TY  - JOUR
AU  - Pajic, Tanja
AU  - Stevanović, Katarina
AU  - Todorović, Nataša
AU  - Krmpot, Aleksandar J.
AU  - Živić, Miroslav
AU  - Savić-Šević, Svetlana
AU  - Lević, Steva M.
AU  - Stanić, Marina
AU  - Pantelić, Dejan
AU  - Jelenković, Brana
AU  - Rabasović, Mihailo
PY  - 2024
UR  - http://rimsi.imsi.bg.ac.rs/handle/123456789/3213
AB  - Studying the membrane physiology of filamentous fungi is key to understanding their interactions with the environment and crucial for developing new therapeutic strategies for disease-causing pathogens. However, their plasma membrane has been inaccessible for a micron-sized patch-clamp pipette for pA current recordings due to the rigid chitinous cell wall. Here, we report the first femtosecond IR laser nanosurgery of the cell wall of the filamentous fungi, which enabled patch-clamp measurements on protoplasts released from hyphae. A reproducible and highly precise (diffraction-limited, submicron resolution) method for obtaining viable released protoplasts was developed. Protoplast release from the nanosurgery-generated incisions in the cell wall was achieved from different regions of the hyphae. The plasma membrane of the obtained protoplasts formed tight and high-resistance (GΩ) contacts with the recording pipette. The entire nanosurgical procedure followed by the patch-clamp technique could be completed in less than 1 hour. Compared to previous studies using heterologously expressed channels, this technique provides the opportunity to identify new ionic currents and to study the properties of the ion channels in the protoplasts of filamentous fungi in their native environment.
PB  - Springer Nature
T2  - Microsystems & Nanoengineering
T1  - In vivo femtosecond laser nanosurgery of the cell wall enabling patch-clamp measurements on filamentous fungi
SP  - 47
VL  - 10
DO  - 10.1038/s41378-024-00664-x
ER  - 

@article{
author = "Pajic, Tanja and Stevanović, Katarina and Todorović, Nataša and Krmpot, Aleksandar J. and Živić, Miroslav and Savić-Šević, Svetlana and Lević, Steva M. and Stanić, Marina and Pantelić, Dejan and Jelenković, Brana and Rabasović, Mihailo",
year = "2024",
abstract = "Studying the membrane physiology of filamentous fungi is key to understanding their interactions with the environment and crucial for developing new therapeutic strategies for disease-causing pathogens. However, their plasma membrane has been inaccessible for a micron-sized patch-clamp pipette for pA current recordings due to the rigid chitinous cell wall. Here, we report the first femtosecond IR laser nanosurgery of the cell wall of the filamentous fungi, which enabled patch-clamp measurements on protoplasts released from hyphae. A reproducible and highly precise (diffraction-limited, submicron resolution) method for obtaining viable released protoplasts was developed. Protoplast release from the nanosurgery-generated incisions in the cell wall was achieved from different regions of the hyphae. The plasma membrane of the obtained protoplasts formed tight and high-resistance (GΩ) contacts with the recording pipette. The entire nanosurgical procedure followed by the patch-clamp technique could be completed in less than 1 hour. Compared to previous studies using heterologously expressed channels, this technique provides the opportunity to identify new ionic currents and to study the properties of the ion channels in the protoplasts of filamentous fungi in their native environment.",
publisher = "Springer Nature",
journal = "Microsystems & Nanoengineering",
title = "In vivo femtosecond laser nanosurgery of the cell wall enabling patch-clamp measurements on filamentous fungi",
pages = "47",
volume = "10",
doi = "10.1038/s41378-024-00664-x"
}

Pajic, T., Stevanović, K., Todorović, N., Krmpot, A. J., Živić, M., Savić-Šević, S., Lević, S. M., Stanić, M., Pantelić, D., Jelenković, B.,& Rabasović, M.. (2024). In vivo femtosecond laser nanosurgery of the cell wall enabling patch-clamp measurements on filamentous fungi. in Microsystems & Nanoengineering
Springer Nature., 10, 47.
https://doi.org/10.1038/s41378-024-00664-x

Pajic T, Stevanović K, Todorović N, Krmpot AJ, Živić M, Savić-Šević S, Lević SM, Stanić M, Pantelić D, Jelenković B, Rabasović M. In vivo femtosecond laser nanosurgery of the cell wall enabling patch-clamp measurements on filamentous fungi. in Microsystems & Nanoengineering. 2024;10:47.
doi:10.1038/s41378-024-00664-x .

Pajic, Tanja, Stevanović, Katarina, Todorović, Nataša, Krmpot, Aleksandar J., Živić, Miroslav, Savić-Šević, Svetlana, Lević, Steva M., Stanić, Marina, Pantelić, Dejan, Jelenković, Brana, Rabasović, Mihailo, "In vivo femtosecond laser nanosurgery of the cell wall enabling patch-clamp measurements on filamentous fungi" in Microsystems & Nanoengineering, 10 (2024):47,
https://doi.org/10.1038/s41378-024-00664-x . .

TY  - JOUR
AU  - Stanić, Marina
AU  - Krizak, Strahinja
AU  - Jovanović, Mirna
AU  - Pajic, Tanja
AU  - Ciric, Ana
AU  - Žižić, Milan
AU  - Zakrzewska, Joanna
AU  - Cvetić-Antić, Tijana
AU  - Todorović, Nataša
AU  - Zivić, Miroslav
PY  - 2017
UR  - http://rimsi.imsi.bg.ac.rs/handle/123456789/1082
AB  - Increasing resistance of fungal strains to known fungicides has prompted identification of new candidates for fungicides among substances previously used for other purposes. We have tested the effects of known anion channel inhibitors anthracene-9-carboxylic acid (A9C) and niflumic acid (NFA) on growth, energy metabolism and anionic current of mycelium of fungus Phycomyces blakesleeanus. Both inhibitors significantly decreased growth and respiration of mycelium, but complete inhibition was only achieved by 100 and 500 mu M NFA for growth and respiration, respectively. A9C had no effect on respiration of human NCI-H460 cell line and very little effect on cucumber root sprout clippings, which nominates this inhibitor for further investigation as a potential new fungicide. Effects of A9C and NFA on respiration of isolated mitochondria of P. blakesleeanus were significantly smaller, which indicates that their inhibitory effect on respiration of mycelium is indirect. NMR spectroscopy showed that both A9C and NFA decrease the levels of ATP and polyphosphates in the mycelium of P. blakesleeanus, but only A9C caused intracellular acidification. Outwardly rectifying, fast inactivating instantaneous anionic current (ORIC) was also reduced to 33 +/- 5 and 21 +/- 3% of its pre-treatment size by A9C and NFA, respectively, but only in the absence of ATP. It can be assumed from our results that the regulation of ORIC is tightly linked to cellular energy metabolism in P. blakesleeanus, and the decrease in ATP and polyphosphate levels could be a direct cause of growth inhibition.
PB  - Microbiology Soc, London
T2  - Microbiology-Sgm
T1  - Growth inhibition of fungus Phycomyces blakesleeanus by anion channel inhibitors anthracene-9-carboxylic and niflumic acid attained through decrease in cellular respiration and energy metabolites
EP  - 372
IS  - 3
SP  - 364
VL  - 163
DO  - 10.1099/mic.0.000429
ER  - 

@article{
author = "Stanić, Marina and Krizak, Strahinja and Jovanović, Mirna and Pajic, Tanja and Ciric, Ana and Žižić, Milan and Zakrzewska, Joanna and Cvetić-Antić, Tijana and Todorović, Nataša and Zivić, Miroslav",
year = "2017",
abstract = "Increasing resistance of fungal strains to known fungicides has prompted identification of new candidates for fungicides among substances previously used for other purposes. We have tested the effects of known anion channel inhibitors anthracene-9-carboxylic acid (A9C) and niflumic acid (NFA) on growth, energy metabolism and anionic current of mycelium of fungus Phycomyces blakesleeanus. Both inhibitors significantly decreased growth and respiration of mycelium, but complete inhibition was only achieved by 100 and 500 mu M NFA for growth and respiration, respectively. A9C had no effect on respiration of human NCI-H460 cell line and very little effect on cucumber root sprout clippings, which nominates this inhibitor for further investigation as a potential new fungicide. Effects of A9C and NFA on respiration of isolated mitochondria of P. blakesleeanus were significantly smaller, which indicates that their inhibitory effect on respiration of mycelium is indirect. NMR spectroscopy showed that both A9C and NFA decrease the levels of ATP and polyphosphates in the mycelium of P. blakesleeanus, but only A9C caused intracellular acidification. Outwardly rectifying, fast inactivating instantaneous anionic current (ORIC) was also reduced to 33 +/- 5 and 21 +/- 3% of its pre-treatment size by A9C and NFA, respectively, but only in the absence of ATP. It can be assumed from our results that the regulation of ORIC is tightly linked to cellular energy metabolism in P. blakesleeanus, and the decrease in ATP and polyphosphate levels could be a direct cause of growth inhibition.",
publisher = "Microbiology Soc, London",
journal = "Microbiology-Sgm",
title = "Growth inhibition of fungus Phycomyces blakesleeanus by anion channel inhibitors anthracene-9-carboxylic and niflumic acid attained through decrease in cellular respiration and energy metabolites",
pages = "372-364",
number = "3",
volume = "163",
doi = "10.1099/mic.0.000429"
}

Stanić, M., Krizak, S., Jovanović, M., Pajic, T., Ciric, A., Žižić, M., Zakrzewska, J., Cvetić-Antić, T., Todorović, N.,& Zivić, M.. (2017). Growth inhibition of fungus Phycomyces blakesleeanus by anion channel inhibitors anthracene-9-carboxylic and niflumic acid attained through decrease in cellular respiration and energy metabolites. in Microbiology-Sgm
Microbiology Soc, London., 163(3), 364-372.
https://doi.org/10.1099/mic.0.000429

Stanić M, Krizak S, Jovanović M, Pajic T, Ciric A, Žižić M, Zakrzewska J, Cvetić-Antić T, Todorović N, Zivić M. Growth inhibition of fungus Phycomyces blakesleeanus by anion channel inhibitors anthracene-9-carboxylic and niflumic acid attained through decrease in cellular respiration and energy metabolites. in Microbiology-Sgm. 2017;163(3):364-372.
doi:10.1099/mic.0.000429 .

Stanić, Marina, Krizak, Strahinja, Jovanović, Mirna, Pajic, Tanja, Ciric, Ana, Žižić, Milan, Zakrzewska, Joanna, Cvetić-Antić, Tijana, Todorović, Nataša, Zivić, Miroslav, "Growth inhibition of fungus Phycomyces blakesleeanus by anion channel inhibitors anthracene-9-carboxylic and niflumic acid attained through decrease in cellular respiration and energy metabolites" in Microbiology-Sgm, 163, no. 3 (2017):364-372,
https://doi.org/10.1099/mic.0.000429 . .