Summary:
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.