title: Investigating the kinetochore complex in Schizosaccharomyces pombe using advanced fluorescence microscopy techniques
title: Untersuchung des Kinetochorkomplexes in Schizosaccharomyces pombe mittels moderner Fluoreszenzmikroskopietechniken
titleType: AlternativeTitle
Creators: name: Vojnovic, Ilijana
Publisher: name: Philipps-Universität Marburg
Year: 2022
Subjects: expansion microscopy
Einzelmoleküllokalisationsmikroskopie
microbiology
single-molecule localization microscopy
Expansionsmikroskopie
Schizosaccharomyces pombe
cell division
Life sciences
Biowissenschaften, Biologie
Kinetochor
kinetochore
Zellteilung
Mikrobiologie

description: Major insights into various biological processes and structures could be achieved using fluorescence microscopy, which is a non-invasive, live- and fixed cell compatible, high contrast imaging technique. Here, the molecule of interest can be specifically labeled with a fluorescent marker using a multitude of different labeling techniques, best suited for the individual biological research question. The location of the molecule of interest can be deduced from the emitted fluorescent signal of the marker due to their immediate proximity. However, structures smaller than the diffraction limit of light of about 200 nm can not be resolved by conventional fluorescence microscopy. Other techniques, such as e.g. electron microscopy (EM) or X-ray crystallography allow for higher resolutions, but lack the target specific read-out or are not compatible with in vivo studies. Nevertheless, by utilizing advanced optical components and illumination patterns and designing fluorophores with tightly controllable photophysics, the diffraction limit of light could be circumvented leading to improved resolutions. From these super-resolution techniques, only single-molecule localization microscopy (SMLM) allows for a quantitative analysis of the target molecule due to the spatiotemporal detection of the fluorescent marker. The segregation of sister-chromatids to the corresponding daughter cells is a vital and irreversible process, which needs to be tightly regulated. Here, a multi-protein complex called the kinetochore (KT), which serves as a force-sensing linker between the centromere in chromosomes and kinetochore microtubules (kMTs) originating from the spindle pole body (SPB), plays a pivotal role as errors in this process lead to aneuploidy or cell death. Thus, understanding the architecture and regulation of this complex is essential. However, even though certain subcomplexes of the KT could be resolved by EM or X-ray crystallography in vitro, the full KT nanostructure was not resolved in vivo yet. Hence in chapter 2, the in vivo nanoscale structure of the fission yeast KT complex was investigated using SMLM. The fission yeast Schizosaccharomyces pombe was used as the model organism of choice, due to its small regional centromeres. It acts as an intermediate between the point centromere in the budding yeast Saccharomyces cerevisiae, on which only one KT assembles, and the larger regional centromeres in humans. To investigate the KT in fission yeast, a structure smaller than the diffraction limit of light, different SMLM imaging and labeling strategies in microbes were developed or applied fitting this research question. However, for the creation of the KT map at least two different super-resolved targets are required: one reference protein at the centromere and one protein of interest (POI) a time in the KT complex. As no combination of commonly used photoswitchable organic dyes for SMLM proved to be applicable in fission yeast, the focus was shifted towards photoactivatable and - convertable fluorescent proteins (FPs) as alternative fluorescent markers. Knowing this, the KT structure was investigated using a multi-color SMLM approach based on FPs utilizing an orthogonal sequential illumination pattern and a KT protein database was generated. Developing novel image analysis tools and controls allowed for the extraction of intra- KT distances and POI copy numbers. Based on these parameters, first conclusions on the structure, preferred KT assembly pathways and stoichiometries were drawn and a model of the fission yeast KT was proposed. Finally, to investigate the KT structure in even greater detail, a new imaging technique combining expansion microscopy (ExM) and SMLM, termed single-molecule expansion microscopy in fission yeast (SExY) was developed in chapter 3, which increases the imaging resolution of SMLM by the corresponding expansion factor (EF) of the sample. For this, the fixed sample was first embedded in a hydrogel and then expanded upon incubation in aqueous media. To achieve an even expansion, the proteins were covalently linked to the gel mesh and obstacles like protein connections, cell walls or membranes were dissolved in homogenization steps prior to expansion. Then, the sample was imaged using the SMLM based imaging technique photoactivated localization microscopy (PALM), which lead to single-digit nanometer resolutions. Since KT proteins are low abundant, we optimized for an increase in protein retention yield, which we could improve by half compared to the initial protocol. We also optimized for an isotropic expansion of the sample, which we controlled by determining the EFs of different cell organelles and the distribution of cytosolic FPs compared to non-expanded cells. With the final SExY protocol at hand we were than able to visualize KT proteins as well as other nuclear targets in vivo at a single digit nanometer range for the first time.