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• The IV International Symposium on Fungal Stress and the XIII International Fungal Biology Conference Alder-Rangel, Alene; Bailão, Alexandre Melo; Herrera-Estrella, Alfredo; Rangel, Amanda E.A; Gácser, Attila; Gasch, Audrey P.; Campos, Claudia B.L; Peters, Christina; Camelim, Francine; Verde, Fulvia; Gadd, Geoffrey Michael; Braus, Gerhard; Eisermann, Iris; Quinn, Janet; Latgé, Jean-Paul; Aguirre, Jesus; Bennett, Joan W.; Heitman, Joseph; Nosanchuk, Joshua D.; Partida-Martínez, Laila P.; Bassilana, Martine; Acheampong, Mavis A.; Riquelme, Meritxell; Feldbrügge, Michael; Keller, Nancy P.; Keyhani, Nemat O.; Gunde-Cimerman, Nina; Nascimento, Raquel; Arkowitz, Robert A.; Mouriño-Pérez, Rosa Reyna; Naz, Sehar Afshan; Avery, Simon V.; Basso, Thiago Olitta; Terpitz, Ulrich; Lin, Xiaorong; Rangel, Drauzio E.N in Fungal Biology (2023).
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  For the first time, the International Symposium on Fungal Stress was joined by the XIII International Fungal Biology Conference. The International Symposium on Fungal Stress (ISFUS), always held in Brazil, is now in its fourth edition, as an event of recognized quality in the international community of mycological research. The event held in São José dos Campos, SP, Brazil, in September 2022, featured 33 renowned speakers from 12 countries, including: Austria, Brazil, France, Germany, Ghana, Hungary, México, Pakistan, Spain, Slovenia, USA, and UK. In addition to the scientific contribution of the event in bringing together national and international researchers and their work in a strategic area, it helps maintain and strengthen international cooperation for scientific development in Brazil.

  @article{alderrangel2023international, abstract = {For the first time, the International Symposium on Fungal Stress was joined by the XIII International Fungal Biology Conference. The International Symposium on Fungal Stress (ISFUS), always held in Brazil, is now in its fourth edition, as an event of recognized quality in the international community of mycological research. The event held in São José dos Campos, SP, Brazil, in September 2022, featured 33 renowned speakers from 12 countries, including: Austria, Brazil, France, Germany, Ghana, Hungary, México, Pakistan, Spain, Slovenia, USA, and UK. In addition to the scientific contribution of the event in bringing together national and international researchers and their work in a strategic area, it helps maintain and strengthen international cooperation for scientific development in Brazil.}, author = {Alder-Rangel, Alene and Bailão, Alexandre Melo and Herrera-Estrella, Alfredo and Rangel, Amanda E.A and Gácser, Attila and Gasch, Audrey P. and Campos, Claudia B.L and Peters, Christina and Camelim, Francine and Verde, Fulvia and Gadd, Geoffrey Michael and Braus, Gerhard and Eisermann, Iris and Quinn, Janet and Latgé, Jean-Paul and Aguirre, Jesus and Bennett, Joan W. and Heitman, Joseph and Nosanchuk, Joshua D. and Partida-Martínez, Laila P. and Bassilana, Martine and Acheampong, Mavis A. and Riquelme, Meritxell and Feldbrügge, Michael and Keller, Nancy P. and Keyhani, Nemat O. and Gunde-Cimerman, Nina and Nascimento, Raquel and Arkowitz, Robert A. and Mouriño-Pérez, Rosa Reyna and Naz, Sehar Afshan and Avery, Simon V. and Basso, Thiago Olitta and Terpitz, Ulrich and Lin, Xiaorong and Rangel, Drauzio E.N}, journal = {Fungal Biology}, keywords = {terpitz}, title = {The IV International Symposium on Fungal Stress and the XIII International Fungal Biology Conference}, year = 2023 }

  %0 Journal Article %1 alderrangel2023international %A Alder-Rangel, Alene %A Bailão, Alexandre Melo %A Herrera-Estrella, Alfredo %A Rangel, Amanda E.A %A Gácser, Attila %A Gasch, Audrey P. %A Campos, Claudia B.L %A Peters, Christina %A Camelim, Francine %A Verde, Fulvia %A Gadd, Geoffrey Michael %A Braus, Gerhard %A Eisermann, Iris %A Quinn, Janet %A Latgé, Jean-Paul %A Aguirre, Jesus %A Bennett, Joan W. %A Heitman, Joseph %A Nosanchuk, Joshua D. %A Partida-Martínez, Laila P. %A Bassilana, Martine %A Acheampong, Mavis A. %A Riquelme, Meritxell %A Feldbrügge, Michael %A Keller, Nancy P. %A Keyhani, Nemat O. %A Gunde-Cimerman, Nina %A Nascimento, Raquel %A Arkowitz, Robert A. %A Mouriño-Pérez, Rosa Reyna %A Naz, Sehar Afshan %A Avery, Simon V. %A Basso, Thiago Olitta %A Terpitz, Ulrich %A Lin, Xiaorong %A Rangel, Drauzio E.N %D 2023 %J Fungal Biology %R https://doi.org/10.1016/j.funbio.2023.04.006 %T The IV International Symposium on Fungal Stress and the XIII International Fungal Biology Conference %U https://www.sciencedirect.com/science/article/pii/S1878614623000521 %X For the first time, the International Symposium on Fungal Stress was joined by the XIII International Fungal Biology Conference. The International Symposium on Fungal Stress (ISFUS), always held in Brazil, is now in its fourth edition, as an event of recognized quality in the international community of mycological research. The event held in São José dos Campos, SP, Brazil, in September 2022, featured 33 renowned speakers from 12 countries, including: Austria, Brazil, France, Germany, Ghana, Hungary, México, Pakistan, Spain, Slovenia, USA, and UK. In addition to the scientific contribution of the event in bringing together national and international researchers and their work in a strategic area, it helps maintain and strengthen international cooperation for scientific development in Brazil.

• Candida albicans Induces Cross-Kingdom miRNA Trafficking in Human Monocytes To Promote Fungal Growth Halder, Luke D.; Babych, Svitlana; Palme, Diana I.; Mansouri-Ghahnavieh, Elham; Ivanov, Lia; Ashonibare, Victory; Langenhorst, Daniela; Prusty, Bhupesh; Rambach, Günter; Wich, Melissa; Trinks, Nora; Blango, Matthew G.; Kornitzer, Daniel; Terpitz, Ulrich; Speth, Cornelia; Jungnickel, Berit; Beyersdorf, Niklas; Zipfel, Peter F.; Brakhage, Axel A.; Skerka, Christine; Lorenz, Michael in mBio (2022). 13 e03563–21.
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  Over the last decade, communication between immune cells by extracellular vesicle-associated miRNAs has emerged as an important regulator of the coordinated immune response. Therefore, a thorough understanding of the conversation occurring via miRNAs, especially during infection, may provide novel insights into both the host reaction to the microbe as well as the microbial response. In response to infections, human immune cells release extracellular vesicles (EVs) that carry a situationally adapted cocktail of proteins and nucleic acids, including microRNAs (miRNAs), to coordinate the immune response. In this study, we identified hsa-miR-21-5p and hsa-miR-24-3p as the most common miRNAs in exosomes released by human monocytes in response to the pathogenic fungus Candida albicans. Functional analysis of miRNAs revealed that hsa-miR-24-3p, but not hsa-miR-21-5p, acted across species and kingdoms, entering C. albicans and inducing fungal cell growth by inhibiting translation of the cyclin-dependent kinase inhibitor Sol1. Packaging of hsa-miR-24-3p into monocyte exosomes required binding of fungal soluble β-glucan to complement receptor 3 (CR3) and binding of mannan to Toll-like receptor 4 (TLR4), resulting in receptor colocalization. Together, our in vitro and in vivo findings reveal a novel cross-species evasion mechanism by which C. albicans exploits a human miRNA to promote fungal growth and survival in the host. IMPORTANCE Over the last decade, communication between immune cells by extracellular vesicle-associated miRNAs has emerged as an important regulator of the coordinated immune response. Therefore, a thorough understanding of the conversation occurring via miRNAs, especially during infection, may provide novel insights into both the host reaction to the microbe as well as the microbial response. This study provides evidence that the pathogenic fungus C. albicans communicates with human monocytes and induces the release of a human miRNA that promotes fungal growth. This mechanism represents an unexpected cross-species interaction and implies that an inhibition of specific miRNAs offers new possibilities for the treatment of human fungal infections.

  @article{halder2022candida, abstract = {Over the last decade, communication between immune cells by extracellular vesicle-associated miRNAs has emerged as an important regulator of the coordinated immune response. Therefore, a thorough understanding of the conversation occurring via miRNAs, especially during infection, may provide novel insights into both the host reaction to the microbe as well as the microbial response. In response to infections, human immune cells release extracellular vesicles (EVs) that carry a situationally adapted cocktail of proteins and nucleic acids, including microRNAs (miRNAs), to coordinate the immune response. In this study, we identified hsa-miR-21-5p and hsa-miR-24-3p as the most common miRNAs in exosomes released by human monocytes in response to the pathogenic fungus Candida albicans. Functional analysis of miRNAs revealed that hsa-miR-24-3p, but not hsa-miR-21-5p, acted across species and kingdoms, entering C. albicans and inducing fungal cell growth by inhibiting translation of the cyclin-dependent kinase inhibitor Sol1. Packaging of hsa-miR-24-3p into monocyte exosomes required binding of fungal soluble β-glucan to complement receptor 3 (CR3) and binding of mannan to Toll-like receptor 4 (TLR4), resulting in receptor colocalization. Together, our in vitro and in vivo findings reveal a novel cross-species evasion mechanism by which C. albicans exploits a human miRNA to promote fungal growth and survival in the host. IMPORTANCE Over the last decade, communication between immune cells by extracellular vesicle-associated miRNAs has emerged as an important regulator of the coordinated immune response. Therefore, a thorough understanding of the conversation occurring via miRNAs, especially during infection, may provide novel insights into both the host reaction to the microbe as well as the microbial response. This study provides evidence that the pathogenic fungus C. albicans communicates with human monocytes and induces the release of a human miRNA that promotes fungal growth. This mechanism represents an unexpected cross-species interaction and implies that an inhibition of specific miRNAs offers new possibilities for the treatment of human fungal infections.}, author = {Halder, Luke D. and Babych, Svitlana and Palme, Diana I. and Mansouri-Ghahnavieh, Elham and Ivanov, Lia and Ashonibare, Victory and Langenhorst, Daniela and Prusty, Bhupesh and Rambach, Günter and Wich, Melissa and Trinks, Nora and Blango, Matthew G. and Kornitzer, Daniel and Terpitz, Ulrich and Speth, Cornelia and Jungnickel, Berit and Beyersdorf, Niklas and Zipfel, Peter F. and Brakhage, Axel A. and Skerka, Christine and Lorenz, Michael}, journal = {mBio}, keywords = {terpitz}, pages = {e03563-21}, series = 1, title = {Candida albicans Induces Cross-Kingdom miRNA Trafficking in Human Monocytes To Promote Fungal Growth}, volume = 13, year = 2022 }

  %0 Journal Article %1 halder2022candida %A Halder, Luke D. %A Babych, Svitlana %A Palme, Diana I. %A Mansouri-Ghahnavieh, Elham %A Ivanov, Lia %A Ashonibare, Victory %A Langenhorst, Daniela %A Prusty, Bhupesh %A Rambach, Günter %A Wich, Melissa %A Trinks, Nora %A Blango, Matthew G. %A Kornitzer, Daniel %A Terpitz, Ulrich %A Speth, Cornelia %A Jungnickel, Berit %A Beyersdorf, Niklas %A Zipfel, Peter F. %A Brakhage, Axel A. %A Skerka, Christine %A Lorenz, Michael %B 1 %D 2022 %J mBio %P e03563-21 %T Candida albicans Induces Cross-Kingdom miRNA Trafficking in Human Monocytes To Promote Fungal Growth %U https://journals.asm.org/doi/abs/10.1128/mbio.03563-21 %V 13 %X Over the last decade, communication between immune cells by extracellular vesicle-associated miRNAs has emerged as an important regulator of the coordinated immune response. Therefore, a thorough understanding of the conversation occurring via miRNAs, especially during infection, may provide novel insights into both the host reaction to the microbe as well as the microbial response. In response to infections, human immune cells release extracellular vesicles (EVs) that carry a situationally adapted cocktail of proteins and nucleic acids, including microRNAs (miRNAs), to coordinate the immune response. In this study, we identified hsa-miR-21-5p and hsa-miR-24-3p as the most common miRNAs in exosomes released by human monocytes in response to the pathogenic fungus Candida albicans. Functional analysis of miRNAs revealed that hsa-miR-24-3p, but not hsa-miR-21-5p, acted across species and kingdoms, entering C. albicans and inducing fungal cell growth by inhibiting translation of the cyclin-dependent kinase inhibitor Sol1. Packaging of hsa-miR-24-3p into monocyte exosomes required binding of fungal soluble β-glucan to complement receptor 3 (CR3) and binding of mannan to Toll-like receptor 4 (TLR4), resulting in receptor colocalization. Together, our in vitro and in vivo findings reveal a novel cross-species evasion mechanism by which C. albicans exploits a human miRNA to promote fungal growth and survival in the host. IMPORTANCE Over the last decade, communication between immune cells by extracellular vesicle-associated miRNAs has emerged as an important regulator of the coordinated immune response. Therefore, a thorough understanding of the conversation occurring via miRNAs, especially during infection, may provide novel insights into both the host reaction to the microbe as well as the microbial response. This study provides evidence that the pathogenic fungus C. albicans communicates with human monocytes and induces the release of a human miRNA that promotes fungal growth. This mechanism represents an unexpected cross-species interaction and implies that an inhibition of specific miRNAs offers new possibilities for the treatment of human fungal infections.
• The Photoreaction of the Proton-Pumping Rhodopsin 1 From the Maize Pathogenic Basidiomycete Ustilago maydis La Greca, Mariafrancesca; Chen, Jheng-Liang; Schubert, Luiz; Kozuch, Jacek; Berneiser, Tim; Terpitz, Ulrich; Heberle, Joachim; Schlesinger, Ramona in Frontiers in Molecular Biosciences (2022). 9
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  Microbial rhodopsins have recently been discovered in pathogenic fungi and have been postulated to be involved in signaling during the course of an infection. Here, we report on the spectroscopic characterization of a light-driven proton pump rhodopsin (UmRh1) from the smut pathogen Ustilago maydis, the causative agent of tumors in maize plants. Electrophysiology, time-resolved UV/Vis and vibrational spectroscopy indicate a pH-dependent photocycle. We also characterized the impact of the auxin hormone indole-3-acetic acid that was shown to influence the pump activity of UmRh1 on individual photocycle intermediates. A facile pumping activity test was established of UmRh1 expressed in Pichia pastoris cells, for probing proton pumping out of the living yeast cells during illumination. We show similarities and distinct differences to the well-known bacteriorhodopsin from archaea and discuss the putative role of UmRh1 in pathogenesis.

  @article{lagreca2022photoreaction, abstract = {Microbial rhodopsins have recently been discovered in pathogenic fungi and have been postulated to be involved in signaling during the course of an infection. Here, we report on the spectroscopic characterization of a light-driven proton pump rhodopsin (UmRh1) from the smut pathogen Ustilago maydis, the causative agent of tumors in maize plants. Electrophysiology, time-resolved UV/Vis and vibrational spectroscopy indicate a pH-dependent photocycle. We also characterized the impact of the auxin hormone indole-3-acetic acid that was shown to influence the pump activity of UmRh1 on individual photocycle intermediates. A facile pumping activity test was established of UmRh1 expressed in Pichia pastoris cells, for probing proton pumping out of the living yeast cells during illumination. We show similarities and distinct differences to the well-known bacteriorhodopsin from archaea and discuss the putative role of UmRh1 in pathogenesis.}, author = {La Greca, Mariafrancesca and Chen, Jheng-Liang and Schubert, Luiz and Kozuch, Jacek and Berneiser, Tim and Terpitz, Ulrich and Heberle, Joachim and Schlesinger, Ramona}, journal = {Frontiers in Molecular Biosciences}, keywords = {terpitz}, title = {The Photoreaction of the Proton-Pumping Rhodopsin 1 From the Maize Pathogenic Basidiomycete Ustilago maydis}, volume = 9, year = 2022 }

  %0 Journal Article %1 lagreca2022photoreaction %A La Greca, Mariafrancesca %A Chen, Jheng-Liang %A Schubert, Luiz %A Kozuch, Jacek %A Berneiser, Tim %A Terpitz, Ulrich %A Heberle, Joachim %A Schlesinger, Ramona %D 2022 %J Frontiers in Molecular Biosciences %T The Photoreaction of the Proton-Pumping Rhodopsin 1 From the Maize Pathogenic Basidiomycete Ustilago maydis %U https://www.frontiersin.org/article/10.3389/fmolb.2022.826990 %V 9 %X Microbial rhodopsins have recently been discovered in pathogenic fungi and have been postulated to be involved in signaling during the course of an infection. Here, we report on the spectroscopic characterization of a light-driven proton pump rhodopsin (UmRh1) from the smut pathogen Ustilago maydis, the causative agent of tumors in maize plants. Electrophysiology, time-resolved UV/Vis and vibrational spectroscopy indicate a pH-dependent photocycle. We also characterized the impact of the auxin hormone indole-3-acetic acid that was shown to influence the pump activity of UmRh1 on individual photocycle intermediates. A facile pumping activity test was established of UmRh1 expressed in Pichia pastoris cells, for probing proton pumping out of the living yeast cells during illumination. We show similarities and distinct differences to the well-known bacteriorhodopsin from archaea and discuss the putative role of UmRh1 in pathogenesis.
• Vicia faba TPC1, a genetically encoded variant of the vacuole Two Pore Channel 1, is hyperexcitable Lu, Jinping; Dreyer, Ingo; Dickinson, Miles Sasha; Panzer, Sabine; Jaslan, Dawid; Navarro-Retamal, Carlos; Geiger, Dietmar; Terpitz, Ulrich; Becker, Dirk; Stroud, Robert M.; Marten, Irene; Hedrich, Rainer in bioRxiv (2022). 2021.12.22.473873-.
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  To fire action-potential-like electrical signals, the vacuole membrane requires the depolarization-activated two-pore channel TPC1, also called Slowly activating Vacuolar SV channel. The TPC1/SV channel, encoded by the TPC1 gene, functions as a voltage-dependent and Ca2+-regulated potassium channel. TPC1 currents are activated by a rise in cytoplasmic Ca2+ but inhibited by luminal Ca2+. In search for species-dependent functional TPC1 channel variants, we studied polymorphic amino acids contributing to luminal Ca2+ sensitivity. We found that the acidic residues E457, E605 and D606 of the Ca2+-sensitive Arabidopsis AtTPC1 channel were neutralized by either asparagine or alanine in Vicia faba and many other Fabaceae as well. When expressed in the Arabidopsis loss-of-AtTPC1 function background, the wild type VfTPC1 was hypersensitive to vacuole depolarization and insensitive to blocking luminal Ca2+. When AtTPC1 was mutated for the three VfTPC1-homologous polymorphic site residues, the Arabidopsis At-VfTPC1 channel mutant gained VfTPC1-like voltage and luminal Ca2+ insensitivity that together made vacuoles hyperexcitable. These findings indicate that natural TPC1 channel variants in plant families exist which differ in vacuole excitability and very likely respond to changes in environmental settings of their ecological niche.Significance statement Vacuolar electrical excitability and stress-related Ca2+ signaling depends on the activity of the vacuolar cation channel TPC1. Until now, the regulatory features of AtTPC1 from the model plant Arabidopsis thaliana was believed to apply to the TPC1 channels of other species. However, here we now show that, surprisingly, the VfTPC1 channel of the economic broad bean, in contrast to AtTPC1, proves to be hyperactive and confers hyperexcitability to the vacuole. The different gating behavior is most likely related to an impaired Ca2+ sensor site in the vacuolar pore vestibule, rising the probability to open at more negative membrane voltages. These natural variants of the TPC1 channel could help the plant adapt and respond to environmental challenges.Competing Interest StatementThe authors have declared no competing interest.

  @article{lu2022ltemgtvicia, abstract = {To fire action-potential-like electrical signals, the vacuole membrane requires the depolarization-activated two-pore channel TPC1, also called Slowly activating Vacuolar SV channel. The TPC1/SV channel, encoded by the TPC1 gene, functions as a voltage-dependent and Ca2+-regulated potassium channel. TPC1 currents are activated by a rise in cytoplasmic Ca2+ but inhibited by luminal Ca2+. In search for species-dependent functional TPC1 channel variants, we studied polymorphic amino acids contributing to luminal Ca2+ sensitivity. We found that the acidic residues E457, E605 and D606 of the Ca2+-sensitive Arabidopsis AtTPC1 channel were neutralized by either asparagine or alanine in Vicia faba and many other Fabaceae as well. When expressed in the Arabidopsis loss-of-AtTPC1 function background, the wild type VfTPC1 was hypersensitive to vacuole depolarization and insensitive to blocking luminal Ca2+. When AtTPC1 was mutated for the three VfTPC1-homologous polymorphic site residues, the Arabidopsis At-VfTPC1 channel mutant gained VfTPC1-like voltage and luminal Ca2+ insensitivity that together made vacuoles hyperexcitable. These findings indicate that natural TPC1 channel variants in plant families exist which differ in vacuole excitability and very likely respond to changes in environmental settings of their ecological niche.Significance statement Vacuolar electrical excitability and stress-related Ca2+ signaling depends on the activity of the vacuolar cation channel TPC1. Until now, the regulatory features of AtTPC1 from the model plant Arabidopsis thaliana was believed to apply to the TPC1 channels of other species. However, here we now show that, surprisingly, the VfTPC1 channel of the economic broad bean, in contrast to AtTPC1, proves to be hyperactive and confers hyperexcitability to the vacuole. The different gating behavior is most likely related to an impaired Ca2+ sensor site in the vacuolar pore vestibule, rising the probability to open at more negative membrane voltages. These natural variants of the TPC1 channel could help the plant adapt and respond to environmental challenges.Competing Interest StatementThe authors have declared no competing interest.}, author = {Lu, Jinping and Dreyer, Ingo and Dickinson, Miles Sasha and Panzer, Sabine and Jaslan, Dawid and Navarro-Retamal, Carlos and Geiger, Dietmar and Terpitz, Ulrich and Becker, Dirk and Stroud, Robert M. and Marten, Irene and Hedrich, Rainer}, journal = {bioRxiv}, keywords = {terpitz}, month = {jan}, pages = {2021.12.22.473873–}, title = {Vicia faba TPC1, a genetically encoded variant of the vacuole Two Pore Channel 1, is hyperexcitable}, year = 2022 }

  %0 Journal Article %1 lu2022ltemgtvicia %A Lu, Jinping %A Dreyer, Ingo %A Dickinson, Miles Sasha %A Panzer, Sabine %A Jaslan, Dawid %A Navarro-Retamal, Carlos %A Geiger, Dietmar %A Terpitz, Ulrich %A Becker, Dirk %A Stroud, Robert M. %A Marten, Irene %A Hedrich, Rainer %D 2022 %J bioRxiv %P 2021.12.22.473873-- %R 10.1101/2021.12.22.473873 %T Vicia faba TPC1, a genetically encoded variant of the vacuole Two Pore Channel 1, is hyperexcitable %U http://biorxiv.org/content/early/2022/03/04/2021.12.22.473873.abstract %X To fire action-potential-like electrical signals, the vacuole membrane requires the depolarization-activated two-pore channel TPC1, also called Slowly activating Vacuolar SV channel. The TPC1/SV channel, encoded by the TPC1 gene, functions as a voltage-dependent and Ca2+-regulated potassium channel. TPC1 currents are activated by a rise in cytoplasmic Ca2+ but inhibited by luminal Ca2+. In search for species-dependent functional TPC1 channel variants, we studied polymorphic amino acids contributing to luminal Ca2+ sensitivity. We found that the acidic residues E457, E605 and D606 of the Ca2+-sensitive Arabidopsis AtTPC1 channel were neutralized by either asparagine or alanine in Vicia faba and many other Fabaceae as well. When expressed in the Arabidopsis loss-of-AtTPC1 function background, the wild type VfTPC1 was hypersensitive to vacuole depolarization and insensitive to blocking luminal Ca2+. When AtTPC1 was mutated for the three VfTPC1-homologous polymorphic site residues, the Arabidopsis At-VfTPC1 channel mutant gained VfTPC1-like voltage and luminal Ca2+ insensitivity that together made vacuoles hyperexcitable. These findings indicate that natural TPC1 channel variants in plant families exist which differ in vacuole excitability and very likely respond to changes in environmental settings of their ecological niche.Significance statement Vacuolar electrical excitability and stress-related Ca2+ signaling depends on the activity of the vacuolar cation channel TPC1. Until now, the regulatory features of AtTPC1 from the model plant Arabidopsis thaliana was believed to apply to the TPC1 channels of other species. However, here we now show that, surprisingly, the VfTPC1 channel of the economic broad bean, in contrast to AtTPC1, proves to be hyperactive and confers hyperexcitability to the vacuole. The different gating behavior is most likely related to an impaired Ca2+ sensor site in the vacuolar pore vestibule, rising the probability to open at more negative membrane voltages. These natural variants of the TPC1 channel could help the plant adapt and respond to environmental challenges.Competing Interest StatementThe authors have declared no competing interest.

• Subdiffraction-resolution fluorescence imaging of immunological synapse formation between NK cells and A. fumigatus by expansion microscopy Trinks, Nora; Reinhard, Sebastian; Drobny, Matthias; Heilig, Linda; Löffler, Jürgen; Sauer, Markus; Terpitz, Ulrich in Communications Biology (2021). 4(1) 1151-.
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  Expansion microscopy (ExM) enables super-resolution fluorescence imaging on standard microscopes by physical expansion of the sample. However, the investigation of interactions between different organisms such as mammalian and fungal cells by ExM remains challenging because different cell types require different expansion protocols to ensure identical, ideally isotropic expansion of both partners. Here, we introduce an ExM method that enables super-resolved visualization of the interaction between NK cells and Aspergillus fumigatus hyphae. 4-fold expansion in combination with confocal fluorescence imaging allows us to resolve details of cytoskeleton rearrangement as well as NK cells’ lytic granules triggered by contact with an RFP-expressing A. fumigatus strain. In particular, subdiffraction-resolution images show polarized degranulation upon contact formation and the presence of LAMP1 surrounding perforin at the NK cell-surface post degranulation. Our data demonstrate that optimized ExM protocols enable the investigation of immunological synapse formation between two different species with so far unmatched spatial resolution.

  @article{trinks2021subdiffractionresolution, abstract = {Expansion microscopy (ExM) enables super-resolution fluorescence imaging on standard microscopes by physical expansion of the sample. However, the investigation of interactions between different organisms such as mammalian and fungal cells by ExM remains challenging because different cell types require different expansion protocols to ensure identical, ideally isotropic expansion of both partners. Here, we introduce an ExM method that enables super-resolved visualization of the interaction between NK cells and Aspergillus fumigatus hyphae. 4-fold expansion in combination with confocal fluorescence imaging allows us to resolve details of cytoskeleton rearrangement as well as NK cells’ lytic granules triggered by contact with an RFP-expressing A. fumigatus strain. In particular, subdiffraction-resolution images show polarized degranulation upon contact formation and the presence of LAMP1 surrounding perforin at the NK cell-surface post degranulation. Our data demonstrate that optimized ExM protocols enable the investigation of immunological synapse formation between two different species with so far unmatched spatial resolution.}, author = {Trinks, Nora and Reinhard, Sebastian and Drobny, Matthias and Heilig, Linda and Löffler, Jürgen and Sauer, Markus and Terpitz, Ulrich}, journal = {Communications Biology}, keywords = {terpitz}, number = 1, pages = {1151–}, title = {Subdiffraction-resolution fluorescence imaging of immunological synapse formation between NK cells and A. fumigatus by expansion microscopy}, volume = 4, year = 2021 }

  %0 Journal Article %1 trinks2021subdiffractionresolution %A Trinks, Nora %A Reinhard, Sebastian %A Drobny, Matthias %A Heilig, Linda %A Löffler, Jürgen %A Sauer, Markus %A Terpitz, Ulrich %D 2021 %J Communications Biology %N 1 %P 1151-- %R 10.1038/s42003-021-02669-y %T Subdiffraction-resolution fluorescence imaging of immunological synapse formation between NK cells and A. fumigatus by expansion microscopy %U https://doi.org/10.1038/s42003-021-02669-y %V 4 %X Expansion microscopy (ExM) enables super-resolution fluorescence imaging on standard microscopes by physical expansion of the sample. However, the investigation of interactions between different organisms such as mammalian and fungal cells by ExM remains challenging because different cell types require different expansion protocols to ensure identical, ideally isotropic expansion of both partners. Here, we introduce an ExM method that enables super-resolved visualization of the interaction between NK cells and Aspergillus fumigatus hyphae. 4-fold expansion in combination with confocal fluorescence imaging allows us to resolve details of cytoskeleton rearrangement as well as NK cells’ lytic granules triggered by contact with an RFP-expressing A. fumigatus strain. In particular, subdiffraction-resolution images show polarized degranulation upon contact formation and the presence of LAMP1 surrounding perforin at the NK cell-surface post degranulation. Our data demonstrate that optimized ExM protocols enable the investigation of immunological synapse formation between two different species with so far unmatched spatial resolution.
• Modified Rhodopsins From Aureobasidium pullulans Excel With Very High Proton-Transport Rates Panzer, Sabine; Zhang, Chong; Konte, Tilen; Bräuer, Celine; Diemar, Anne; Yogendran, Parathy; Yu-Strzelczyk, Jing; Nagel, Georg; Gao, Shiqiang; Terpitz, Ulrich in Frontiers in Molecular Biosciences (2021). 8
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  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.

  @article{panzer2021modified, 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.}, author = {Panzer, Sabine and Zhang, Chong and Konte, Tilen and Bräuer, Celine and Diemar, Anne and Yogendran, Parathy and Yu-Strzelczyk, Jing and Nagel, Georg and Gao, Shiqiang and Terpitz, Ulrich}, journal = {Frontiers in Molecular Biosciences}, keywords = {terpitz}, series = 941, title = {Modified Rhodopsins From Aureobasidium pullulans Excel With Very High Proton-Transport Rates}, volume = 8, year = 2021 }

  %0 Journal Article %1 panzer2021modified %A Panzer, Sabine %A Zhang, Chong %A Konte, Tilen %A Bräuer, Celine %A Diemar, Anne %A Yogendran, Parathy %A Yu-Strzelczyk, Jing %A Nagel, Georg %A Gao, Shiqiang %A Terpitz, Ulrich %B 941 %D 2021 %J Frontiers in Molecular Biosciences %T Modified Rhodopsins From Aureobasidium pullulans Excel With Very High Proton-Transport Rates %U https://www.frontiersin.org/article/10.3389/fmolb.2021.750528 %V 8 %X 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.
• Superaufgelöste Mikroskopie: Pilze unter Beobachtung Sputh, Sebastian; Panzer, Sabine; Stigloher, Christian; Terpitz, Ulrich in BIOspektrum (2021). 27(4) 380–382.
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  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).

  @article{sputh2021superaufgelste, 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).}, author = {Sputh, Sebastian and Panzer, Sabine and Stigloher, Christian and Terpitz, Ulrich}, journal = {BIOspektrum}, keywords = {terpitz}, number = 4, pages = {380–382}, title = {Superaufgelöste Mikroskopie: Pilze unter Beobachtung}, volume = 27, year = 2021 }

  %0 Journal Article %1 sputh2021superaufgelste %A Sputh, Sebastian %A Panzer, Sabine %A Stigloher, Christian %A Terpitz, Ulrich %D 2021 %J BIOspektrum %N 4 %P 380--382 %R 10.1007/s12268-021-1592-6 %T Superaufgelöste Mikroskopie: Pilze unter Beobachtung %U https://doi.org/10.1007/s12268-021-1592-6 %V 27 %X 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).
• Acidosis-induced activation of anion channel SLAH3 in the flooding-related stress response of Arabidopsis Lehmann, Julian; Jørgensen, Morten E.; Fratz, Stefanie; Müller, Heike M.; Kusch, Jana; Scherzer, Sönke; Navarro-Retamal, Carlos; Mayer, Dominik; Böhm, Jennifer; Konrad, Kai R.; Terpitz, Ulrich; Dreyer, Ingo; Mueller, Thomas D.; Sauer, Markus; Hedrich, Rainer; Geiger, Dietmar; Maierhofer, Tobias in Current Biology (2021).
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  Summary Plants, as sessile organisms, gained the ability to sense and respond to biotic and abiotic stressors to survive severe changes in their environments. The change in our climate comes with extreme dry periods but also episodes of flooding. The latter stress condition causes anaerobiosis-triggered cytosolic acidosis and impairs plant function. The molecular mechanism that enables plant cells to sense acidity and convey this signal via membrane depolarization was previously unknown. Here, we show that acidosis-induced anion efflux from Arabidopsis (Arabidopsis thaliana) roots is dependent on the S-type anion channel AtSLAH3. Heterologous expression of SLAH3 in Xenopus oocytes revealed that the anion channel is directly activated by a small, physiological drop in cytosolic pH. Acidosis-triggered activation of SLAH3 is mediated by protonation of histidine 330 and 454. Super-resolution microscopy analysis showed that the increase in cellular proton concentration switches SLAH3 from an electrically silent channel dimer into its active monomeric form. Our results show that, upon acidification, protons directly switch SLAH3 to its open configuration, bypassing kinase-dependent activation. Moreover, under flooding conditions, the stress response of Arabidopsis wild-type (WT) plants was significantly higher compared to SLAH3 loss-of-function mutants. Our genetic evidence of SLAH3 pH sensor function may guide the development of crop varieties with improved stress tolerance.

  @article{lehmann2021acidosisinduced, abstract = {Summary Plants, as sessile organisms, gained the ability to sense and respond to biotic and abiotic stressors to survive severe changes in their environments. The change in our climate comes with extreme dry periods but also episodes of flooding. The latter stress condition causes anaerobiosis-triggered cytosolic acidosis and impairs plant function. The molecular mechanism that enables plant cells to sense acidity and convey this signal via membrane depolarization was previously unknown. Here, we show that acidosis-induced anion efflux from Arabidopsis (Arabidopsis thaliana) roots is dependent on the S-type anion channel AtSLAH3. Heterologous expression of SLAH3 in Xenopus oocytes revealed that the anion channel is directly activated by a small, physiological drop in cytosolic pH. Acidosis-triggered activation of SLAH3 is mediated by protonation of histidine 330 and 454. Super-resolution microscopy analysis showed that the increase in cellular proton concentration switches SLAH3 from an electrically silent channel dimer into its active monomeric form. Our results show that, upon acidification, protons directly switch SLAH3 to its open configuration, bypassing kinase-dependent activation. Moreover, under flooding conditions, the stress response of Arabidopsis wild-type (WT) plants was significantly higher compared to SLAH3 loss-of-function mutants. Our genetic evidence of SLAH3 pH sensor function may guide the development of crop varieties with improved stress tolerance.}, author = {Lehmann, Julian and Jørgensen, Morten E. and Fratz, Stefanie and Müller, Heike M. and Kusch, Jana and Scherzer, Sönke and Navarro-Retamal, Carlos and Mayer, Dominik and Böhm, Jennifer and Konrad, Kai R. and Terpitz, Ulrich and Dreyer, Ingo and Mueller, Thomas D. and Sauer, Markus and Hedrich, Rainer and Geiger, Dietmar and Maierhofer, Tobias}, journal = {Current Biology}, keywords = {terpitz}, title = {Acidosis-induced activation of anion channel SLAH3 in the flooding-related stress response of Arabidopsis}, year = 2021 }

  %0 Journal Article %1 lehmann2021acidosisinduced %A Lehmann, Julian %A Jørgensen, Morten E. %A Fratz, Stefanie %A Müller, Heike M. %A Kusch, Jana %A Scherzer, Sönke %A Navarro-Retamal, Carlos %A Mayer, Dominik %A Böhm, Jennifer %A Konrad, Kai R. %A Terpitz, Ulrich %A Dreyer, Ingo %A Mueller, Thomas D. %A Sauer, Markus %A Hedrich, Rainer %A Geiger, Dietmar %A Maierhofer, Tobias %D 2021 %J Current Biology %R https://doi.org/10.1016/j.cub.2021.06.018 %T Acidosis-induced activation of anion channel SLAH3 in the flooding-related stress response of Arabidopsis %U https://www.sciencedirect.com/science/article/pii/S0960982221008137 %X Summary Plants, as sessile organisms, gained the ability to sense and respond to biotic and abiotic stressors to survive severe changes in their environments. The change in our climate comes with extreme dry periods but also episodes of flooding. The latter stress condition causes anaerobiosis-triggered cytosolic acidosis and impairs plant function. The molecular mechanism that enables plant cells to sense acidity and convey this signal via membrane depolarization was previously unknown. Here, we show that acidosis-induced anion efflux from Arabidopsis (Arabidopsis thaliana) roots is dependent on the S-type anion channel AtSLAH3. Heterologous expression of SLAH3 in Xenopus oocytes revealed that the anion channel is directly activated by a small, physiological drop in cytosolic pH. Acidosis-triggered activation of SLAH3 is mediated by protonation of histidine 330 and 454. Super-resolution microscopy analysis showed that the increase in cellular proton concentration switches SLAH3 from an electrically silent channel dimer into its active monomeric form. Our results show that, upon acidification, protons directly switch SLAH3 to its open configuration, bypassing kinase-dependent activation. Moreover, under flooding conditions, the stress response of Arabidopsis wild-type (WT) plants was significantly higher compared to SLAH3 loss-of-function mutants. Our genetic evidence of SLAH3 pH sensor function may guide the development of crop varieties with improved stress tolerance.
• Coregulation of gene expression by White collar 1 and phytochrome in Ustilago maydis Brych, Annika; Haas, Fabian B.; Parzefall, Katharina; Panzer, Sabine; Schermuly, Jeanette; Altmüller, Janine; Engelsdorf, Timo; Terpitz, Ulrich; Rensing, Stefan A.; Kiontke, Stephan; Batschauer, Alfred in Fungal Genetics and Biology (2021). 103570-.
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  Ustilago maydis encodes ten predicted light-sensing proteins. The biological functions of only a few of them are elucidated. Among the characterized ones are two DNA-photolyases and two rhodopsins that act as DNA-repair enzymes or green light-driven proton pumps, respectively. Here we report on the role of two other photoreceptors in U. maydis, namely White collar 1 (Wco1) and Phytochrome 1 (Phy1). We show that they bind flavins or biliverdin as chromophores, respectively. Both photoreceptors undergo a photocycle in vitro. Wco1 is the dominant blue light receptor in the saprophytic phase, controlling all of the 324 differentially expressed genes in blue light. U. maydis also responds to red and far-red light. However, the number of red or far-red light-controlled genes is less compared to blue light-regulated ones. Moreover, most of the red and far-red light-controlled genes not only depend on Phy1 but also on Wco1, indicating partial coregulation of gene expression by both photoreceptors. GFP-fused Wco1 is preferentially located in the nucleus, Phy1 in the cytosol, thus providing no hint that these photoreceptors directly interact or operate within the same complex. This is the first report on a functional characterization and coaction of White collar 1 and phytochrome orthologs in basidiomycetes.

  @article{brych2021coregulation, abstract = {Ustilago maydis encodes ten predicted light-sensing proteins. The biological functions of only a few of them are elucidated. Among the characterized ones are two DNA-photolyases and two rhodopsins that act as DNA-repair enzymes or green light-driven proton pumps, respectively. Here we report on the role of two other photoreceptors in U. maydis, namely White collar 1 (Wco1) and Phytochrome 1 (Phy1). We show that they bind flavins or biliverdin as chromophores, respectively. Both photoreceptors undergo a photocycle in vitro. Wco1 is the dominant blue light receptor in the saprophytic phase, controlling all of the 324 differentially expressed genes in blue light. U. maydis also responds to red and far-red light. However, the number of red or far-red light-controlled genes is less compared to blue light-regulated ones. Moreover, most of the red and far-red light-controlled genes not only depend on Phy1 but also on Wco1, indicating partial coregulation of gene expression by both photoreceptors. GFP-fused Wco1 is preferentially located in the nucleus, Phy1 in the cytosol, thus providing no hint that these photoreceptors directly interact or operate within the same complex. This is the first report on a functional characterization and coaction of White collar 1 and phytochrome orthologs in basidiomycetes.}, author = {Brych, Annika and Haas, Fabian B. and Parzefall, Katharina and Panzer, Sabine and Schermuly, Jeanette and Altmüller, Janine and Engelsdorf, Timo and Terpitz, Ulrich and Rensing, Stefan A. and Kiontke, Stephan and Batschauer, Alfred}, journal = {Fungal Genetics and Biology}, keywords = {terpitz}, pages = {103570–}, title = {Coregulation of gene expression by White collar 1 and phytochrome in Ustilago maydis}, year = 2021 }

  %0 Journal Article %1 brych2021coregulation %A Brych, Annika %A Haas, Fabian B. %A Parzefall, Katharina %A Panzer, Sabine %A Schermuly, Jeanette %A Altmüller, Janine %A Engelsdorf, Timo %A Terpitz, Ulrich %A Rensing, Stefan A. %A Kiontke, Stephan %A Batschauer, Alfred %D 2021 %J Fungal Genetics and Biology %P 103570-- %R https://doi.org/10.1016/j.fgb.2021.103570 %T Coregulation of gene expression by White collar 1 and phytochrome in Ustilago maydis %U https://www.sciencedirect.com/science/article/pii/S1087184521000542 %X Ustilago maydis encodes ten predicted light-sensing proteins. The biological functions of only a few of them are elucidated. Among the characterized ones are two DNA-photolyases and two rhodopsins that act as DNA-repair enzymes or green light-driven proton pumps, respectively. Here we report on the role of two other photoreceptors in U. maydis, namely White collar 1 (Wco1) and Phytochrome 1 (Phy1). We show that they bind flavins or biliverdin as chromophores, respectively. Both photoreceptors undergo a photocycle in vitro. Wco1 is the dominant blue light receptor in the saprophytic phase, controlling all of the 324 differentially expressed genes in blue light. U. maydis also responds to red and far-red light. However, the number of red or far-red light-controlled genes is less compared to blue light-regulated ones. Moreover, most of the red and far-red light-controlled genes not only depend on Phy1 but also on Wco1, indicating partial coregulation of gene expression by both photoreceptors. GFP-fused Wco1 is preferentially located in the nucleus, Phy1 in the cytosol, thus providing no hint that these photoreceptors directly interact or operate within the same complex. This is the first report on a functional characterization and coaction of White collar 1 and phytochrome orthologs in basidiomycetes.
• Direct Visualization of Fungal Burden in Filamentous Fungus-Infected Silkworms Yu, Yidong; Wolf, Ann-Katrin; Thusek, Sina; Heinekamp, Thorsten; Bromley, Michael; Krappmann, Sven; Terpitz, Ulrich; Voigt, Kerstin; Brakhage, Axel A.; Beilhack, Andreas (2021).
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  Invasive fungal infections (IFIs) are difficult to diagnose and to treat and, despite several available antifungal drugs, cause high mortality rates. In the past decades, the incidence of IFIs has continuously increased. More recently, SARS-CoV-2-associated lethal IFIs have been reported worldwide in critically ill patients. Combating IFIs requires a more profound understanding of fungal pathogenicity to facilitate the development of novel antifungal strategies. Animal models are indispensable for studying fungal infections and to develop new antifungals. However, using mammalian animal models faces various hurdles including ethical issues and high costs, which makes large-scale infection experiments extremely challenging. To overcome these limitations, we optimized an invertebrate model and introduced a simple calcofluor white (CW) staining protocol to macroscopically and microscopically monitor disease progression in silkworms (Bombyx mori) infected with the human pathogenic filamentous fungi Aspergillus fumigatus and Lichtheimia corymbifera. This advanced silkworm A. fumigatus infection model could validate knockout mutants with either attenuated, strongly attenuated or unchanged virulence. Finally, CW staining allowed us to efficiently visualize antifungal treatment outcomes in infected silkworms. Conclusively, we here present a powerful animal model combined with a straightforward staining protocol to expedite large-scale in vivo research of fungal pathogenicity and to investigate novel antifungal candidates.

  @misc{yu2021direct, abstract = {Invasive fungal infections (IFIs) are difficult to diagnose and to treat and, despite several available antifungal drugs, cause high mortality rates. In the past decades, the incidence of IFIs has continuously increased. More recently, SARS-CoV-2-associated lethal IFIs have been reported worldwide in critically ill patients. Combating IFIs requires a more profound understanding of fungal pathogenicity to facilitate the development of novel antifungal strategies. Animal models are indispensable for studying fungal infections and to develop new antifungals. However, using mammalian animal models faces various hurdles including ethical issues and high costs, which makes large-scale infection experiments extremely challenging. To overcome these limitations, we optimized an invertebrate model and introduced a simple calcofluor white (CW) staining protocol to macroscopically and microscopically monitor disease progression in silkworms (Bombyx mori) infected with the human pathogenic filamentous fungi Aspergillus fumigatus and Lichtheimia corymbifera. This advanced silkworm A. fumigatus infection model could validate knockout mutants with either attenuated, strongly attenuated or unchanged virulence. Finally, CW staining allowed us to efficiently visualize antifungal treatment outcomes in infected silkworms. Conclusively, we here present a powerful animal model combined with a straightforward staining protocol to expedite large-scale in vivo research of fungal pathogenicity and to investigate novel antifungal candidates.}, author = {Yu, Yidong and Wolf, Ann-Katrin and Thusek, Sina and Heinekamp, Thorsten and Bromley, Michael and Krappmann, Sven and Terpitz, Ulrich and Voigt, Kerstin and Brakhage, Axel A. and Beilhack, Andreas}, booktitle = {Journal of Fungi}, keywords = {terpitz}, number = 2, title = {Direct Visualization of Fungal Burden in Filamentous Fungus-Infected Silkworms}, volume = 7, year = 2021 }

  %0 Generic %1 yu2021direct %A Yu, Yidong %A Wolf, Ann-Katrin %A Thusek, Sina %A Heinekamp, Thorsten %A Bromley, Michael %A Krappmann, Sven %A Terpitz, Ulrich %A Voigt, Kerstin %A Brakhage, Axel A. %A Beilhack, Andreas %B Journal of Fungi %D 2021 %N 2 %R 10.3390/jof7020136 %T Direct Visualization of Fungal Burden in Filamentous Fungus-Infected Silkworms %V 7 %X Invasive fungal infections (IFIs) are difficult to diagnose and to treat and, despite several available antifungal drugs, cause high mortality rates. In the past decades, the incidence of IFIs has continuously increased. More recently, SARS-CoV-2-associated lethal IFIs have been reported worldwide in critically ill patients. Combating IFIs requires a more profound understanding of fungal pathogenicity to facilitate the development of novel antifungal strategies. Animal models are indispensable for studying fungal infections and to develop new antifungals. However, using mammalian animal models faces various hurdles including ethical issues and high costs, which makes large-scale infection experiments extremely challenging. To overcome these limitations, we optimized an invertebrate model and introduced a simple calcofluor white (CW) staining protocol to macroscopically and microscopically monitor disease progression in silkworms (Bombyx mori) infected with the human pathogenic filamentous fungi Aspergillus fumigatus and Lichtheimia corymbifera. This advanced silkworm A. fumigatus infection model could validate knockout mutants with either attenuated, strongly attenuated or unchanged virulence. Finally, CW staining allowed us to efficiently visualize antifungal treatment outcomes in infected silkworms. Conclusively, we here present a powerful animal model combined with a straightforward staining protocol to expedite large-scale in vivo research of fungal pathogenicity and to investigate novel antifungal candidates.
• The impact of episporic modification of Lichtheimia corymbifera on virulence and interaction with phagocytes Hassan, Mohamed I. Abdelwahab; Keller, Monique; Hillger, Michael; Binder, Ulrike; Reuter, Stefanie; Herold, Kristina; Telagathoti, Anusha; Dahse, Hans-Martin; Wicht, Saiedeh; Trinks, Nora; Nietzsche, Sandor; Deckert-Gaudig, Tanja; Deckert, Volker; Mrowka, Ralf; Terpitz, Ulrich; {Peter Saluz}, Hans; Voigt, Kerstin in Computational and Structural Biotechnology Journal (2021). 19 880–896.
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  Fungal infections caused by the ancient lineage Mucorales are emerging and increasingly reported in humans. Comprehensive surveys on promising attributes from a multitude of possible virulence factors are limited and so far, focused on Mucor and Rhizopus. This study addresses a systematic approach to monitor phagocytosis after physical and enzymatic modification of the outer spore wall of Lichtheimia corymbifera, one of the major causative agents of mucormycosis. Episporic modifications were performed and their consequences on phagocytosis, intracellular survival and virulence by murine alveolar macrophages and in an invertebrate infection model were elucidated. While depletion of lipids did not affect the phagocytosis of both strains, delipidation led to attenuation of LCA strain but appears to be dispensable for infection with LCV strain in the settings used in this study. Combined glucano-proteolytic treatment was necessary to achieve a significant decrease of virulence of the LCV strain in Galleria mellonella during maintenance of the full potential for spore germination as shown by a novel automated germination assay. Proteolytic and glucanolytic treatments largely increased phagocytosis compared to alive resting and swollen spores. Whilst resting spores barely (1–2%) fuse to lysosomes after invagination in to phagosomes, spore trypsinization led to a 10-fold increase of phagolysosomal fusion as measured by intracellular acidification. This is the first report of a polyphasic measurement of the consequences of episporic modification of a mucormycotic pathogen in spore germination, spore surface ultrastructure, phagocytosis, stimulation of Toll-like receptors (TLRs), phagolysosomal fusion and intracellular acidification, apoptosis, generation of reactive oxygen species (ROS) and virulence.

  @article{HASSAN2021880, abstract = {Fungal infections caused by the ancient lineage Mucorales are emerging and increasingly reported in humans. Comprehensive surveys on promising attributes from a multitude of possible virulence factors are limited and so far, focused on Mucor and Rhizopus. This study addresses a systematic approach to monitor phagocytosis after physical and enzymatic modification of the outer spore wall of Lichtheimia corymbifera, one of the major causative agents of mucormycosis. Episporic modifications were performed and their consequences on phagocytosis, intracellular survival and virulence by murine alveolar macrophages and in an invertebrate infection model were elucidated. While depletion of lipids did not affect the phagocytosis of both strains, delipidation led to attenuation of LCA strain but appears to be dispensable for infection with LCV strain in the settings used in this study. Combined glucano-proteolytic treatment was necessary to achieve a significant decrease of virulence of the LCV strain in Galleria mellonella during maintenance of the full potential for spore germination as shown by a novel automated germination assay. Proteolytic and glucanolytic treatments largely increased phagocytosis compared to alive resting and swollen spores. Whilst resting spores barely (1–2%) fuse to lysosomes after invagination in to phagosomes, spore trypsinization led to a 10-fold increase of phagolysosomal fusion as measured by intracellular acidification. This is the first report of a polyphasic measurement of the consequences of episporic modification of a mucormycotic pathogen in spore germination, spore surface ultrastructure, phagocytosis, stimulation of Toll-like receptors (TLRs), phagolysosomal fusion and intracellular acidification, apoptosis, generation of reactive oxygen species (ROS) and virulence.}, author = {Hassan, Mohamed I. Abdelwahab and Keller, Monique and Hillger, Michael and Binder, Ulrike and Reuter, Stefanie and Herold, Kristina and Telagathoti, Anusha and Dahse, Hans-Martin and Wicht, Saiedeh and Trinks, Nora and Nietzsche, Sandor and Deckert-Gaudig, Tanja and Deckert, Volker and Mrowka, Ralf and Terpitz, Ulrich and {Peter Saluz}, Hans and Voigt, Kerstin}, journal = {Computational and Structural Biotechnology Journal}, keywords = {terpitz}, pages = {880 - 896}, title = {The impact of episporic modification of Lichtheimia corymbifera on virulence and interaction with phagocytes}, volume = 19, year = 2021 }

  %0 Journal Article %1 HASSAN2021880 %A Hassan, Mohamed I. Abdelwahab %A Keller, Monique %A Hillger, Michael %A Binder, Ulrike %A Reuter, Stefanie %A Herold, Kristina %A Telagathoti, Anusha %A Dahse, Hans-Martin %A Wicht, Saiedeh %A Trinks, Nora %A Nietzsche, Sandor %A Deckert-Gaudig, Tanja %A Deckert, Volker %A Mrowka, Ralf %A Terpitz, Ulrich %A {Peter Saluz}, Hans %A Voigt, Kerstin %D 2021 %J Computational and Structural Biotechnology Journal %P 880 - 896 %R https://doi.org/10.1016/j.csbj.2021.01.023 %T The impact of episporic modification of Lichtheimia corymbifera on virulence and interaction with phagocytes %U https://doi.org/10.1016/j.csbj.2021.01.023 %V 19 %X Fungal infections caused by the ancient lineage Mucorales are emerging and increasingly reported in humans. Comprehensive surveys on promising attributes from a multitude of possible virulence factors are limited and so far, focused on Mucor and Rhizopus. This study addresses a systematic approach to monitor phagocytosis after physical and enzymatic modification of the outer spore wall of Lichtheimia corymbifera, one of the major causative agents of mucormycosis. Episporic modifications were performed and their consequences on phagocytosis, intracellular survival and virulence by murine alveolar macrophages and in an invertebrate infection model were elucidated. While depletion of lipids did not affect the phagocytosis of both strains, delipidation led to attenuation of LCA strain but appears to be dispensable for infection with LCV strain in the settings used in this study. Combined glucano-proteolytic treatment was necessary to achieve a significant decrease of virulence of the LCV strain in Galleria mellonella during maintenance of the full potential for spore germination as shown by a novel automated germination assay. Proteolytic and glucanolytic treatments largely increased phagocytosis compared to alive resting and swollen spores. Whilst resting spores barely (1–2%) fuse to lysosomes after invagination in to phagosomes, spore trypsinization led to a 10-fold increase of phagolysosomal fusion as measured by intracellular acidification. This is the first report of a polyphasic measurement of the consequences of episporic modification of a mucormycotic pathogen in spore germination, spore surface ultrastructure, phagocytosis, stimulation of Toll-like receptors (TLRs), phagolysosomal fusion and intracellular acidification, apoptosis, generation of reactive oxygen species (ROS) and virulence.

• Reconstituting NK Cells After Allogeneic Stem Cell Transplantation Show Impaired Response to the Fungal Pathogen Aspergillus fumigatus Weiss, Esther; Schlegel, Jan; Terpitz, Ulrich; Weber, Michael; Linde, Jörg; Schmitt, Anna-Lena; Hünniger, Kerstin; Marischen, Lothar; Gamon, Florian; Bauer, Joachim; Löffler, Claudia; Kurzai, Oliver; Morton, Charles Oliver; Sauer, Markus; Einsele, Hermann; Loeffler, Juergen in Frontiers in Immunology (2020). 11
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  Delayed natural killer (NK) cell reconstitution after allogeneic stem cell transplantation (alloSCT) is associated with a higher risk of developing invasive aspergillosis. The interaction of NK cells with the human pathogen Aspergillus (A.) fumigatus is mediated by the fungal recognition receptor CD56, which is relocated to the fungal interface after contact. Blocking of CD56 signaling inhibits the fungal mediated chemokine secretion of MIP-1α, MIP-1β, and RANTES and reduces cell activation, indicating a functional role of CD56 in fungal recognition. We collected peripheral blood from recipients of an allograft at defined time points after alloSCT (day 60, 90, 120, 180). NK cells were isolated, directly challenged with live A. fumigatus germ tubes, and cell function was analyzed and compared to healthy age and gender-matched individuals. After alloSCT, NK cells displayed a higher percentage of CD56^brightCD16^dim cells throughout the time of blood collection. However, CD56 binding and relocalization to the fungal contact side were decreased. We were able to correlate this deficiency to the administration of corticosteroid therapy that further negatively influenced the secretion of MIP-1α, MIP-1β, and RANTES. As a consequence, the treatment of healthy NK cells ex vivo with corticosteroids abrogated chemokine secretion measured by multiplex immunoassay. Furthermore, we analyzed NK cells regarding their actin cytoskeleton by Structured Illumination Microscopy (SIM) and flow cytometry and demonstrate an actin dysfunction of NK cells shown by reduced F-actin content after fungal co-cultivation early after alloSCT. This dysfunction remains until 180 days post-alloSCT, concluding that further actin-dependent cellular processes may be negatively influenced after alloSCT. To investigate the molecular pathomechansism, we compared CD56 receptor mobility on the plasma membrane of healthy and alloSCT primary NK cells by single-molecule tracking. The results were very robust and reproducible between tested conditions which point to a different molecular mechanism and emphasize the importance of proper CD56 mobility.

  @article{weiss2020reconstituting, abstract = {Delayed natural killer (NK) cell reconstitution after allogeneic stem cell transplantation (alloSCT) is associated with a higher risk of developing invasive aspergillosis. The interaction of NK cells with the human pathogen Aspergillus (A.) fumigatus is mediated by the fungal recognition receptor CD56, which is relocated to the fungal interface after contact. Blocking of CD56 signaling inhibits the fungal mediated chemokine secretion of MIP-1α, MIP-1β, and RANTES and reduces cell activation, indicating a functional role of CD56 in fungal recognition. We collected peripheral blood from recipients of an allograft at defined time points after alloSCT (day 60, 90, 120, 180). NK cells were isolated, directly challenged with live A. fumigatus germ tubes, and cell function was analyzed and compared to healthy age and gender-matched individuals. After alloSCT, NK cells displayed a higher percentage of CD56^brightCD16^dim cells throughout the time of blood collection. However, CD56 binding and relocalization to the fungal contact side were decreased. We were able to correlate this deficiency to the administration of corticosteroid therapy that further negatively influenced the secretion of MIP-1α, MIP-1β, and RANTES. As a consequence, the treatment of healthy NK cells ex vivo with corticosteroids abrogated chemokine secretion measured by multiplex immunoassay. Furthermore, we analyzed NK cells regarding their actin cytoskeleton by Structured Illumination Microscopy (SIM) and flow cytometry and demonstrate an actin dysfunction of NK cells shown by reduced F-actin content after fungal co-cultivation early after alloSCT. This dysfunction remains until 180 days post-alloSCT, concluding that further actin-dependent cellular processes may be negatively influenced after alloSCT. To investigate the molecular pathomechansism, we compared CD56 receptor mobility on the plasma membrane of healthy and alloSCT primary NK cells by single-molecule tracking. The results were very robust and reproducible between tested conditions which point to a different molecular mechanism and emphasize the importance of proper CD56 mobility.}, author = {Weiss, Esther and Schlegel, Jan and Terpitz, Ulrich and Weber, Michael and Linde, Jörg and Schmitt, Anna-Lena and Hünniger, Kerstin and Marischen, Lothar and Gamon, Florian and Bauer, Joachim and Löffler, Claudia and Kurzai, Oliver and Morton, Charles Oliver and Sauer, Markus and Einsele, Hermann and Loeffler, Juergen}, journal = {Frontiers in Immunology}, keywords = {terpitz}, series = 2117, title = {Reconstituting NK Cells After Allogeneic Stem Cell Transplantation Show Impaired Response to the Fungal Pathogen Aspergillus fumigatus}, volume = 11, year = 2020 }

  %0 Journal Article %1 weiss2020reconstituting %A Weiss, Esther %A Schlegel, Jan %A Terpitz, Ulrich %A Weber, Michael %A Linde, Jörg %A Schmitt, Anna-Lena %A Hünniger, Kerstin %A Marischen, Lothar %A Gamon, Florian %A Bauer, Joachim %A Löffler, Claudia %A Kurzai, Oliver %A Morton, Charles Oliver %A Sauer, Markus %A Einsele, Hermann %A Loeffler, Juergen %B 2117 %D 2020 %J Frontiers in Immunology %T Reconstituting NK Cells After Allogeneic Stem Cell Transplantation Show Impaired Response to the Fungal Pathogen Aspergillus fumigatus %U https://www.frontiersin.org/article/10.3389/fimmu.2020.02117 %V 11 %X Delayed natural killer (NK) cell reconstitution after allogeneic stem cell transplantation (alloSCT) is associated with a higher risk of developing invasive aspergillosis. The interaction of NK cells with the human pathogen Aspergillus (A.) fumigatus is mediated by the fungal recognition receptor CD56, which is relocated to the fungal interface after contact. Blocking of CD56 signaling inhibits the fungal mediated chemokine secretion of MIP-1α, MIP-1β, and RANTES and reduces cell activation, indicating a functional role of CD56 in fungal recognition. We collected peripheral blood from recipients of an allograft at defined time points after alloSCT (day 60, 90, 120, 180). NK cells were isolated, directly challenged with live A. fumigatus germ tubes, and cell function was analyzed and compared to healthy age and gender-matched individuals. After alloSCT, NK cells displayed a higher percentage of CD56^brightCD16^dim cells throughout the time of blood collection. However, CD56 binding and relocalization to the fungal contact side were decreased. We were able to correlate this deficiency to the administration of corticosteroid therapy that further negatively influenced the secretion of MIP-1α, MIP-1β, and RANTES. As a consequence, the treatment of healthy NK cells ex vivo with corticosteroids abrogated chemokine secretion measured by multiplex immunoassay. Furthermore, we analyzed NK cells regarding their actin cytoskeleton by Structured Illumination Microscopy (SIM) and flow cytometry and demonstrate an actin dysfunction of NK cells shown by reduced F-actin content after fungal co-cultivation early after alloSCT. This dysfunction remains until 180 days post-alloSCT, concluding that further actin-dependent cellular processes may be negatively influenced after alloSCT. To investigate the molecular pathomechansism, we compared CD56 receptor mobility on the plasma membrane of healthy and alloSCT primary NK cells by single-molecule tracking. The results were very robust and reproducible between tested conditions which point to a different molecular mechanism and emphasize the importance of proper CD56 mobility.
• Expansion Microscopy for Cell Biology Analysis in Fungi Götz, Ralph; Panzer, Sabine; Trinks, Nora; Eilts, Janna; Wagener, Johannes; Turrà, David; Di Pietro, Antonio; Sauer, Markus; Terpitz, Ulrich in Frontiers in Microbiology (2020). 11
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  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.

  @article{gotz2020expansion, 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.}, author = {Götz, Ralph and Panzer, Sabine and Trinks, Nora and Eilts, Janna and Wagener, Johannes and Turrà, David and Di Pietro, Antonio and Sauer, Markus and Terpitz, Ulrich}, journal = {Frontiers in Microbiology}, keywords = {terpitz}, series = 574, title = {Expansion Microscopy for Cell Biology Analysis in Fungi}, volume = 11, year = 2020 }

  %0 Journal Article %1 gotz2020expansion %A Götz, Ralph %A Panzer, Sabine %A Trinks, Nora %A Eilts, Janna %A Wagener, Johannes %A Turrà, David %A Di Pietro, Antonio %A Sauer, Markus %A Terpitz, Ulrich %B 574 %D 2020 %J Frontiers in Microbiology %T Expansion Microscopy for Cell Biology Analysis in Fungi %U https://www.frontiersin.org/article/10.3389/fmicb.2020.00574 %V 11 %X 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.

• Opsin 1 and Opsin 2 of the Corn Smut Fungus Ustilago maydis Are Green Light-Driven Proton Pumps Panzer, Sabine; Brych, Annika; Batschauer, Alfred; Terpitz, Ulrich in Frontiers in Microbiology (2019). 10
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  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, especially by acetic acid and indole-3-acetic acid. 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.

  @article{panzer2019opsin, 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, especially by acetic acid and indole-3-acetic acid. 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.}, author = {Panzer, Sabine and Brych, Annika and Batschauer, Alfred and Terpitz, Ulrich}, journal = {Frontiers in Microbiology}, keywords = {terpitz}, series = 735, title = {Opsin 1 and Opsin 2 of the Corn Smut Fungus Ustilago maydis Are Green Light-Driven Proton Pumps}, volume = 10, year = 2019 }

  %0 Journal Article %1 panzer2019opsin %A Panzer, Sabine %A Brych, Annika %A Batschauer, Alfred %A Terpitz, Ulrich %B 735 %D 2019 %J Frontiers in Microbiology %T Opsin 1 and Opsin 2 of the Corn Smut Fungus Ustilago maydis Are Green Light-Driven Proton Pumps %U https://www.frontiersin.org/article/10.3389/fmicb.2019.00735 %V 10 %X 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, especially by acetic acid and indole-3-acetic acid. 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.

• Protein Activity of the Fusarium fujikuroi Rhodopsins CarO and OpsA and Their Relation to Fungus–Plant Interaction Adam, Alexander; Deimel, Stephan; Pardo-Medina, Javier; García-Martínez, Jorge; Konte, Tilen; Limón, M.; Avalos, Javier; Terpitz, Ulrich in International Journal of Molecular Sciences (2018). 19 215.
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  Fungi possess diverse photosensory proteins that allow them to perceive different light wavelengths and to adapt to changing light conditions in their environment. The biological and physiological roles of the green light-sensing rhodopsins in fungi are not yet resolved. The rice plant pathogen Fusarium fujikuroi exhibits two different rhodopsins, CarO and OpsA. CarO was previously characterized as a light-driven proton pump. We further analyzed the pumping behavior of CarO by patch-clamp experiments. Our data show that CarO pumping activity is strongly augmented in the presence of the plant hormone indole-3-acetic acid and in sodium acetate, in a dose-dependent manner under slightly acidic conditions. By contrast, under these and other tested conditions, the Neurospora rhodopsin (NR)-like rhodopsin OpsA did not exhibit any pump activity. Basic local alignment search tool (BLAST) searches in the genomes of ascomycetes revealed the occurrence of rhodopsin-encoding genes mainly in phyto-associated or phytopathogenic fungi, suggesting a possible correlation of the presence of rhodopsins with fungal ecology. In accordance, rice plants infected with a CarO-deficient F. fujikuroi strain showed more severe bakanae symptoms than the reference strain, indicating a potential role of the CarO rhodopsin in the regulation of plant infection by this fungus.

  @article{adam2018protein, abstract = {Fungi possess diverse photosensory proteins that allow them to perceive different light wavelengths and to adapt to changing light conditions in their environment. The biological and physiological roles of the green light-sensing rhodopsins in fungi are not yet resolved. The rice plant pathogen Fusarium fujikuroi exhibits two different rhodopsins, CarO and OpsA. CarO was previously characterized as a light-driven proton pump. We further analyzed the pumping behavior of CarO by patch-clamp experiments. Our data show that CarO pumping activity is strongly augmented in the presence of the plant hormone indole-3-acetic acid and in sodium acetate, in a dose-dependent manner under slightly acidic conditions. By contrast, under these and other tested conditions, the Neurospora rhodopsin (NR)-like rhodopsin OpsA did not exhibit any pump activity. Basic local alignment search tool (BLAST) searches in the genomes of ascomycetes revealed the occurrence of rhodopsin-encoding genes mainly in phyto-associated or phytopathogenic fungi, suggesting a possible correlation of the presence of rhodopsins with fungal ecology. In accordance, rice plants infected with a CarO-deficient F. fujikuroi strain showed more severe bakanae symptoms than the reference strain, indicating a potential role of the CarO rhodopsin in the regulation of plant infection by this fungus.}, author = {Adam, Alexander and Deimel, Stephan and Pardo-Medina, Javier and García-Martínez, Jorge and Konte, Tilen and Limón, M. and Avalos, Javier and Terpitz, Ulrich}, journal = {International Journal of Molecular Sciences}, keywords = {terpitz}, pages = 215, series = 1, title = {Protein Activity of the Fusarium fujikuroi Rhodopsins CarO and OpsA and Their Relation to Fungus–Plant Interaction}, volume = 19, year = 2018 }

  %0 Journal Article %1 adam2018protein %A Adam, Alexander %A Deimel, Stephan %A Pardo-Medina, Javier %A García-Martínez, Jorge %A Konte, Tilen %A Limón, M. %A Avalos, Javier %A Terpitz, Ulrich %B 1 %D 2018 %J International Journal of Molecular Sciences %P 215 %T Protein Activity of the Fusarium fujikuroi Rhodopsins CarO and OpsA and Their Relation to Fungus–Plant Interaction %U http://www.mdpi.com/1422-0067/19/1/215 %V 19 %X Fungi possess diverse photosensory proteins that allow them to perceive different light wavelengths and to adapt to changing light conditions in their environment. The biological and physiological roles of the green light-sensing rhodopsins in fungi are not yet resolved. The rice plant pathogen Fusarium fujikuroi exhibits two different rhodopsins, CarO and OpsA. CarO was previously characterized as a light-driven proton pump. We further analyzed the pumping behavior of CarO by patch-clamp experiments. Our data show that CarO pumping activity is strongly augmented in the presence of the plant hormone indole-3-acetic acid and in sodium acetate, in a dose-dependent manner under slightly acidic conditions. By contrast, under these and other tested conditions, the Neurospora rhodopsin (NR)-like rhodopsin OpsA did not exhibit any pump activity. Basic local alignment search tool (BLAST) searches in the genomes of ascomycetes revealed the occurrence of rhodopsin-encoding genes mainly in phyto-associated or phytopathogenic fungi, suggesting a possible correlation of the presence of rhodopsins with fungal ecology. In accordance, rice plants infected with a CarO-deficient F. fujikuroi strain showed more severe bakanae symptoms than the reference strain, indicating a potential role of the CarO rhodopsin in the regulation of plant infection by this fungus.
• HyphaTracker: An ImageJ toolbox for time-resolved analysis of spore germination in filamentous fungi Brunk, Michael; Sputh, Sebastian; Doose, Sören; van de Linde, Sebastian; Terpitz, Ulrich in Scientific Reports (2018). 8(1) 605-.
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  The dynamics of early fungal development and its interference with physiological signals and environmental factors is yet poorly understood. Especially computational analysis tools for the evaluation of the process of early spore germination and germ tube formation are still lacking. For the time-resolved analysis of conidia germination of the filamentous ascomycete Fusarium fujikuroi we developed a straightforward toolbox implemented in ImageJ. It allows for processing of microscopic acquisitions (movies) of conidial germination starting with drift correction and data reduction prior to germling analysis. From the image time series germling related region of interests (ROIs) are extracted, which are analysed for their area, circularity, and timing. ROIs originating from germlings crossing other hyphae or the image boundaries are omitted during analysis. Each conidium/hypha is identified and related to its origin, thus allowing subsequent categorization. The efficiency of HyphaTracker was proofed and the accuracy was tested on simulated germlings at different signal-to-noise ratios. Bright-field microscopic images of conidial germination of rhodopsin-deficient F. fujikuroi mutants and their respective control strains were analysed with HyphaTracker. Consistent with our observation in earlier studies the CarO deficient mutant germinated earlier and grew faster than other, CarO expressing strains.

  @article{brunk2018hyphatracker, abstract = {The dynamics of early fungal development and its interference with physiological signals and environmental factors is yet poorly understood. Especially computational analysis tools for the evaluation of the process of early spore germination and germ tube formation are still lacking. For the time-resolved analysis of conidia germination of the filamentous ascomycete Fusarium fujikuroi we developed a straightforward toolbox implemented in ImageJ. It allows for processing of microscopic acquisitions (movies) of conidial germination starting with drift correction and data reduction prior to germling analysis. From the image time series germling related region of interests (ROIs) are extracted, which are analysed for their area, circularity, and timing. ROIs originating from germlings crossing other hyphae or the image boundaries are omitted during analysis. Each conidium/hypha is identified and related to its origin, thus allowing subsequent categorization. The efficiency of HyphaTracker was proofed and the accuracy was tested on simulated germlings at different signal-to-noise ratios. Bright-field microscopic images of conidial germination of rhodopsin-deficient F. fujikuroi mutants and their respective control strains were analysed with HyphaTracker. Consistent with our observation in earlier studies the CarO deficient mutant germinated earlier and grew faster than other, CarO expressing strains.}, author = {Brunk, Michael and Sputh, Sebastian and Doose, Sören and van de Linde, Sebastian and Terpitz, Ulrich}, journal = {Scientific Reports}, keywords = {terpitz}, number = 1, pages = {605–}, title = {HyphaTracker: An ImageJ toolbox for time-resolved analysis of spore germination in filamentous fungi}, volume = 8, year = 2018 }

  %0 Journal Article %1 brunk2018hyphatracker %A Brunk, Michael %A Sputh, Sebastian %A Doose, Sören %A van de Linde, Sebastian %A Terpitz, Ulrich %D 2018 %J Scientific Reports %N 1 %P 605-- %R 10.1038/s41598-017-19103-1 %T HyphaTracker: An ImageJ toolbox for time-resolved analysis of spore germination in filamentous fungi %U https://doi.org/10.1038/s41598-017-19103-1 %V 8 %X The dynamics of early fungal development and its interference with physiological signals and environmental factors is yet poorly understood. Especially computational analysis tools for the evaluation of the process of early spore germination and germ tube formation are still lacking. For the time-resolved analysis of conidia germination of the filamentous ascomycete Fusarium fujikuroi we developed a straightforward toolbox implemented in ImageJ. It allows for processing of microscopic acquisitions (movies) of conidial germination starting with drift correction and data reduction prior to germling analysis. From the image time series germling related region of interests (ROIs) are extracted, which are analysed for their area, circularity, and timing. ROIs originating from germlings crossing other hyphae or the image boundaries are omitted during analysis. Each conidium/hypha is identified and related to its origin, thus allowing subsequent categorization. The efficiency of HyphaTracker was proofed and the accuracy was tested on simulated germlings at different signal-to-noise ratios. Bright-field microscopic images of conidial germination of rhodopsin-deficient F. fujikuroi mutants and their respective control strains were analysed with HyphaTracker. Consistent with our observation in earlier studies the CarO deficient mutant germinated earlier and grew faster than other, CarO expressing strains.
• Bioorthogonal Click Chemistry Enables Site-specific Fluorescence Labeling of Functional NMDA Receptors for Super-Resolution Imaging Neubert, Franziska; Beliu, Gerti; Terpitz, Ulrich; Werner, Christian; Geis, Christian; Sauer, Markus; Doose, Sören in Angewandte Chemie International Edition (2018). 57(50) 16364–16369.
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  Abstract Super-resolution microscopy requires small fluorescent labels. We report the application of genetic code expansion in combination with bioorthogonal click chemistry to label the NR1 domain of the NMDA receptor. We generated NR1 mutants incorporating an unnatural amino acid at various positions in order to attach small organic fluorophores such as Cy5-tetrazine site-specifically to the extracellular domain of the receptor. Mutants were optimized with regard to protein expression, labeling efficiency and receptor functionality as tested by fluorescence microscopy and whole-cell patch clamp. The results show that bioorthogonal click chemistry in combination with small organic dyes is superior to available immunocytochemistry protocols for receptor labeling in live and fixed cells and enables single-molecule sensitive super-resolution microscopy experiments.

  @article{neubert2018bioorthogonal, abstract = {Abstract Super-resolution microscopy requires small fluorescent labels. We report the application of genetic code expansion in combination with bioorthogonal click chemistry to label the NR1 domain of the NMDA receptor. We generated NR1 mutants incorporating an unnatural amino acid at various positions in order to attach small organic fluorophores such as Cy5-tetrazine site-specifically to the extracellular domain of the receptor. Mutants were optimized with regard to protein expression, labeling efficiency and receptor functionality as tested by fluorescence microscopy and whole-cell patch clamp. The results show that bioorthogonal click chemistry in combination with small organic dyes is superior to available immunocytochemistry protocols for receptor labeling in live and fixed cells and enables single-molecule sensitive super-resolution microscopy experiments.}, author = {Neubert, Franziska and Beliu, Gerti and Terpitz, Ulrich and Werner, Christian and Geis, Christian and Sauer, Markus and Doose, Sören}, booktitle = {Angewandte Chemie International Edition}, journal = {Angewandte Chemie International Edition}, keywords = {werner}, month = {oct}, number = 50, pages = {16364–16369}, publisher = {John Wiley & Sons, Ltd}, title = {Bioorthogonal Click Chemistry Enables Site-specific Fluorescence Labeling of Functional NMDA Receptors for Super-Resolution Imaging}, volume = 57, year = 2018 }

  %0 Journal Article %1 neubert2018bioorthogonal %A Neubert, Franziska %A Beliu, Gerti %A Terpitz, Ulrich %A Werner, Christian %A Geis, Christian %A Sauer, Markus %A Doose, Sören %B Angewandte Chemie International Edition %D 2018 %I John Wiley & Sons, Ltd %J Angewandte Chemie International Edition %N 50 %P 16364--16369 %R 10.1002/anie.201808951 %T Bioorthogonal Click Chemistry Enables Site-specific Fluorescence Labeling of Functional NMDA Receptors for Super-Resolution Imaging %U https://doi.org/10.1002/anie.201808951 %V 57 %X Abstract Super-resolution microscopy requires small fluorescent labels. We report the application of genetic code expansion in combination with bioorthogonal click chemistry to label the NR1 domain of the NMDA receptor. We generated NR1 mutants incorporating an unnatural amino acid at various positions in order to attach small organic fluorophores such as Cy5-tetrazine site-specifically to the extracellular domain of the receptor. Mutants were optimized with regard to protein expression, labeling efficiency and receptor functionality as tested by fluorescence microscopy and whole-cell patch clamp. The results show that bioorthogonal click chemistry in combination with small organic dyes is superior to available immunocytochemistry protocols for receptor labeling in live and fixed cells and enables single-molecule sensitive super-resolution microscopy experiments.

• CD56 Is a Pathogen Recognition Receptor on Human Natural Killer Cells Ziegler, Sabrina; Weiss, Esther; Schmitt, Anna-Lena; Schlegel, Jan; Burgert, Anne; Terpitz, Ulrich; Sauer, Markus; Moretta, Lorenzo; Sivori, Simona; Leonhardt, Ines; Kurzai, Oliver; Einsele, Hermann; Loeffler, Juergen in Scientific Reports (2017). 7(1) 6138-.
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  Aspergillus (A.) fumigatus is an opportunistic fungal mold inducing invasive aspergillosis (IA) in immunocompromised patients. Although antifungal activity of human natural killer (NK) cells was shown in previous studies, the underlying cellular mechanisms and pathogen recognition receptors (PRRs) are still unknown. Using flow cytometry we were able to show that the fluorescence positivity of the surface receptor CD56 significantly decreased upon fungal contact. To visualize the interaction site of NK cells and A. fumigatus we used SEM, CLSM and dSTORM techniques, which clearly demonstrated that NK cells directly interact with A. fumigatus via CD56 and that CD56 is re-organized and accumulated at this interaction site time-dependently. The inhibition of the cytoskeleton showed that the receptor re-organization was an active process dependent on actin re-arrangements. Furthermore, we could show that CD56 plays a role in the fungus mediated NK cell activation, since blocking of CD56 surface receptor reduced fungal mediated NK cell activation and reduced cytokine secretion. These results confirmed the direct interaction of NK cells and A. fumigatus, leading to the conclusion that CD56 is a pathogen recognition receptor. These findings give new insights into the functional role of CD56 in the pathogen recognition during the innate immune response.

  @article{ziegler2017pathogen, abstract = {Aspergillus (A.) fumigatus is an opportunistic fungal mold inducing invasive aspergillosis (IA) in immunocompromised patients. Although antifungal activity of human natural killer (NK) cells was shown in previous studies, the underlying cellular mechanisms and pathogen recognition receptors (PRRs) are still unknown. Using flow cytometry we were able to show that the fluorescence positivity of the surface receptor CD56 significantly decreased upon fungal contact. To visualize the interaction site of NK cells and A. fumigatus we used SEM, CLSM and dSTORM techniques, which clearly demonstrated that NK cells directly interact with A. fumigatus via CD56 and that CD56 is re-organized and accumulated at this interaction site time-dependently. The inhibition of the cytoskeleton showed that the receptor re-organization was an active process dependent on actin re-arrangements. Furthermore, we could show that CD56 plays a role in the fungus mediated NK cell activation, since blocking of CD56 surface receptor reduced fungal mediated NK cell activation and reduced cytokine secretion. These results confirmed the direct interaction of NK cells and A. fumigatus, leading to the conclusion that CD56 is a pathogen recognition receptor. These findings give new insights into the functional role of CD56 in the pathogen recognition during the innate immune response.}, author = {Ziegler, Sabrina and Weiss, Esther and Schmitt, Anna-Lena and Schlegel, Jan and Burgert, Anne and Terpitz, Ulrich and Sauer, Markus and Moretta, Lorenzo and Sivori, Simona and Leonhardt, Ines and Kurzai, Oliver and Einsele, Hermann and Loeffler, Juergen}, journal = {Scientific Reports}, keywords = {terpitz}, number = 1, pages = {6138–}, title = {CD56 Is a Pathogen Recognition Receptor on Human Natural Killer Cells}, volume = 7, year = 2017 }

  %0 Journal Article %1 ziegler2017pathogen %A Ziegler, Sabrina %A Weiss, Esther %A Schmitt, Anna-Lena %A Schlegel, Jan %A Burgert, Anne %A Terpitz, Ulrich %A Sauer, Markus %A Moretta, Lorenzo %A Sivori, Simona %A Leonhardt, Ines %A Kurzai, Oliver %A Einsele, Hermann %A Loeffler, Juergen %D 2017 %J Scientific Reports %N 1 %P 6138-- %R 10.1038/s41598-017-06238-4 %T CD56 Is a Pathogen Recognition Receptor on Human Natural Killer Cells %U https://doi.org/10.1038/s41598-017-06238-4 %V 7 %X Aspergillus (A.) fumigatus is an opportunistic fungal mold inducing invasive aspergillosis (IA) in immunocompromised patients. Although antifungal activity of human natural killer (NK) cells was shown in previous studies, the underlying cellular mechanisms and pathogen recognition receptors (PRRs) are still unknown. Using flow cytometry we were able to show that the fluorescence positivity of the surface receptor CD56 significantly decreased upon fungal contact. To visualize the interaction site of NK cells and A. fumigatus we used SEM, CLSM and dSTORM techniques, which clearly demonstrated that NK cells directly interact with A. fumigatus via CD56 and that CD56 is re-organized and accumulated at this interaction site time-dependently. The inhibition of the cytoskeleton showed that the receptor re-organization was an active process dependent on actin re-arrangements. Furthermore, we could show that CD56 plays a role in the fungus mediated NK cell activation, since blocking of CD56 surface receptor reduced fungal mediated NK cell activation and reduced cytokine secretion. These results confirmed the direct interaction of NK cells and A. fumigatus, leading to the conclusion that CD56 is a pathogen recognition receptor. These findings give new insights into the functional role of CD56 in the pathogen recognition during the innate immune response.

• Optochemokine Tandem for Light-Control of Intracellular Ca²⁺ Feldbauer, Katrin; Schlegel, Jan; Weissbecker, Juliane; Sauer, Frank; Wood, Phillip G.; Bamberg, Ernst; Terpitz, Ulrich in PLoS ONE (2016). 11(10) e0165344-.
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  <p>An optochemokine tandem was developed to control the release of calcium from endosomes into the cytosol by light and to analyze the internalization kinetics of G-protein coupled receptors (GPCRs) by electrophysiology. A previously constructed rhodopsin tandem was re-engineered to combine the light-gated Ca²⁺-permeable cation channel Channelrhodopsin-2(L132C), CatCh, with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. The GPCR was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. As shown by patch-clamp measurements and confocal laser scanning microscopy, heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signaling pathway. The kinetics of internalization could be followed electrophysiologically via the amplitude of the CatCh signal. The light-induced release of Ca²⁺ by tandem endosomes into the cytosol via CatCh was visualized using the Ca²⁺-sensitive dyes rhod2 and rhod2-AM showing an increase of intracellular Ca²⁺ in response to light.</p>

  @article{feldbauer2016optochemokine, abstract = {<p>An optochemokine tandem was developed to control the release of calcium from endosomes into the cytosol by light and to analyze the internalization kinetics of G-protein coupled receptors (GPCRs) by electrophysiology. A previously constructed rhodopsin tandem was re-engineered to combine the light-gated Ca²⁺-permeable cation channel Channelrhodopsin-2(L132C), CatCh, with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. The GPCR was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. As shown by patch-clamp measurements and confocal laser scanning microscopy, heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signaling pathway. The kinetics of internalization could be followed electrophysiologically via the amplitude of the CatCh signal. The light-induced release of Ca²⁺ by tandem endosomes into the cytosol via CatCh was visualized using the Ca²⁺-sensitive dyes rhod2 and rhod2-AM showing an increase of intracellular Ca²⁺ in response to light.</p>}, author = {Feldbauer, Katrin and Schlegel, Jan and Weissbecker, Juliane and Sauer, Frank and Wood, Phillip G. and Bamberg, Ernst and Terpitz, Ulrich}, journal = {PLoS ONE}, keywords = {terpitz}, month = {oct}, number = 10, pages = {e0165344–}, publisher = {Public Library of Science}, title = {Optochemokine Tandem for Light-Control of Intracellular Ca²⁺}, volume = 11, year = 2016 }

  %0 Journal Article %1 feldbauer2016optochemokine %A Feldbauer, Katrin %A Schlegel, Jan %A Weissbecker, Juliane %A Sauer, Frank %A Wood, Phillip G. %A Bamberg, Ernst %A Terpitz, Ulrich %D 2016 %I Public Library of Science %J PLoS ONE %N 10 %P e0165344-- %T Optochemokine Tandem for Light-Control of Intracellular Ca²⁺ %U http://dx.doi.org/10.1371/journal.pone.0165344 %V 11 %X <p>An optochemokine tandem was developed to control the release of calcium from endosomes into the cytosol by light and to analyze the internalization kinetics of G-protein coupled receptors (GPCRs) by electrophysiology. A previously constructed rhodopsin tandem was re-engineered to combine the light-gated Ca²⁺-permeable cation channel Channelrhodopsin-2(L132C), CatCh, with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. The GPCR was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. As shown by patch-clamp measurements and confocal laser scanning microscopy, heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signaling pathway. The kinetics of internalization could be followed electrophysiologically via the amplitude of the CatCh signal. The light-induced release of Ca²⁺ by tandem endosomes into the cytosol via CatCh was visualized using the Ca²⁺-sensitive dyes rhod2 and rhod2-AM showing an increase of intracellular Ca²⁺ in response to light.</p>
• Reconstruction of the high-osmolarity glycerol (HOG) signaling pathway from the halophilic fungus Wallemia ichthyophaga in Saccharomyces cerevisiae Konte, Tilen; Terpitz, Ulrich; Plemenitas, Ana in Frontiers in Microbiology (2016). 7(901)
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  The basidiomycetous fungus Wallemia ichthyophaga grows between 1.7 M and 5.1 M NaCl and is the most halophilic eukaryote described to date. Like in other fungi, in W. ichthyophaga, changes in environmental salinity are detected mainly by the evolutionarily conserved high-osmolarity glycerol (HOG) signaling pathway. In Saccharomyces cerevisiae, the HOG pathway has been extensively studied in connection to osmotic regulation, with a valuable knock-out strain collection established. In the present study, we reconstructed the architecture of the HOG pathway of W. ichthyophaga in suitable S. cerevisiae knock-out strains, through heterologous expression of the W. ichthyophaga HOG pathway proteins. Compared to S. cerevisiae, where the Pbs2 (ScPbs2) kinase of the HOG pathway is activated via the SHO1 and SLN1 branches, the interactions between the W. ichthyophaga Pbs2 (WiPbs2) kinase and the W. ichthyophaga SHO1 branch orthologs are not conserved: as well as evidence of poor interactions between the WiSho1 Src-homology 3 (SH3) domain and the WiPbs2 proline-rich motif, the absence of a considerable part of the osmosensing apparatus in the genome of W. ichthyophaga suggests that the SHO1 branch components are not involved in HOG signaling in this halophilic fungus. In contrast, the conserved activation of WiPbs2 by the S. cerevisiae ScSsk2/ScSsk22 kinase and the sensitivity of W. ichthyophaga cells to fludioxonil, emphasize the significance of two-component (SLN1-like) signaling via Group III histidine kinase. Combined with the protein modeling data, our study reveals conserved and nonconserved protein interactions in the HOG signaling pathway of W. ichthyophaga and therefore significantly improves the knowledge of hyperosmotic signal processing in this halophilic fungus.

  @article{10.3389/fmicb.2016.00901, abstract = {The basidiomycetous fungus Wallemia ichthyophaga grows between 1.7 M and 5.1 M NaCl and is the most halophilic eukaryote described to date. Like in other fungi, in W. ichthyophaga, changes in environmental salinity are detected mainly by the evolutionarily conserved high-osmolarity glycerol (HOG) signaling pathway. In Saccharomyces cerevisiae, the HOG pathway has been extensively studied in connection to osmotic regulation, with a valuable knock-out strain collection established. In the present study, we reconstructed the architecture of the HOG pathway of W. ichthyophaga in suitable S. cerevisiae knock-out strains, through heterologous expression of the W. ichthyophaga HOG pathway proteins. Compared to S. cerevisiae, where the Pbs2 (ScPbs2) kinase of the HOG pathway is activated via the SHO1 and SLN1 branches, the interactions between the W. ichthyophaga Pbs2 (WiPbs2) kinase and the W. ichthyophaga SHO1 branch orthologs are not conserved: as well as evidence of poor interactions between the WiSho1 Src-homology 3 (SH3) domain and the WiPbs2 proline-rich motif, the absence of a considerable part of the osmosensing apparatus in the genome of W. ichthyophaga suggests that the SHO1 branch components are not involved in HOG signaling in this halophilic fungus. In contrast, the conserved activation of WiPbs2 by the S. cerevisiae ScSsk2/ScSsk22 kinase and the sensitivity of W. ichthyophaga cells to fludioxonil, emphasize the significance of two-component (SLN1-like) signaling via Group III histidine kinase. Combined with the protein modeling data, our study reveals conserved and nonconserved protein interactions in the HOG signaling pathway of W. ichthyophaga and therefore significantly improves the knowledge of hyperosmotic signal processing in this halophilic fungus.}, author = {Konte, Tilen and Terpitz, Ulrich and Plemenitas, Ana}, journal = {Frontiers in Microbiology}, keywords = {terpitz}, number = 901, title = {Reconstruction of the high-osmolarity glycerol (HOG) signaling pathway from the halophilic fungus Wallemia ichthyophaga in Saccharomyces cerevisiae}, volume = 7, year = 2016 }

  %0 Journal Article %1 10.3389/fmicb.2016.00901 %A Konte, Tilen %A Terpitz, Ulrich %A Plemenitas, Ana %D 2016 %J Frontiers in Microbiology %N 901 %R 10.3389/fmicb.2016.00901 %T Reconstruction of the high-osmolarity glycerol (HOG) signaling pathway from the halophilic fungus Wallemia ichthyophaga in Saccharomyces cerevisiae %U http://www.frontiersin.org/extreme_microbiology/10.3389/fmicb.2016.00901/abstract %V 7 %X The basidiomycetous fungus Wallemia ichthyophaga grows between 1.7 M and 5.1 M NaCl and is the most halophilic eukaryote described to date. Like in other fungi, in W. ichthyophaga, changes in environmental salinity are detected mainly by the evolutionarily conserved high-osmolarity glycerol (HOG) signaling pathway. In Saccharomyces cerevisiae, the HOG pathway has been extensively studied in connection to osmotic regulation, with a valuable knock-out strain collection established. In the present study, we reconstructed the architecture of the HOG pathway of W. ichthyophaga in suitable S. cerevisiae knock-out strains, through heterologous expression of the W. ichthyophaga HOG pathway proteins. Compared to S. cerevisiae, where the Pbs2 (ScPbs2) kinase of the HOG pathway is activated via the SHO1 and SLN1 branches, the interactions between the W. ichthyophaga Pbs2 (WiPbs2) kinase and the W. ichthyophaga SHO1 branch orthologs are not conserved: as well as evidence of poor interactions between the WiSho1 Src-homology 3 (SH3) domain and the WiPbs2 proline-rich motif, the absence of a considerable part of the osmosensing apparatus in the genome of W. ichthyophaga suggests that the SHO1 branch components are not involved in HOG signaling in this halophilic fungus. In contrast, the conserved activation of WiPbs2 by the S. cerevisiae ScSsk2/ScSsk22 kinase and the sensitivity of W. ichthyophaga cells to fludioxonil, emphasize the significance of two-component (SLN1-like) signaling via Group III histidine kinase. Combined with the protein modeling data, our study reveals conserved and nonconserved protein interactions in the HOG signaling pathway of W. ichthyophaga and therefore significantly improves the knowledge of hyperosmotic signal processing in this halophilic fungus.

• Ein Rhodopsin des Pilzes Fusarium fujikuroi reguliert die Sporenkeimung Terpitz, U. in Naturwissenschaftliche Rundschau (2015). 68(9) 465–466.
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  @article{terpitz2015rhodopsin, author = {Terpitz, U.}, journal = {Naturwissenschaftliche Rundschau}, keywords = {terpitz}, number = 9, pages = {465-466}, title = {Ein Rhodopsin des Pilzes Fusarium fujikuroi reguliert die Sporenkeimung}, volume = 68, year = 2015 }

  %0 Journal Article %1 terpitz2015rhodopsin %A Terpitz, U. %D 2015 %J Naturwissenschaftliche Rundschau %N 9 %P 465-466 %T Ein Rhodopsin des Pilzes Fusarium fujikuroi reguliert die Sporenkeimung %V 68
• The CarO rhodopsin of the fungus Fusarium fujikuroi is a light-driven proton pump that retards spore germination García-Martínez, Jorge; Brunk, Michael; Avalos, Javier; Terpitz, Ulrich in Sci. Rep. (2015). 5
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  Rhodopsins are membrane-embedded photoreceptors found in all major taxonomic kingdoms using retinal as their chromophore. They play well-known functions in different biological systems, but their roles in fungi remain unknown. The filamentous fungus Fusarium fujikuroi contains two putative rhodopsins, CarO and OpsA. The gene carO is light-regulated, and the predicted polypeptide contains all conserved residues required for proton pumping. We aimed to elucidate the expression and cellular location of the fungal rhodopsin CarO, its presumed proton-pumping activity and the possible effect of such function on F. fujikuroi growth. In electrophysiology experiments we confirmed that CarO is a green-light driven proton pump. Visualization of fluorescent CarO-YFP expressed in F. fujikuroi under control of its native promoter revealed higher accumulation in spores (conidia) produced by light-exposed mycelia. Germination analyses of conidia from carO− mutant and carO+ control strains showed a faster development of light-exposed carO− germlings. In conclusion, CarO is an active proton pump, abundant in light-formed conidia, whose activity slows down early hyphal development under light. Interestingly, CarO-related rhodopsins are typically found in plant-associated fungi, where green light dominates the phyllosphere. Our data provide the first reliable clue on a possible biological role of a fungal rhodopsin.

  @article{garciamartinez2015rhodopsin, abstract = {Rhodopsins are membrane-embedded photoreceptors found in all major taxonomic kingdoms using retinal as their chromophore. They play well-known functions in different biological systems, but their roles in fungi remain unknown. The filamentous fungus Fusarium fujikuroi contains two putative rhodopsins, CarO and OpsA. The gene carO is light-regulated, and the predicted polypeptide contains all conserved residues required for proton pumping. We aimed to elucidate the expression and cellular location of the fungal rhodopsin CarO, its presumed proton-pumping activity and the possible effect of such function on F. fujikuroi growth. In electrophysiology experiments we confirmed that CarO is a green-light driven proton pump. Visualization of fluorescent CarO-YFP expressed in F. fujikuroi under control of its native promoter revealed higher accumulation in spores (conidia) produced by light-exposed mycelia. Germination analyses of conidia from carO− mutant and carO+ control strains showed a faster development of light-exposed carO− germlings. In conclusion, CarO is an active proton pump, abundant in light-formed conidia, whose activity slows down early hyphal development under light. Interestingly, CarO-related rhodopsins are typically found in plant-associated fungi, where green light dominates the phyllosphere. Our data provide the first reliable clue on a possible biological role of a fungal rhodopsin.}, author = {García-Martínez, Jorge and Brunk, Michael and Avalos, Javier and Terpitz, Ulrich}, journal = {Sci. Rep.}, keywords = {terpitz}, month = {jan}, publisher = {Macmillan Publishers Limited. All rights reserved}, title = {The CarO rhodopsin of the fungus Fusarium fujikuroi is a light-driven proton pump that retards spore germination}, volume = 5, year = 2015 }

  %0 Journal Article %1 garciamartinez2015rhodopsin %A García-Martínez, Jorge %A Brunk, Michael %A Avalos, Javier %A Terpitz, Ulrich %D 2015 %I Macmillan Publishers Limited. All rights reserved %J Sci. Rep. %T The CarO rhodopsin of the fungus Fusarium fujikuroi is a light-driven proton pump that retards spore germination %U http://dx.doi.org/10.1038/srep07798 %V 5 %X Rhodopsins are membrane-embedded photoreceptors found in all major taxonomic kingdoms using retinal as their chromophore. They play well-known functions in different biological systems, but their roles in fungi remain unknown. The filamentous fungus Fusarium fujikuroi contains two putative rhodopsins, CarO and OpsA. The gene carO is light-regulated, and the predicted polypeptide contains all conserved residues required for proton pumping. We aimed to elucidate the expression and cellular location of the fungal rhodopsin CarO, its presumed proton-pumping activity and the possible effect of such function on F. fujikuroi growth. In electrophysiology experiments we confirmed that CarO is a green-light driven proton pump. Visualization of fluorescent CarO-YFP expressed in F. fujikuroi under control of its native promoter revealed higher accumulation in spores (conidia) produced by light-exposed mycelia. Germination analyses of conidia from carO− mutant and carO+ control strains showed a faster development of light-exposed carO− germlings. In conclusion, CarO is an active proton pump, abundant in light-formed conidia, whose activity slows down early hyphal development under light. Interestingly, CarO-related rhodopsins are typically found in plant-associated fungi, where green light dominates the phyllosphere. Our data provide the first reliable clue on a possible biological role of a fungal rhodopsin.

• Prototype for Automatable, Dielectrophoretically-Accessed Intracellular Membrane-Potential Measurements by Metal Electrodes Terpitz, Ulrich; Sukhorukov, Vladimir L.; Zimmermann, Dirk in ASSAY and Drug Development Technologies. (2013). 11(1) 9–16.
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  Functional access to membrane proteins, for example, ion channels, of individual cells is an important prerequisite in drug discovery studies. The highly sophisticated patch-clamp method is widely used for electrogenic membrane proteins, but is demanding for the operator, and its automation remains challenging. The dielectrophoretically-accessed, intracellular membrane–potential measurement (DAIMM) method is a new technique showing high potential for automation of electrophysiological data recording in the whole-cell configuration. A cell suspension is brought between a mm-scaled planar electrode and a μm-scaled tip electrode, placed opposite to each other. Due to the asymmetric electrode configuration, the application of alternating electric fields (1–5 MHz) provokes a dielectrophoretic force acting on the target cell. As a consequence, the cell is accelerated and pierced by the tip electrode, hence functioning as the internal (working) electrode. We used the light-gated cation channel Channelrhodopsin-2 as a reporter protein expressed in HEK293 cells to characterize the DAIMM method in comparison with the patch-clamp technique.

  @article{terpitz2012prototype, abstract = {Functional access to membrane proteins, for example, ion channels, of individual cells is an important prerequisite in drug discovery studies. The highly sophisticated patch-clamp method is widely used for electrogenic membrane proteins, but is demanding for the operator, and its automation remains challenging. The dielectrophoretically-accessed, intracellular membrane–potential measurement (DAIMM) method is a new technique showing high potential for automation of electrophysiological data recording in the whole-cell configuration. A cell suspension is brought between a mm-scaled planar electrode and a μm-scaled tip electrode, placed opposite to each other. Due to the asymmetric electrode configuration, the application of alternating electric fields (1–5 MHz) provokes a dielectrophoretic force acting on the target cell. As a consequence, the cell is accelerated and pierced by the tip electrode, hence functioning as the internal (working) electrode. We used the light-gated cation channel Channelrhodopsin-2 as a reporter protein expressed in HEK293 cells to characterize the DAIMM method in comparison with the patch-clamp technique.}, author = {Terpitz, Ulrich and Sukhorukov, Vladimir L. and Zimmermann, Dirk}, journal = {ASSAY and Drug Development Technologies.}, keywords = {vladimir}, month = {February}, number = 1, pages = {9-16}, title = {Prototype for Automatable, Dielectrophoretically-Accessed Intracellular Membrane-Potential Measurements by Metal Electrodes}, volume = 11, year = 2013 }

  %0 Journal Article %1 terpitz2012prototype %A Terpitz, Ulrich %A Sukhorukov, Vladimir L. %A Zimmermann, Dirk %D 2013 %J ASSAY and Drug Development Technologies. %N 1 %P 9-16 %T Prototype for Automatable, Dielectrophoretically-Accessed Intracellular Membrane-Potential Measurements by Metal Electrodes %U http://dx.doi.org/10.1089/adt.2012.455 %V 11 %X Functional access to membrane proteins, for example, ion channels, of individual cells is an important prerequisite in drug discovery studies. The highly sophisticated patch-clamp method is widely used for electrogenic membrane proteins, but is demanding for the operator, and its automation remains challenging. The dielectrophoretically-accessed, intracellular membrane–potential measurement (DAIMM) method is a new technique showing high potential for automation of electrophysiological data recording in the whole-cell configuration. A cell suspension is brought between a mm-scaled planar electrode and a μm-scaled tip electrode, placed opposite to each other. Due to the asymmetric electrode configuration, the application of alternating electric fields (1–5 MHz) provokes a dielectrophoretic force acting on the target cell. As a consequence, the cell is accelerated and pierced by the tip electrode, hence functioning as the internal (working) electrode. We used the light-gated cation channel Channelrhodopsin-2 as a reporter protein expressed in HEK293 cells to characterize the DAIMM method in comparison with the patch-clamp technique.

• Dielectric Analysis and Multi-cell Electrofusion of the Yeast Pichia pastoris for Electrophysiological Studies Terpitz, Ulrich; Letschert, Sebastian; Bonda, Ulrich; Spahn, Christoph; Guan, Chonglin; Sauer, Markus; Zimmermann, Ulrich; Bamberg, Ernst; Zimmermann, Dirk; Sukhorukov, Vladimir in Journal of Membrane Biology (2012). 1–12.
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  The yeast Pichia pastoris has become the most favored eukaryotic host for heterologous protein expression. P. pastoris strains capable of overexpressing various membrane proteins are now available. Due to their small size and the fungal cell wall, however, P. pastoris cells are hardly suitable for direct electrophysiological studies. To overcome these limitations, the present study aimed to produce giant protoplasts of P. pastoris by means of multi-cell electrofusion. Using a P. pastoris strain expressing channelrhodopsin-2 (ChR2), we first developed an improved enzymatic method for cell wall digestion and preparation of wall-less protoplasts. We thoroughly analyzed the dielectric properties of protoplasts by means of electrorotation and dielectrophoresis. Based on the dielectric data of tiny parental protoplasts (2–4 μm diameter), we elaborated efficient electrofusion conditions yielding consistently stable multinucleated protoplasts of P. pastoris with diameters of up to 35 μm. The giant protoplasts were suitable for electrophysiological measurements, as proved by whole-cell patch clamp recordings of light-induced, ChR2-mediated currents, which was impossible with parental protoplasts. The approach presented here offers a potentially valuable technique for the functional analysis of low-signal channels and transporters, expressed heterologously in P. pastoris and related host systems.

  @article{springerlink:10.1007/s00232-012-9484-9, abstract = {The yeast Pichia pastoris has become the most favored eukaryotic host for heterologous protein expression. P. pastoris strains capable of overexpressing various membrane proteins are now available. Due to their small size and the fungal cell wall, however, P. pastoris cells are hardly suitable for direct electrophysiological studies. To overcome these limitations, the present study aimed to produce giant protoplasts of P. pastoris by means of multi-cell electrofusion. Using a P. pastoris strain expressing channelrhodopsin-2 (ChR2), we first developed an improved enzymatic method for cell wall digestion and preparation of wall-less protoplasts. We thoroughly analyzed the dielectric properties of protoplasts by means of electrorotation and dielectrophoresis. Based on the dielectric data of tiny parental protoplasts (2–4 μm diameter), we elaborated efficient electrofusion conditions yielding consistently stable multinucleated protoplasts of P. pastoris with diameters of up to 35 μm. The giant protoplasts were suitable for electrophysiological measurements, as proved by whole-cell patch clamp recordings of light-induced, ChR2-mediated currents, which was impossible with parental protoplasts. The approach presented here offers a potentially valuable technique for the functional analysis of low-signal channels and transporters, expressed heterologously in P. pastoris and related host systems.}, author = {Terpitz, Ulrich and Letschert, Sebastian and Bonda, Ulrich and Spahn, Christoph and Guan, Chonglin and Sauer, Markus and Zimmermann, Ulrich and Bamberg, Ernst and Zimmermann, Dirk and Sukhorukov, Vladimir}, journal = {Journal of Membrane Biology}, keywords = {vladimir}, note = {10.1007/s00232-012-9484-9}, pages = {1-12}, publisher = {Springer New York}, title = {Dielectric Analysis and Multi-cell Electrofusion of the Yeast Pichia pastoris for Electrophysiological Studies}, year = 2012 }

  %0 Journal Article %1 springerlink:10.1007/s00232-012-9484-9 %A Terpitz, Ulrich %A Letschert, Sebastian %A Bonda, Ulrich %A Spahn, Christoph %A Guan, Chonglin %A Sauer, Markus %A Zimmermann, Ulrich %A Bamberg, Ernst %A Zimmermann, Dirk %A Sukhorukov, Vladimir %D 2012 %I Springer New York %J Journal of Membrane Biology %P 1-12 %T Dielectric Analysis and Multi-cell Electrofusion of the Yeast Pichia pastoris for Electrophysiological Studies %U http://dx.doi.org/10.1007/s00232-012-9484-9 %X The yeast Pichia pastoris has become the most favored eukaryotic host for heterologous protein expression. P. pastoris strains capable of overexpressing various membrane proteins are now available. Due to their small size and the fungal cell wall, however, P. pastoris cells are hardly suitable for direct electrophysiological studies. To overcome these limitations, the present study aimed to produce giant protoplasts of P. pastoris by means of multi-cell electrofusion. Using a P. pastoris strain expressing channelrhodopsin-2 (ChR2), we first developed an improved enzymatic method for cell wall digestion and preparation of wall-less protoplasts. We thoroughly analyzed the dielectric properties of protoplasts by means of electrorotation and dielectrophoresis. Based on the dielectric data of tiny parental protoplasts (2–4 μm diameter), we elaborated efficient electrofusion conditions yielding consistently stable multinucleated protoplasts of P. pastoris with diameters of up to 35 μm. The giant protoplasts were suitable for electrophysiological measurements, as proved by whole-cell patch clamp recordings of light-induced, ChR2-mediated currents, which was impossible with parental protoplasts. The approach presented here offers a potentially valuable technique for the functional analysis of low-signal channels and transporters, expressed heterologously in P. pastoris and related host systems.

• A gene-fusion strategy for stoichiometric and co-localized expression of light-gated membrane proteins Kleinlogel, Sonja; Terpitz, Ulrich; Legrum, Barbara; Gokbuget, Deniz; Boyden, Edward S; Bamann, Christian; Wood, Phillip G; Bamberg, Ernst in Nat Meth (2011). 8(12) 1083–1088.
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  The precise co-localization and stoichiometric expression of two different light-gated membrane proteins can vastly improve the physiological usefulness of optogenetics for the modulation of cell excitability with light. Here we present a gene-fusion strategy for the stable 1:1 expression of any two microbial rhodopsins in a single polypeptide chain. By joining the excitatory channelrhodopsin-2 with the inhibitory ion pumps halorhodopsin or bacteriorhodopsin, we demonstrate light-regulated quantitative bi-directional control of the membrane potential in HEK293 cells and neurons in vitro. We also present synergistic rhodopsin combinations of channelrhodopsin-2 with Volvox carteri channelrhodopsin-1 or slow channelrhodopsin-2 mutants, to achieve enhanced spectral or kinetic properties, respectively. Finally, we demonstrate the utility of our fusion strategy to determine ion-turnovers of as yet uncharacterized rhodopsins, exemplified for archaerhodopsin and CatCh, or to correct pump cycles, exemplified for halorhodopsin.

  @article{kleinlogel2011genefusion, abstract = {The precise co-localization and stoichiometric expression of two different light-gated membrane proteins can vastly improve the physiological usefulness of optogenetics for the modulation of cell excitability with light. Here we present a gene-fusion strategy for the stable 1:1 expression of any two microbial rhodopsins in a single polypeptide chain. By joining the excitatory channelrhodopsin-2 with the inhibitory ion pumps halorhodopsin or bacteriorhodopsin, we demonstrate light-regulated quantitative bi-directional control of the membrane potential in HEK293 cells and neurons in vitro. We also present synergistic rhodopsin combinations of channelrhodopsin-2 with Volvox carteri channelrhodopsin-1 or slow channelrhodopsin-2 mutants, to achieve enhanced spectral or kinetic properties, respectively. Finally, we demonstrate the utility of our fusion strategy to determine ion-turnovers of as yet uncharacterized rhodopsins, exemplified for archaerhodopsin and CatCh, or to correct pump cycles, exemplified for halorhodopsin.}, author = {Kleinlogel, Sonja and Terpitz, Ulrich and Legrum, Barbara and Gokbuget, Deniz and Boyden, Edward S and Bamann, Christian and Wood, Phillip G and Bamberg, Ernst}, journal = {Nat Meth}, keywords = {terpitz}, month = {dec}, number = 12, pages = {1083–1088}, publisher = {Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.}, title = {A gene-fusion strategy for stoichiometric and co-localized expression of light-gated membrane proteins}, volume = 8, year = 2011 }

  %0 Journal Article %1 kleinlogel2011genefusion %A Kleinlogel, Sonja %A Terpitz, Ulrich %A Legrum, Barbara %A Gokbuget, Deniz %A Boyden, Edward S %A Bamann, Christian %A Wood, Phillip G %A Bamberg, Ernst %D 2011 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nat Meth %N 12 %P 1083--1088 %T A gene-fusion strategy for stoichiometric and co-localized expression of light-gated membrane proteins %U http://dx.doi.org/10.1038/nmeth.1766 %V 8 %X The precise co-localization and stoichiometric expression of two different light-gated membrane proteins can vastly improve the physiological usefulness of optogenetics for the modulation of cell excitability with light. Here we present a gene-fusion strategy for the stable 1:1 expression of any two microbial rhodopsins in a single polypeptide chain. By joining the excitatory channelrhodopsin-2 with the inhibitory ion pumps halorhodopsin or bacteriorhodopsin, we demonstrate light-regulated quantitative bi-directional control of the membrane potential in HEK293 cells and neurons in vitro. We also present synergistic rhodopsin combinations of channelrhodopsin-2 with Volvox carteri channelrhodopsin-1 or slow channelrhodopsin-2 mutants, to achieve enhanced spectral or kinetic properties, respectively. Finally, we demonstrate the utility of our fusion strategy to determine ion-turnovers of as yet uncharacterized rhodopsins, exemplified for archaerhodopsin and CatCh, or to correct pump cycles, exemplified for halorhodopsin.

• Isolation of guard cells from fresh epidermis using a piezo-power microdissection system with vibration-attenuated needles Terpitz, Ulrich; Zimmermann, Dirk in BIOTECHNIQUES (2010). 48(1) 68–70.
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  The Eppendorf Piezo-Power Microdissection (PPMD) system uses a tungsten needle (MicroChisel) oscillating in a forward-backward (vertical) mode to cut cells from surrounding tissue. This technology competes with laser-based dissection systems, which offer high accuracy and precision, but are more expensive and require fixed tissue. In contrast, PPMD systems can dissect freshly prepared tissue, but their accuracy and precision is lower due to unwanted lateral vibrations of the MicroChisel. Especially in tissues where elasticity is high, these vibrations can limit the cutting resolution or hamper the dissection. Here we describe a cost-efficient and simple glass capillary-encapsulation modification of MicroChisels for effective attenuation of lateral vibrations. The use of modified MicroChisels enables accurate and precise tissue dissection from highly elastic material.

  @article{Terpitz2010, abstract = {The Eppendorf Piezo-Power Microdissection (PPMD) system uses a tungsten needle (MicroChisel) oscillating in a forward-backward (vertical) mode to cut cells from surrounding tissue. This technology competes with laser-based dissection systems, which offer high accuracy and precision, but are more expensive and require fixed tissue. In contrast, PPMD systems can dissect freshly prepared tissue, but their accuracy and precision is lower due to unwanted lateral vibrations of the MicroChisel. Especially in tissues where elasticity is high, these vibrations can limit the cutting resolution or hamper the dissection. Here we describe a cost-efficient and simple glass capillary-encapsulation modification of MicroChisels for effective attenuation of lateral vibrations. The use of modified MicroChisels enables accurate and precise tissue dissection from highly elastic material.}, address = {52 VANDERBILT AVE, NEW YORK, NY 10017 USA}, author = {Terpitz, Ulrich and Zimmermann, Dirk}, journal = {BIOTECHNIQUES}, keywords = {terpitz}, month = {JAN}, number = 1, pages = {68-70}, publisher = {INFORMA HEALTHCARE}, title = {Isolation of guard cells from fresh epidermis using a piezo-power microdissection system with vibration-attenuated needles}, type = {Article}, volume = 48, year = 2010 }

  %0 Journal Article %1 Terpitz2010 %A Terpitz, Ulrich %A Zimmermann, Dirk %C 52 VANDERBILT AVE, NEW YORK, NY 10017 USA %D 2010 %I INFORMA HEALTHCARE %J BIOTECHNIQUES %N 1 %P 68-70 %T Isolation of guard cells from fresh epidermis using a piezo-power microdissection system with vibration-attenuated needles %U http://dx.doi.org/10.2144/000113346 %V 48 %X The Eppendorf Piezo-Power Microdissection (PPMD) system uses a tungsten needle (MicroChisel) oscillating in a forward-backward (vertical) mode to cut cells from surrounding tissue. This technology competes with laser-based dissection systems, which offer high accuracy and precision, but are more expensive and require fixed tissue. In contrast, PPMD systems can dissect freshly prepared tissue, but their accuracy and precision is lower due to unwanted lateral vibrations of the MicroChisel. Especially in tissues where elasticity is high, these vibrations can limit the cutting resolution or hamper the dissection. Here we describe a cost-efficient and simple glass capillary-encapsulation modification of MicroChisels for effective attenuation of lateral vibrations. The use of modified MicroChisels enables accurate and precise tissue dissection from highly elastic material.

• A combined patch-clamp and electrorotation study of the voltage- and frequency-dependent membrane capacitance caused by structurally dissimilar lipophilic anions Zimmermann, D.; Kiesel, M.; Terpitz, U.; Zhou, A.; Reuss, R.; Kraus, J.; Schenk, W. A.; Bamberg, E.; Sukhorukov, V. L. in JOURNAL OF MEMBRANE BIOLOGY (2008). 221(2) 107–121.
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  Interactions of structurally dissimilar anionic compounds with the plasma membrane of HEK293 cells were analyzed by patch clamp and electrorotation. The combined approach provides complementary information on the lipophilicity, preferential affinity of the anions to the inner/outer membrane leaflet, adsorption depth and transmembrane mobility. The anionic species studied here included the well-known lipophilic anions dipicrylamine (DPA(-)), tetraphenylborate (TPB-) and {[}W-2(CO)(10)(S2CH)](-), the putative lipophilic anion B(CF3)(4)(-) and three new heterocyclic W(CO)(5) derivatives. All tested anions partitioned strongly into the cell membrane, as indicated by the capacitance increase in patch-clamped cells. The capacitance increment exhibited a bell-shaped dependence on membrane voltage. The midpoint potentials of the maximum capacitance increment were negative, indicating the exclusion of lipophilic anions from the outer membrane leaflet. The adsorption depth of the large organic anions DPA(-), TPB- and B(CF3)(4)(-) increased and that of W(CO)(5) derivatives decreased with increasing concentration of mobile charges. In agreement with the patch-clamp data, electrorotation of cells treated with DPA(-) and W(CO)(5) derivatives revealed a large dispersion of membrane capacitance in the kilohertz to megahertz range due to the translocation of mobile charges. In contrast, in the presence of TPB- and B(CF3)(4)(-) no mobile charges could be detected by electrorotation, despite their strong membrane adsorption. Our data suggest that the presence of oxygen atoms in the outer molecular shell is an important factor for the fast translocation ability of lipophilic anions.

  @article{Zimmermann2008a, abstract = {Interactions of structurally dissimilar anionic compounds with the plasma membrane of HEK293 cells were analyzed by patch clamp and electrorotation. The combined approach provides complementary information on the lipophilicity, preferential affinity of the anions to the inner/outer membrane leaflet, adsorption depth and transmembrane mobility. The anionic species studied here included the well-known lipophilic anions dipicrylamine (DPA(-)), tetraphenylborate (TPB-) and {[}W-2(CO)(10)(S2CH)](-), the putative lipophilic anion B(CF3)(4)(-) and three new heterocyclic W(CO)(5) derivatives. All tested anions partitioned strongly into the cell membrane, as indicated by the capacitance increase in patch-clamped cells. The capacitance increment exhibited a bell-shaped dependence on membrane voltage. The midpoint potentials of the maximum capacitance increment were negative, indicating the exclusion of lipophilic anions from the outer membrane leaflet. The adsorption depth of the large organic anions DPA(-), TPB- and B(CF3)(4)(-) increased and that of W(CO)(5) derivatives decreased with increasing concentration of mobile charges. In agreement with the patch-clamp data, electrorotation of cells treated with DPA(-) and W(CO)(5) derivatives revealed a large dispersion of membrane capacitance in the kilohertz to megahertz range due to the translocation of mobile charges. In contrast, in the presence of TPB- and B(CF3)(4)(-) no mobile charges could be detected by electrorotation, despite their strong membrane adsorption. Our data suggest that the presence of oxygen atoms in the outer molecular shell is an important factor for the fast translocation ability of lipophilic anions.}, address = {233 SPRING STREET, NEW YORK, NY 10013 USA}, author = {Zimmermann, D. and Kiesel, M. and Terpitz, U. and Zhou, A. and Reuss, R. and Kraus, J. and Schenk, W. A. and Bamberg, E. and Sukhorukov, V. L.}, journal = {JOURNAL OF MEMBRANE BIOLOGY}, keywords = {vladimir}, month = {JAN}, number = 2, pages = {107-121}, publisher = {SPRINGER}, title = {A combined patch-clamp and electrorotation study of the voltage- and frequency-dependent membrane capacitance caused by structurally dissimilar lipophilic anions}, type = {Article}, volume = 221, year = 2008 }

  %0 Journal Article %1 Zimmermann2008a %A Zimmermann, D. %A Kiesel, M. %A Terpitz, U. %A Zhou, A. %A Reuss, R. %A Kraus, J. %A Schenk, W. A. %A Bamberg, E. %A Sukhorukov, V. L. %C 233 SPRING STREET, NEW YORK, NY 10013 USA %D 2008 %I SPRINGER %J JOURNAL OF MEMBRANE BIOLOGY %N 2 %P 107-121 %T A combined patch-clamp and electrorotation study of the voltage- and frequency-dependent membrane capacitance caused by structurally dissimilar lipophilic anions %U http://dx.doi.org/10.1007/s00232-007-9090-4 %V 221 %X Interactions of structurally dissimilar anionic compounds with the plasma membrane of HEK293 cells were analyzed by patch clamp and electrorotation. The combined approach provides complementary information on the lipophilicity, preferential affinity of the anions to the inner/outer membrane leaflet, adsorption depth and transmembrane mobility. The anionic species studied here included the well-known lipophilic anions dipicrylamine (DPA(-)), tetraphenylborate (TPB-) and {[}W-2(CO)(10)(S2CH)](-), the putative lipophilic anion B(CF3)(4)(-) and three new heterocyclic W(CO)(5) derivatives. All tested anions partitioned strongly into the cell membrane, as indicated by the capacitance increase in patch-clamped cells. The capacitance increment exhibited a bell-shaped dependence on membrane voltage. The midpoint potentials of the maximum capacitance increment were negative, indicating the exclusion of lipophilic anions from the outer membrane leaflet. The adsorption depth of the large organic anions DPA(-), TPB- and B(CF3)(4)(-) increased and that of W(CO)(5) derivatives decreased with increasing concentration of mobile charges. In agreement with the patch-clamp data, electrorotation of cells treated with DPA(-) and W(CO)(5) derivatives revealed a large dispersion of membrane capacitance in the kilohertz to megahertz range due to the translocation of mobile charges. In contrast, in the presence of TPB- and B(CF3)(4)(-) no mobile charges could be detected by electrorotation, despite their strong membrane adsorption. Our data suggest that the presence of oxygen atoms in the outer molecular shell is an important factor for the fast translocation ability of lipophilic anions.
• Electrofused giant protoplasts of Saccharomyces cerevisiae as a novel system for electrophysiological studies on membrane proteins Terpitz, Ulrich; Raimunda, Daniel; Westhoff, Markus; Sukhorukov, Vladimir L.; Beauge, Luis; Bamberg, Ernst; Zimmermann, Dirk in BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES (2008). 1778(6) 1493–1500.
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  Giant protoplasts of Saccharomyces cerevisiae of 10-35 mu m in diameter were generated by multi-cell electrofusion. Thereby two different preparation strategies were evaluated with a focus on size distribution and ``patchability{''} of electrofused protoplasts. In general, parental protoplasts were suitable for electrofusion 1-12 h after isolation. The electrophysiological properties of electrofused giant protoplasts could be analyzed by the whole-cell patch clamp technique. The area-specific membrane capacitance (0.66 +/- 0.07 mu F/cm(2)) and conductance (23-44 mu S/cm(2)) of giant protoplasts were consistent with the corresponding data for parental protoplasts. Measurements with fluorescein-filled patch pipettes allowed to exclude any internal compartmentalisation of giant protoplasts by plasma membranes, since uniform (diffusion-controlled) dye uptake was only observed in the whole-cell configuration, but not in the cell-attached formation. The homogeneous structure of giant protoplasts was further confirmed by the observation that no plasma membrane associated fluorescence was seen in the interior of giant cells after electrofusion of protoplasts expressing the light-activated cation channel Channelrhodopsin-2 (ChR2) linked to yellow fluorescent protein (YFP). Patch clamp analysis of the heterologously expressed ChR2-YFP showed typical blue light dependent, inwardly-directed currents for both electrofused giant and parental protoplasts. Most importantly, neither channel characteristics nor channel expression density was altered by electric field treatment. Summarising, multi-cell electrofusion increases considerably the absolute number of membrane proteins accessible in patch clamp experiments, thus presumably providing a convenient tool for the biophysical investigation of low-signal transporters and channels. (C) 2008 Elsevier B.V. All rights reserved.

  @article{Terpitz2008, abstract = {Giant protoplasts of Saccharomyces cerevisiae of 10-35 mu m in diameter were generated by multi-cell electrofusion. Thereby two different preparation strategies were evaluated with a focus on size distribution and ``patchability{''} of electrofused protoplasts. In general, parental protoplasts were suitable for electrofusion 1-12 h after isolation. The electrophysiological properties of electrofused giant protoplasts could be analyzed by the whole-cell patch clamp technique. The area-specific membrane capacitance (0.66 +/- 0.07 mu F/cm(2)) and conductance (23-44 mu S/cm(2)) of giant protoplasts were consistent with the corresponding data for parental protoplasts. Measurements with fluorescein-filled patch pipettes allowed to exclude any internal compartmentalisation of giant protoplasts by plasma membranes, since uniform (diffusion-controlled) dye uptake was only observed in the whole-cell configuration, but not in the cell-attached formation. The homogeneous structure of giant protoplasts was further confirmed by the observation that no plasma membrane associated fluorescence was seen in the interior of giant cells after electrofusion of protoplasts expressing the light-activated cation channel Channelrhodopsin-2 (ChR2) linked to yellow fluorescent protein (YFP). Patch clamp analysis of the heterologously expressed ChR2-YFP showed typical blue light dependent, inwardly-directed currents for both electrofused giant and parental protoplasts. Most importantly, neither channel characteristics nor channel expression density was altered by electric field treatment. Summarising, multi-cell electrofusion increases considerably the absolute number of membrane proteins accessible in patch clamp experiments, thus presumably providing a convenient tool for the biophysical investigation of low-signal transporters and channels. (C) 2008 Elsevier B.V. All rights reserved.}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {Terpitz, Ulrich and Raimunda, Daniel and Westhoff, Markus and Sukhorukov, Vladimir L. and Beauge, Luis and Bamberg, Ernst and Zimmermann, Dirk}, journal = {BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES}, keywords = {vladimir}, month = {JUN}, number = 6, pages = {1493-1500}, publisher = {ELSEVIER SCIENCE BV}, title = {Electrofused giant protoplasts of Saccharomyces cerevisiae as a novel system for electrophysiological studies on membrane proteins}, type = {Article}, volume = 1778, year = 2008 }

  %0 Journal Article %1 Terpitz2008 %A Terpitz, Ulrich %A Raimunda, Daniel %A Westhoff, Markus %A Sukhorukov, Vladimir L. %A Beauge, Luis %A Bamberg, Ernst %A Zimmermann, Dirk %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 2008 %I ELSEVIER SCIENCE BV %J BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES %N 6 %P 1493-1500 %T Electrofused giant protoplasts of Saccharomyces cerevisiae as a novel system for electrophysiological studies on membrane proteins %U http://dx.doi.org/10.1016/j.bbamem.2008.03.015 %V 1778 %X Giant protoplasts of Saccharomyces cerevisiae of 10-35 mu m in diameter were generated by multi-cell electrofusion. Thereby two different preparation strategies were evaluated with a focus on size distribution and ``patchability{''} of electrofused protoplasts. In general, parental protoplasts were suitable for electrofusion 1-12 h after isolation. The electrophysiological properties of electrofused giant protoplasts could be analyzed by the whole-cell patch clamp technique. The area-specific membrane capacitance (0.66 +/- 0.07 mu F/cm(2)) and conductance (23-44 mu S/cm(2)) of giant protoplasts were consistent with the corresponding data for parental protoplasts. Measurements with fluorescein-filled patch pipettes allowed to exclude any internal compartmentalisation of giant protoplasts by plasma membranes, since uniform (diffusion-controlled) dye uptake was only observed in the whole-cell configuration, but not in the cell-attached formation. The homogeneous structure of giant protoplasts was further confirmed by the observation that no plasma membrane associated fluorescence was seen in the interior of giant cells after electrofusion of protoplasts expressing the light-activated cation channel Channelrhodopsin-2 (ChR2) linked to yellow fluorescent protein (YFP). Patch clamp analysis of the heterologously expressed ChR2-YFP showed typical blue light dependent, inwardly-directed currents for both electrofused giant and parental protoplasts. Most importantly, neither channel characteristics nor channel expression density was altered by electric field treatment. Summarising, multi-cell electrofusion increases considerably the absolute number of membrane proteins accessible in patch clamp experiments, thus presumably providing a convenient tool for the biophysical investigation of low-signal transporters and channels. (C) 2008 Elsevier B.V. All rights reserved.
• Effects on capacitance by overexpression of membrane proteins Zimmermann, D.; Zhou, A.; Kiesel, M.; Feldbauer, K.; Terpitz, U.; Haase, W.; Schneider-Hohendorf, T.; Bamberg, E.; Sukhorukov, V. L. in BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS (2008). 369(4) 1022–1026.
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  Functional Channelrhodopsin-2 (ChR2) overexpression of about 10(4) channels/mu m(2) in the plasma membrane of HEK293 cells was studied by patch-clamp and freeze-fracture electron microscopy. Simultaneous electrorotation measurements revealed that ChR2 expression was accompanied by a marked increase of the area-specific membrane capacitance (C-m). The C-m increase apparently resulted partly from an enlargement of the size and/or number of microvilli. This is suggested by a relatively large C-m of 1.15 +/- 0.08 mu F/cm(2) in ChR2-expressing; cells measured under isotonic conditions. This value was much higher than that of the control HEK293 cells (039 +/- 0.02 mu F/cm(2)). However, even after complete loss of microvilli under strong hypoosmolar conditions (100 mOsm), the ChR2-expressing cells still exhibited a significantly larger C-m (0.85 +/- 0.07 mu F/cm(2)). as compared to non-expressing control cells (0.70 +/- 0.03 mu F/cm(2)). Therefore, a second mechanism of capacitance increase may involve changes in the membrane permittivity and/or thickness due to the embedded ChR2 proteins. (C) 2008 Elsevier Inc. All rights reserved.

  @article{Zimmermann2008, abstract = {Functional Channelrhodopsin-2 (ChR2) overexpression of about 10(4) channels/mu m(2) in the plasma membrane of HEK293 cells was studied by patch-clamp and freeze-fracture electron microscopy. Simultaneous electrorotation measurements revealed that ChR2 expression was accompanied by a marked increase of the area-specific membrane capacitance (C-m). The C-m increase apparently resulted partly from an enlargement of the size and/or number of microvilli. This is suggested by a relatively large C-m of 1.15 +/- 0.08 mu F/cm(2) in ChR2-expressing; cells measured under isotonic conditions. This value was much higher than that of the control HEK293 cells (039 +/- 0.02 mu F/cm(2)). However, even after complete loss of microvilli under strong hypoosmolar conditions (100 mOsm), the ChR2-expressing cells still exhibited a significantly larger C-m (0.85 +/- 0.07 mu F/cm(2)). as compared to non-expressing control cells (0.70 +/- 0.03 mu F/cm(2)). Therefore, a second mechanism of capacitance increase may involve changes in the membrane permittivity and/or thickness due to the embedded ChR2 proteins. (C) 2008 Elsevier Inc. All rights reserved.}, address = {525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA}, author = {Zimmermann, D. and Zhou, A. and Kiesel, M. and Feldbauer, K. and Terpitz, U. and Haase, W. and Schneider-Hohendorf, T. and Bamberg, E. and Sukhorukov, V. L.}, journal = {BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS}, keywords = {vladimir}, month = {MAY 16}, number = 4, pages = {1022-1026}, publisher = {ACADEMIC PRESS INC ELSEVIER SCIENCE}, title = {Effects on capacitance by overexpression of membrane proteins}, type = {Article}, volume = 369, year = 2008 }

  %0 Journal Article %1 Zimmermann2008 %A Zimmermann, D. %A Zhou, A. %A Kiesel, M. %A Feldbauer, K. %A Terpitz, U. %A Haase, W. %A Schneider-Hohendorf, T. %A Bamberg, E. %A Sukhorukov, V. L. %C 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA %D 2008 %I ACADEMIC PRESS INC ELSEVIER SCIENCE %J BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS %N 4 %P 1022-1026 %T Effects on capacitance by overexpression of membrane proteins %U http://dx.doi.org/10.1016/j.bbrc.2008.02.153 %V 369 %X Functional Channelrhodopsin-2 (ChR2) overexpression of about 10(4) channels/mu m(2) in the plasma membrane of HEK293 cells was studied by patch-clamp and freeze-fracture electron microscopy. Simultaneous electrorotation measurements revealed that ChR2 expression was accompanied by a marked increase of the area-specific membrane capacitance (C-m). The C-m increase apparently resulted partly from an enlargement of the size and/or number of microvilli. This is suggested by a relatively large C-m of 1.15 +/- 0.08 mu F/cm(2) in ChR2-expressing; cells measured under isotonic conditions. This value was much higher than that of the control HEK293 cells (039 +/- 0.02 mu F/cm(2)). However, even after complete loss of microvilli under strong hypoosmolar conditions (100 mOsm), the ChR2-expressing cells still exhibited a significantly larger C-m (0.85 +/- 0.07 mu F/cm(2)). as compared to non-expressing control cells (0.70 +/- 0.03 mu F/cm(2)). Therefore, a second mechanism of capacitance increase may involve changes in the membrane permittivity and/or thickness due to the embedded ChR2 proteins. (C) 2008 Elsevier Inc. All rights reserved.

• Diversity and adaptation of soil fungi in an ecosystem with contamination originating from a phosphate fertilizer plant Terpitz, Ulrich; Kothe, Erika in JOURNAL OF APPLIED BOTANY AND FOOD QUALITY-ANGEWANDTE BOTANIK (2006). 80(2) 187–193.
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  In the vicinity of a former phosphate fertilizer production plant, high phosphate contents with up to 463 mg phosphate per kilogram of soil dry matter were found 13 years after closing the plant while pH is only slightly elevated at pH 7 to 8. The deposited phosphate is seen to be moved to deeper soil horizons. The effect on soil microbiota was analyzed with respect to soil respiration, fungal biomass and cultivation of benomyl resistant and ligninolytic fungi. Increasing numbers and diversity of soil fungi were found with distance from the former emittent. This was confirmed by investigation of mycorrhization rates of mycotrophic, ectomycorrhizal birch trees. We tested for adaptation by growth and phosphate acquisition on soil extract media. The best growth was seen on the media containing highest phosphate concentrations showing no in vitro growth inhibition. In contrast to previous findings, however, more polyphosphate granula were seen on soil extract media from distant sites, although phosphate concentration was lowest in these media.

  @article{Terpitz2006, abstract = {In the vicinity of a former phosphate fertilizer production plant, high phosphate contents with up to 463 mg phosphate per kilogram of soil dry matter were found 13 years after closing the plant while pH is only slightly elevated at pH 7 to 8. The deposited phosphate is seen to be moved to deeper soil horizons. The effect on soil microbiota was analyzed with respect to soil respiration, fungal biomass and cultivation of benomyl resistant and ligninolytic fungi. Increasing numbers and diversity of soil fungi were found with distance from the former emittent. This was confirmed by investigation of mycorrhization rates of mycotrophic, ectomycorrhizal birch trees. We tested for adaptation by growth and phosphate acquisition on soil extract media. The best growth was seen on the media containing highest phosphate concentrations showing no in vitro growth inhibition. In contrast to previous findings, however, more polyphosphate granula were seen on soil extract media from distant sites, although phosphate concentration was lowest in these media.}, address = {BACKHAUSSTRASSE 9B, 37081 GOTTINGEN, GERMANY}, author = {Terpitz, Ulrich and Kothe, Erika}, journal = {JOURNAL OF APPLIED BOTANY AND FOOD QUALITY-ANGEWANDTE BOTANIK}, keywords = {terpitz}, month = {NOV}, number = 2, pages = {187-193}, publisher = {DRUCKEREI LIDDY HALM}, title = {Diversity and adaptation of soil fungi in an ecosystem with contamination originating from a phosphate fertilizer plant}, type = {Article}, volume = 80, year = 2006 }

  %0 Journal Article %1 Terpitz2006 %A Terpitz, Ulrich %A Kothe, Erika %C BACKHAUSSTRASSE 9B, 37081 GOTTINGEN, GERMANY %D 2006 %I DRUCKEREI LIDDY HALM %J JOURNAL OF APPLIED BOTANY AND FOOD QUALITY-ANGEWANDTE BOTANIK %N 2 %P 187-193 %T Diversity and adaptation of soil fungi in an ecosystem with contamination originating from a phosphate fertilizer plant %V 80 %X In the vicinity of a former phosphate fertilizer production plant, high phosphate contents with up to 463 mg phosphate per kilogram of soil dry matter were found 13 years after closing the plant while pH is only slightly elevated at pH 7 to 8. The deposited phosphate is seen to be moved to deeper soil horizons. The effect on soil microbiota was analyzed with respect to soil respiration, fungal biomass and cultivation of benomyl resistant and ligninolytic fungi. Increasing numbers and diversity of soil fungi were found with distance from the former emittent. This was confirmed by investigation of mycorrhization rates of mycotrophic, ectomycorrhizal birch trees. We tested for adaptation by growth and phosphate acquisition on soil extract media. The best growth was seen on the media containing highest phosphate concentrations showing no in vitro growth inhibition. In contrast to previous findings, however, more polyphosphate granula were seen on soil extract media from distant sites, although phosphate concentration was lowest in these media.
• Biophysical characterisation of electrofused giant HEK293-cells as a novel electrophysiological expression system Zimmermann, D.; Terpitz, U.; Zhou, A.; Reuss, R.; Mueller, K.; Sukhorukov, V. L.; Gessner, P.; Nagel, G.; Zimmermann, U.; Bamberg, E. in BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS (2006). 348(2) 673–681.
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  Giant HEK293 cells of 30-65 mu m in diameter were produced by three-dimensional multi-cell electrofusion in 75 mOsm sorbitol media. These strong hypotonic conditions facilitated fusion because of the spherical shape and smooth membrane surface of the swollen cells. A regulatory volume decrease (RVD), as observed at higher osmolalities, did not occur at 75 mOsm. In contrast to field-treated, but unfused cells, the increase in volume induced by hypotonic shock was only partly reversible in the case of fused giant cells after their transfer into isotonic medium. The large size of the electrofused cells allowed the study of their electrophysiological properties by application of both whole-cell and giant excised patch-clamp techniques. Recordings on giant cells yielded a value of 1.1 +/- 0.1 mu F/cm(2) for the area-specific membrane capacitance. This value was consistent with that of the parental cells. The area-specific conductivity of giant cells (diameter > 50 mu m) was found to be between 12.8 and 16.1 mu S/cm(2), which is in the range of that of the parental cells. Measurements with patch-pipettes containing fluorescein showed uniform dye uptake in the whole-cell configuration, but not in the cell-attached configuration. The diffusion-controlled uniform uptake of the dye into the cell interior excludes internal compartmentalisation. The finding of a homogeneous fusion was also supported by expression of the yellow fluorescent protein YFP (as part of the fusion-protein ChR2-YFP) in giant cells since no plasma-membrane bound YFP-mediated fluorescence was detected in the interior of the electrofused cells. Functional expression and the electrophysiological characterisation of the light-activated cation channel Channelrhodopsin 2 (ChR2) yielded similar results as for parental cells. Most importantly, the giant cells exhibited a comparable expression density of the channel protein in the plasma membrane as observed in parental cells. This demonstrates that electrofused cells can be used as a heterologous expression system. (c) 2006 Elsevier Inc. All rights reserved.

  @article{Zimmermann2006, abstract = {Giant HEK293 cells of 30-65 mu m in diameter were produced by three-dimensional multi-cell electrofusion in 75 mOsm sorbitol media. These strong hypotonic conditions facilitated fusion because of the spherical shape and smooth membrane surface of the swollen cells. A regulatory volume decrease (RVD), as observed at higher osmolalities, did not occur at 75 mOsm. In contrast to field-treated, but unfused cells, the increase in volume induced by hypotonic shock was only partly reversible in the case of fused giant cells after their transfer into isotonic medium. The large size of the electrofused cells allowed the study of their electrophysiological properties by application of both whole-cell and giant excised patch-clamp techniques. Recordings on giant cells yielded a value of 1.1 +/- 0.1 mu F/cm(2) for the area-specific membrane capacitance. This value was consistent with that of the parental cells. The area-specific conductivity of giant cells (diameter > 50 mu m) was found to be between 12.8 and 16.1 mu S/cm(2), which is in the range of that of the parental cells. Measurements with patch-pipettes containing fluorescein showed uniform dye uptake in the whole-cell configuration, but not in the cell-attached configuration. The diffusion-controlled uniform uptake of the dye into the cell interior excludes internal compartmentalisation. The finding of a homogeneous fusion was also supported by expression of the yellow fluorescent protein YFP (as part of the fusion-protein ChR2-YFP) in giant cells since no plasma-membrane bound YFP-mediated fluorescence was detected in the interior of the electrofused cells. Functional expression and the electrophysiological characterisation of the light-activated cation channel Channelrhodopsin 2 (ChR2) yielded similar results as for parental cells. Most importantly, the giant cells exhibited a comparable expression density of the channel protein in the plasma membrane as observed in parental cells. This demonstrates that electrofused cells can be used as a heterologous expression system. (c) 2006 Elsevier Inc. All rights reserved.}, address = {525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA}, author = {Zimmermann, D. and Terpitz, U. and Zhou, A. and Reuss, R. and Mueller, K. and Sukhorukov, V. L. and Gessner, P. and Nagel, G. and Zimmermann, U. and Bamberg, E.}, journal = {BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS}, keywords = {vladimir}, month = {SEP 22}, number = 2, pages = {673-681}, publisher = {ACADEMIC PRESS INC ELSEVIER SCIENCE}, title = {Biophysical characterisation of electrofused giant HEK293-cells as a novel electrophysiological expression system}, type = {Article}, volume = 348, year = 2006 }

  %0 Journal Article %1 Zimmermann2006 %A Zimmermann, D. %A Terpitz, U. %A Zhou, A. %A Reuss, R. %A Mueller, K. %A Sukhorukov, V. L. %A Gessner, P. %A Nagel, G. %A Zimmermann, U. %A Bamberg, E. %C 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA %D 2006 %I ACADEMIC PRESS INC ELSEVIER SCIENCE %J BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS %N 2 %P 673-681 %T Biophysical characterisation of electrofused giant HEK293-cells as a novel electrophysiological expression system %U http://dx.doi.org/10.1016/j.bbrc.2006.07.112 %V 348 %X Giant HEK293 cells of 30-65 mu m in diameter were produced by three-dimensional multi-cell electrofusion in 75 mOsm sorbitol media. These strong hypotonic conditions facilitated fusion because of the spherical shape and smooth membrane surface of the swollen cells. A regulatory volume decrease (RVD), as observed at higher osmolalities, did not occur at 75 mOsm. In contrast to field-treated, but unfused cells, the increase in volume induced by hypotonic shock was only partly reversible in the case of fused giant cells after their transfer into isotonic medium. The large size of the electrofused cells allowed the study of their electrophysiological properties by application of both whole-cell and giant excised patch-clamp techniques. Recordings on giant cells yielded a value of 1.1 +/- 0.1 mu F/cm(2) for the area-specific membrane capacitance. This value was consistent with that of the parental cells. The area-specific conductivity of giant cells (diameter > 50 mu m) was found to be between 12.8 and 16.1 mu S/cm(2), which is in the range of that of the parental cells. Measurements with patch-pipettes containing fluorescein showed uniform dye uptake in the whole-cell configuration, but not in the cell-attached configuration. The diffusion-controlled uniform uptake of the dye into the cell interior excludes internal compartmentalisation. The finding of a homogeneous fusion was also supported by expression of the yellow fluorescent protein YFP (as part of the fusion-protein ChR2-YFP) in giant cells since no plasma-membrane bound YFP-mediated fluorescence was detected in the interior of the electrofused cells. Functional expression and the electrophysiological characterisation of the light-activated cation channel Channelrhodopsin 2 (ChR2) yielded similar results as for parental cells. Most importantly, the giant cells exhibited a comparable expression density of the channel protein in the plasma membrane as observed in parental cells. This demonstrates that electrofused cells can be used as a heterologous expression system. (c) 2006 Elsevier Inc. All rights reserved.