@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} } @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{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} }