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Studying Large Multi-Protein Complexes Using Single Molecule Localization Microscopy


Virant,  David
Department of Systems and Synthetic Microbiology, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

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Virant, D. (2019). Studying Large Multi-Protein Complexes Using Single Molecule Localization Microscopy. PhD Thesis, Philipps-Universität Marburg, Marburg.

Cite as: https://hdl.handle.net/21.11116/0000-0008-EF23-4
Biology would not be where it is today without fluorescence microscopy. It is arguably one of the most commonly used tools in the biologists toolbox and it has helped scientists study the localization of cellular proteins and other small things for decades, but it is not without its limitations. Due to the diffraction limit, conventional fluorescence microscopy is limited to micrometer-range structures. Science has long relied upon electron microscopy and X-ray crystallography to study phenomena that occur below this limit. However, many of lifes processes occur between these two spatial domains. Super-resolution microscopy, the next stage of evolution of fluorescence microscopy, has the potential to bridge this gap between micro and nano. It combines superior resolutions of down to a few nanometers with the ability to view objects in their natural environments. It is the ideal tool for studying the large, multi-protein complexes that carry out most of lifes functions, but are too complex and fragile to put on an electron microscope or into a synchrotron. A form of super-resolution microscopy called SMLM Microscopy shows especially high promise in this regard. With its ability to detect individual molecules, it combines the high resolution needed for structural studies with the quantitative readout required for obtaining data on the stoichiometry of multi-protein complexes. This thesis describes new tools which expand the toolbox of SMLM with the specific aim of studying multi-protein complexes. First, the development of a novel fluorescent tagging system that is a mix of genetic tagging and immuno-staining. The system, termed BC2, consists of a short, genetically encodable peptide that is targeted by a nanobody (BC2 nanobody). The system brings several advantages. The small tag is not disruptive to the protein it is attached to and the small nanobody can get into tight spaces, making it an excellent tag for dense multi-protein structures. Next, several new variants of some commonly used green-to-red fluorescent proteins. The novel variants, which can be converted with a combination of blue and infrared light are especially useful for live-cell imaging. The developed fluorescent proteins can also be combined with photo-activatable fluorescent proteins to enable imaging of several targets with the same color protein. Finally, an application of the latter technique to study the multi-protein kinetochore complex and gain first glimpses into its spatial organization and the stoichiometry of its subunits.