells to be labelled on a rapid timescale – crucial for real-time studies of dynamic biological processes.
To make the technique even more versatile, Nikić and her fellow researchers managed to enable dual-colour labelling by combining two very fast click-reactions in a way that had not been achieved before.
“To the best of my knowledge, this work represents the first implementation of super-resolution microscopy based on click-chemistry residue-specific protein labelling by means of genetic code expansion. It opens many possibilities for labelling cells in a non-invasive and non-toxic way, which enables the technique to be used on living cells,” Nikić says.
The team has tested the technology by labelling distinct groups of membrane proteins, such as insulin receptors and influenza virus proteins. It has also experimented with intracellular proteins such as cytoskeleton structures and nuclear pore channels – applications that are still being explored by the Lemke research group.
For Nikić, the interdisciplinary nature of the NUPTAG research has been fundamental to the project’s success: “My PhD involved working with various microscopy techniques, but I had no experience with protein engineering or molecular or chemical biology. Having a biologist such as myself working with chemists, biochemists and biophysicists in the Lemke lab has helped to push this project forward.”
Nikić has since received an Emmy Noether grant from German research foundation DFG, and has been appointed as a junior group leader at the Werner Reichardt Centre for Integrative Neuroscience in Tübingen. She is in the process of establishing her own research team to focus on the molecular aspects of tissue damage in multiple sclerosis, further building on the achievements of NUPTAG.