@phdthesis{Trinks2024, author = {Trinks, Nora Isabel}, title = {Super-resolution fluorescence microscopic visualization and analysis of interactions between human immune cells and \(Aspergillus\) \(fumigatus\)}, doi = {10.25972/OPUS-26640}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-266407}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {The mold Aspergillus fumigatus (A. fumigatus) is known as human pathogen and can cause life-threatening infections in humans with a weakened immune system. This is a known complication in patients receiving glucocorticoids, e.g. after hematopoietic stem cell transplantation or solid organ transplantation. Although research in the field of immune cell/fungus interaction has discovered key strategies how immune cells fight against infectious fungi, our knowledge is still incomplete. In order to develop effective treatment options against fungal infections, a detailed understanding of their interactions is crucial. Thus, visualization of immune cell and fungus is an excellent approach to gain further knowledge. For a detailed view of such interaction processes, a high optical resolution on nanometer scale is required. There is a variety of super resolution microscopy techniques, enabling fluorescence imaging beyond the diffraction limit. This work combines the use of three complementary super resolution microscopy techniques, in order to study immune cell/fungus interaction from different points of view. Aim of this work is the introduction of the recently invented imaging technique named expansion microscopy (ExM) for the study of immune cell/fungus interactions. The core aspect of this method is the physical magnification of the specimen, which increases the distance between protein structures that are close to each other and which can therefore be imaged separately. The simultaneous magnification of primary human natural killer (NK) cells and A. fumigatus hyphae was established in this work using ExM. Reorganization of cytoskeletal components of interacting NK cells was demonstrated here, by expansion of the immunological synapse (IS), formed between NK cells and A. fumigatus. In addition, reorganization of the microtubule-organizing center (MTOC) towards fungal hyphae and an accumulation of actin at the IS has been observed. Furthermore, ExM has been used to visualize lytic granules of NK cells after degranulation. After magnification of the specimen, lysosome associated protein 1 (LAMP1) was shown to surround perforin. In absence of the plasma membrane-exposed degranulation marker LAMP1, a "ring-shaped" structure was often observed for fluorescently labeled perforin. Volume calculation of lytic granules demonstrated the benefit of ExM. Compared to pre-expansion images, analyses of post-expansion images showed two volume distributions for degranulated and non-degranulated NK cells. In addition, this work emphasizes the importance of determining the expansion factor for a structure in each species, as variations of expansion factors have been observed. This factor, as well as possible sample distortions should be considered, when ExM is used in order to analyze the interaction between two species. A second focus of this work is the visualization of a chimeric antigen receptor (CAR), targeting an epitope on the cell wall of A. fumigatus. Structured illumination microscopy (SIM) revealed that the CAR is part of the immunological synapse of primary human CAR T cells and CAR-NK-92 cells. At the interaction site, an accumulation of the CAR was observed, as well as the presence of perforin. CAR accumulation at fungal hyphae was further demonstrated by automated live cell imaging of interacting CAR-NK-92 cells, expressing a fluorescent fusion protein. Additionally, the use of direct stochastic optical reconstruction microscopy (dSTORM) gave first insights in CAR expression levels on the basal membrane of CAR-NK-92 cells, with single molecule sensitivity. CAR cluster analyses displayed a heterogeneous CAR density on the basal membrane of transfected NK 92 cells. In summary, this work provides insights into the application of ExM for studying the interaction of primary human NK cells and A. fumigatus for the first time. Furthermore, this thesis presents first insights regarding the characterization of an A. fumigatus-targeting CAR, by applying super-resolution fluorescence microscopy, like SIM and dSTORM.}, subject = {Mikroskopie}, language = {en} } @phdthesis{Panzer2022, author = {Panzer, Sabine}, title = {Spotlight on Fungal Rhodopsins: A Microscopic and Electrophysiological Study}, doi = {10.25972/OPUS-27185}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-271859}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {Microbial rhodopsins are abundant membrane proteins often capable of ion transport and are found in all three domains of life. Thus, many fungi, especially phyto-associated or phyto-pathogenic ones, contain these green-light-sensing photoreceptors. Proteins that perceive other wavelengths are often well characterized in terms of their impact on fungal biology whereas little is known about the function of fungal rhodopsins. In this work, five fungal rhodopsins, UmOps1 and UmOps2 from the corn smut Ustilago maydis as well as ApOps1, ApOps2 and ApOps3 from the black yeast Aureobasidium pullulans, were characterized electrophysiologically using mammalian expression systems and the patch-clamp technique to explore their ion transport properties. The latter three were modified using a membrane trafficking cassette, termed "2.0" that consists of the lucy rho motif, two Kir2.1 Golgi apparatus trafficking signals and a Kir2.1 endoplasmic reticulum export signal, what resulted in better plasma membrane localization. Rhodopsin mutants were created to identify amino acid residues that are key players in the ion transport process. Current enhancement in the presence of weak organic acids, that was already described before for the fungal rhodopsin CarO from Fusarium fujikuroi (Garc{\´i}a-Mart{\´i}nez et al., 2015; Adam et al., 2018), was investigated for the U. maydis rhodopsins as well as for ApOps2 by supplementing acetate in the patch-clamp electrolyte solutions. All five rhodopsins were found to be proton pumps unidirectionally transporting protons out of the cytosol upon green-light exposure with every rhodopsin exhibiting special features or unique characteristics in terms of the photocurrents. To name just a few, UmOps1, for example, showed a striking pH-dependency with massive enhancement of pump currents in the presence of extracellular acidic pH. Moreover, especially ApOps2 and ApOps3 showed very high current densities, however, the ones of ApOps3 were impaired when exchanging intracellular sodium to cesium. Concerning the mutations, it was found, that the electron releasing group in UmOps1 seems to be involved in the striking pH effect and that the mutation of the proton donor site resulted in almost unfunctional proteins. Moreover, a conserved arginine inside ApOps2 was mutated to turn the proton pump into a channel. Regarding the effect of weak organic acids, acetate was able to induce enhanced pump currents in UmOps1 and ApOps2, but not in UmOps2. Due to the capability of current production upon light illumination, microbial rhodopsins are used in the research field of optogenetics that aims to control neuronal activity by light. ApOps2 was used to test its functionality in differentiated NG108-15 cells addressing the question whether it is a promising candidate that can be used as an optogenetic tool. Indeed, this rhodopsin could be functionally expressed in this experimental system. Furthermore, microscopic studies were done to elucidate the localization of selected rhodopsins in fungal cells. Therefore, conventional (confocal laser scanning or structured illumination microscopy) as well as novel super-resolution techniques (expansion or correlated light and electron microscopy) were used. This was done on U. maydis sporidia, the yeast-like form of this fungus, via eGFP-tagged UmOps1 or UmOps2 expressing strains. Moreover, CarO-eYFP expressing F. fujikuroi was imaged microscopically to confirm the plasma membrane and tonoplast localization (Garc{\´i}a-Mart{\´i}nez et al., 2015) with the help of counterstaining experiments. UmOps1 was found to reside in the plasma membrane, UmOps2 localized to the tonoplast and CarO was indeed found in both of these localizations. This work gains further insight into rhodopsin functions and paves the way for further research in terms of the biological role of rhodopsins in fungal life cycles.}, subject = {Opsin}, language = {en} } @phdthesis{Maier2023, author = {Maier, Sophia Edith}, title = {Mapping membrane receptor distribution on resting platelets combining Expansion Microscopy and fluorescence confocal microscopy}, doi = {10.25972/OPUS-30031}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-300317}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Stroke and myocardial infarction are the most prominent and severe consequences of pathological thrombus formation. For prevention and/or treatment of thrombotic events there is a variety of anti-coagulation and antiplatelet medication that all have one side effect in common: the increased risk of bleeding. To design drugs that only intervene in the unwanted aggregation process but do not disturb general hemostasis, it is crucial to decipher the exact clotting pathway which has not been fully understood yet. Platelet membrane receptors play a vital role in the clotting pathway and, thus, the aim of this work is to establish a method to elucidate the interactions, clustering, and reorganization of involved membrane receptors such as GPIIb/IIIa and GPIX as part of the GPIb-IX-V complex. The special challenges regarding visualizing membrane receptor interactions on blood platelets are the high abundancy of the first and the small size of the latter (1—3µm of diameter). The resolution limit of conventional fluorescence microscopy and even super-resolution approaches prevents the successful differentiation of densely packed receptors from one another. Here, this issue is approached with the combination of a recently developed technique called Expansion Microscopy (ExM). The image resolution of a conventional fluorescence microscope is enhanced by simply enlarging the sample physically and thus pulling the receptors apart from each other. This method requires a complex sample preparation and holds lots of obstacles such as variable or anisotropic expansion and low images contrast. To increase ExM accuracy and sensitivity for interrogating blood platelets, it needs optimized sample preparation as well as image analysis pipelines which are the main part of this thesis. The colocalization results show that either fourfold or tenfold expanded, resting platelets allow a clear distinction between dependent, clustered, and independent receptor organizations compared to unexpanded platelets.Combining dual-color Expansion and confocal fluorescence microscopy enables to image in the nanometer range identifying GPIIb/IIIa clustering in resting platelets - a pattern that may play a key role in the clotting pathway}, language = {en} } @phdthesis{Aigner2023, author = {Aigner, Max}, title = {Establishing successful protocols and imaging pipelines for Expansion Microscopy in murine blood platelets}, doi = {10.25972/OPUS-30900}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-309003}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Platelets play an important role in the body, since they are part of the hemostasis system, preventing and stopping blood loss. Nevertheless, when platelet or coagulation system function are impaired, uncontrolled bleedings but also irreversible vessel occlusion followed by ischemic tissue damage can occur. Therefore, understanding platelet function and activation, mechanisms which are controlled by a variety of platelet membrane receptors and other factors is important to advance out knowledge of hemostasis and platelet malfunction. For a complete picture of platelet function and their modulating behavior it is desired to be able to quantify receptor distributions and interactions of these densely packed molecular ensembles in the membrane. This challenges scientists for several reasons. Most importantly, platelets are microscopically small objects, challenging the spatial resolution of conventional light microscopy. Moreover, platelet receptors are highly abundant on the membrane so even super-resolution microscopy struggles with quantitative receptor imaging on platelets. With Expansion microscopy (ExM), a new super-resolution technique was introduced, allowing resolutions to achieve super-resolution without using a super-resolution microscope, but by combining a conventional confocal microscopy with a highly processed sample that has been expanded physically. In this doctoral thesis, I evaluated the potential of this technique for super-resolution platelet imaging by optimizing the sample preparation process and establishing an imaging and image processing pipeline for dual-color 3D images of different membrane receptors. The analysis of receptor colocalization using ExM demonstrated a clear superiority compared to conventional microscopy. Furthermore, I identified a library of fluorescently labeled antibodies against different platelet receptors compatible with ExM and showed the possibility of staining membrane receptors and parts of the cytoskeleton at the same time.}, subject = {Mikroskopie}, language = {en} }