@article{MartensPanzervandenWijngaardetal.2020, author = {Martens, Johannes and Panzer, Sabine and van den Wijngaard, Jeroen and Siebes, Maria and Schreiber, Laura M.}, title = {Influence of contrast agent dispersion on bolus-based MRI myocardial perfusion measurements: A computational fluid dynamics study}, series = {Magnetic Resonance in Medicine}, volume = {84}, journal = {Magnetic Resonance in Medicine}, number = {1}, doi = {10.1002/mrm.28125}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-208698}, pages = {467-483}, year = {2020}, abstract = {Purpose: Bolus-based dynamic contrast agent (CA) perfusion measurements of the heart are subject to systematic errors due to CA bolus dispersion in the coronary arteries. To better understand these effects on quantification of myocardial blood flow and myocardial perfusion reserve (MPR), an in-silico model of the coronary arteries down to the pre-arteriolar vessels has been developed. Methods: In this work, a computational fluid dynamics analysis is performed to investigate these errors on the basis of realistic 3D models of the left and right porcine coronary artery trees, including vessels at the pre-arteriolar level. Using advanced boundary conditions, simulations of blood flow and CA transport are conducted at rest and under stress. These are evaluated with regard to dispersion (assessed by the width of CA concentration time curves and associated vascular transport functions) and errors of myocardial blood flow and myocardial perfusion reserve quantification. Results: Contrast agent dispersion increases with traveled distance as well as vessel diameter, and decreases with higher flow velocities. Overall, the average myocardial blood flow errors are -28\% ± 16\% and -8.5\% ± 3.3\% at rest and stress, respectively, and the average myocardial perfusion reserve error is 26\% ± 22\%. The calculated values are different in the left and right coronary tree. Conclusion: Contrast agent dispersion is dependent on a complex interplay of several different factors characterizing the cardiovascular bed, including vessel size and integrated vascular length. Quantification errors evoked by the observed CA dispersion show nonnegligible distortion in dynamic CA bolus-based perfusion measurements. We expect future improvements of quantitative perfusion measurements to make the systematic errors described here more apparent.}, language = {en} } @article{PanzerZhangKonteetal.2021, author = {Panzer, Sabine and Zhang, Chong and Konte, Tilen and Br{\"a}uer, Celine and Diemar, Anne and Yogendran, Parathy and Yu-Strzelczyk, Jing and Nagel, Georg and Gao, Shiqiang and Terpitz, Ulrich}, title = {Modified Rhodopsins From Aureobasidium pullulans Excel With Very High Proton-Transport Rates}, series = {Frontiers in Molecular Biosciences}, volume = {8}, journal = {Frontiers in Molecular Biosciences}, issn = {2296-889X}, doi = {10.3389/fmolb.2021.750528}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-249248}, year = {2021}, abstract = {Aureobasidium pullulans is a black fungus that can adapt to various stressful conditions like hypersaline, acidic, and alkaline environments. The genome of A. pullulans exhibits three genes coding for putative opsins ApOps1, ApOps2, and ApOps3. We heterologously expressed these genes in mammalian cells and Xenopus oocytes. Localization in the plasma membrane was greatly improved by introducing additional membrane trafficking signals at the N-terminus and the C-terminus. In patch-clamp and two-electrode-voltage clamp experiments, all three proteins showed proton pump activity with maximal activity in green light. Among them, ApOps2 exhibited the most pronounced proton pump activity with current amplitudes occasionally extending 10 pA/pF at 0 mV. Proton pump activity was further supported in the presence of extracellular weak organic acids. Furthermore, we used site-directed mutagenesis to reshape protein functions and thereby implemented light-gated proton channels. We discuss the difference to other well-known proton pumps and the potential of these rhodopsins for optogenetic applications.}, language = {en} } @article{GoetzPanzerTrinksetal.2020, author = {G{\"o}tz, Ralph and Panzer, Sabine and Trinks, Nora and Eilts, Janna and Wagener, Johannes and Turr{\`a}, David and Di Pietro, Antonio and Sauer, Markus and Terpitz, Ulrich}, title = {Expansion Microscopy for Cell Biology Analysis in Fungi}, series = {Frontiers in Microbiology}, volume = {11}, journal = {Frontiers in Microbiology}, issn = {1664-302X}, doi = {10.3389/fmicb.2020.00574}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-202569}, year = {2020}, abstract = {Super-resolution microscopy has evolved as a powerful method for subdiffraction-resolution fluorescence imaging of cells and cellular organelles, but requires sophisticated and expensive installations. Expansion microscopy (ExM), which is based on the physical expansion of the cellular structure of interest, provides a cheap alternative to bypass the diffraction limit and enable super-resolution imaging on a conventional fluorescence microscope. While ExM has shown impressive results for the magnified visualization of proteins and RNAs in cells and tissues, it has not yet been applied in fungi, mainly due to their complex cell wall. Here we developed a method that enables reliable isotropic expansion of ascomycetes and basidiomycetes upon treatment with cell wall degrading enzymes. Confocal laser scanning microscopy (CLSM) and structured illumination microscopy (SIM) images of 4.5-fold expanded sporidia of Ustilago maydis expressing fluorescent fungal rhodopsins and hyphae of Fusarium oxysporum or Aspergillus fumigatus expressing either histone H1-mCherry together with Lifeact-sGFP or mRFP targeted to mitochondria, revealed details of subcellular structures with an estimated spatial resolution of around 30 nm. ExM is thus well suited for cell biology studies in fungi on conventional fluorescence microscopes.}, 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} } @article{PanzerBrychBatschaueretal.2019, author = {Panzer, Sabine and Brych, Annika and Batschauer, Alfred and Terpitz, Ulrich}, title = {Opsin 1 and Opsin 2 of the corn smut fungus ustilago maydis are green light-driven proton pumps}, series = {Frontiers in Microbiology}, volume = {10}, journal = {Frontiers in Microbiology}, doi = {10.3389/fmicb.2019.00735}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201453}, pages = {735}, year = {2019}, abstract = {In fungi, green light is absorbed by rhodopsins, opsin proteins carrying a retinal molecule as chromophore. The basidiomycete Ustilago maydis, a fungal pathogen that infects corn plants, encodes three putative photoactive opsins, called ops1 (UMAG_02629), ops2 (UMAG_00371), and ops3 (UMAG_04125). UmOps1 and UmOps2 are expressed during the whole life cycle, in axenic cultures as well as in planta, whereas UmOps3 was recently shown to be absent in axenic cultures but highly expressed during plant infection. Here we show that expression of UmOps1 and UmOps2 is induced by blue light under control of white collar 1 (Wco1). UmOps1 is mainly localized in the plasma membrane, both when expressed in HEK cells and U. maydis sporidia. In contrast, UmOps2 was mostly found intracellularly in the membranes of vacuoles. Patch-clamp studies demonstrated that both rhodopsins are green light-driven outward rectifying proton pumps. UmOps1 revealed an extraordinary pH dependency with increased activity in more acidic environment. Also, UmOps1 showed a pronounced, concentration-dependent enhancement of pump current caused by weak organic acids (WOAs), especially by acetic acid and indole-3-acetic acid (IAA). In contrast, UmOps2 showed the typical behavior of light-driven, outwardly directed proton pumps, whereas UmOps3 did not exhibit any electrogenity. With this work, insights were gained into the localization and molecular function of two U. maydis rhodopsins, paving the way for further studies on the biological role of these rhodopsins in the life cycle of U. maydis.}, language = {en} } @article{SputhPanzerStigloheretal.2021, author = {Sputh, Sebastian and Panzer, Sabine and Stigloher, Christian and Terpitz, Ulrich}, title = {Superaufgel{\"o}ste Mikroskopie: Pilze unter Beobachtung}, series = {BIOspektrum}, volume = {27}, journal = {BIOspektrum}, number = {4}, issn = {1868-6249}, doi = {10.1007/s12268-021-1592-6}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-270014}, pages = {380-382}, year = {2021}, abstract = {The diffraction limit of light confines fluorescence imaging of subcellular structures in fungi. Different super-resolution methods are available for the analysis of fungi that we briefly discuss. We exploit the filamentous fungus Fusarium fujikuroi expressing a YFP-labeled membrane protein showing the benefit of correlative light- and electron microscopy (CLEM), that combines structured illumination microscopy (SIM) and scanning election microscopy (SEM).}, language = {de} } @article{LuDreyerDickinsonetal.2023, author = {Lu, Jinping and Dreyer, Ingo and Dickinson, Miles Sasha and Panzer, Sabine and Jaślan, Dawid and Navarro-Retamal, Carlos and Geiger, Dietmar and Terpitz, Ulrich and Becker, Dirk and Stroud, Robert M. and Marten, Irene and Hedrich, Rainer}, title = {Vicia faba SV channel VfTPC1 is a hyperexcitable variant of plant vacuole two pore channels}, series = {eLife}, volume = {12}, journal = {eLife}, doi = {10.7554/eLife.86384}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-350264}, year = {2023}, abstract = {To fire action-potential-like electrical signals, the vacuole membrane requires the two-pore channel TPC1, formerly called SV channel. The TPC1/SV channel functions as a depolarization-stimulated, non-selective cation channel that is inhibited by luminal Ca\(^{2+}\). In our search for species-dependent functional TPC1 channel variants with different luminal Ca\(^{2+}\) sensitivity, we found in total three acidic residues present in Ca\(^{2+}\) sensor sites 2 and 3 of the Ca\(^{2+}\)-sensitive AtTPC1 channel from Arabidopsis thaliana that were neutral in its Vicia faba ortholog and also in those of many other Fabaceae. When expressed in the Arabidopsis AtTPC1-loss-of-function background, wild-type VfTPC1 was hypersensitive to vacuole depolarization and only weakly sensitive to blocking luminal Ca\(^{2+}\). When AtTPC1 was mutated for these VfTPC1-homologous polymorphic residues, two neutral substitutions in Ca\(^{2+}\) sensor site 3 alone were already sufficient for the Arabidopsis At-VfTPC1 channel mutant to gain VfTPC1-like voltage and luminal Ca\(^{2+}\) sensitivity that together rendered vacuoles hyperexcitable. Thus, natural TPC1 channel variants exist in plant families which may fine-tune vacuole excitability and adapt it to environmental settings of the particular ecological niche.}, language = {en} }