TY  - JOUR
AU  - Pajić, 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 D
PY  - 2024
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6666
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
VL  - 10
DO  - 10.1038/s41378-024-00664-x
SP  - 47
ER  - 

@article{
author = "Pajić, 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 D",
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",
volume = "10",
doi = "10.1038/s41378-024-00664-x",
pages = "47"
}

Pajić, T., Stevanović, K., Todorović, N., Krmpot, A. J., Živić, M., Savić-Šević, S., Lević, S. M., Stanić, M., Pantelić, D., Jelenković, B.,& Rabasović, M. D.. (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

Pajić T, Stevanović K, Todorović N, Krmpot AJ, Živić M, Savić-Šević S, Lević SM, Stanić M, Pantelić D, Jelenković B, Rabasović MD. 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 .

Pajić, Tanja, Stevanović, Katarina, Todorović, Nataša, Krmpot, Aleksandar J, Živić, Miroslav, Savić-Šević, Svetlana, Lević, Steva M, Stanić, Marina, Pantelić, Dejan, Jelenković, Brana, Rabasović, Mihailo D, "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  - CONF
AU  - Todorović, Nataša
AU  - Živić, Miroslav
AU  - Stevanović, Katarina
AU  - Pajić, Tanja
AU  - Cvetić Antić, Tijana
AU  - Atlagić, Kristina
AU  - Krmpot, Aleksandar
PY  - 2023
UR  - https://microbiology.bg.ac.rs/workshop2023/
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6248
AB  - The use of chemical fungicides is detrimental for soil and aquatic ecosystems.
Although highly effective, they raise concerns about impact on the environment.
Therefore, the need to reduce the use of chemical fungicides and protect
the soil and water, pose the challenge to find novel, eco-friendly, but also
more efficient, means of crop protection from phytopathogenic fungi. We are
aiming to utilize the native Trichoderma species, as biological control agent,
for controlling or eliminating the phytopathogenic fungi. Our research will be
based on cell nano-surgery of fungal cell wall using ultrashort laser pulses and
subsequent patch clamping on the released protoplast membrane. This will
enable the studies of activity and potency of specific class of antimicrobial short
peptides, peptaibols, released by Trichoderma species, which act by forming
ion channels in pathogen membrane as one of the main mechanisms of their
fungicide action. The result of this project will be the developed instrumentation
and unique method based on nano-surgery and patch clamp for studies of ionic
channels in native fungal plasma membrane. Testing a range of autochthonous
Trichoderma sp. isolates will enable formulation of their combinations that work
best. The selected Trichoderma strains with the highest standardized peptaibol
activity in specific combination of conditions, can be used as a starting point for
development of new, more potent biocontrol agents.
PB  - Belgrade: University of Belgrade, Faculty of Biology
C3  - Book of Abstracts: ICGEB Workshop: Trends in microbial solutions for sustainable agriculture; 2023 Sep13-15; Belgrade, Serbia
T1  - Novel research project – BioPhysFun for advancement of characterization of Trichoderma as biological control agent
SP  - 84
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6248
ER  - 

@conference{
author = "Todorović, Nataša and Živić, Miroslav and Stevanović, Katarina and Pajić, Tanja and Cvetić Antić, Tijana and Atlagić, Kristina and Krmpot, Aleksandar",
year = "2023",
abstract = "The use of chemical fungicides is detrimental for soil and aquatic ecosystems.
Although highly effective, they raise concerns about impact on the environment.
Therefore, the need to reduce the use of chemical fungicides and protect
the soil and water, pose the challenge to find novel, eco-friendly, but also
more efficient, means of crop protection from phytopathogenic fungi. We are
aiming to utilize the native Trichoderma species, as biological control agent,
for controlling or eliminating the phytopathogenic fungi. Our research will be
based on cell nano-surgery of fungal cell wall using ultrashort laser pulses and
subsequent patch clamping on the released protoplast membrane. This will
enable the studies of activity and potency of specific class of antimicrobial short
peptides, peptaibols, released by Trichoderma species, which act by forming
ion channels in pathogen membrane as one of the main mechanisms of their
fungicide action. The result of this project will be the developed instrumentation
and unique method based on nano-surgery and patch clamp for studies of ionic
channels in native fungal plasma membrane. Testing a range of autochthonous
Trichoderma sp. isolates will enable formulation of their combinations that work
best. The selected Trichoderma strains with the highest standardized peptaibol
activity in specific combination of conditions, can be used as a starting point for
development of new, more potent biocontrol agents.",
publisher = "Belgrade: University of Belgrade, Faculty of Biology",
journal = "Book of Abstracts: ICGEB Workshop: Trends in microbial solutions for sustainable agriculture; 2023 Sep13-15; Belgrade, Serbia",
title = "Novel research project – BioPhysFun for advancement of characterization of Trichoderma as biological control agent",
pages = "84",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6248"
}

Todorović, N., Živić, M., Stevanović, K., Pajić, T., Cvetić Antić, T., Atlagić, K.,& Krmpot, A.. (2023). Novel research project – BioPhysFun for advancement of characterization of Trichoderma as biological control agent. in Book of Abstracts: ICGEB Workshop: Trends in microbial solutions for sustainable agriculture; 2023 Sep13-15; Belgrade, Serbia
Belgrade: University of Belgrade, Faculty of Biology., 84.
https://hdl.handle.net/21.15107/rcub_ibiss_6248

Todorović N, Živić M, Stevanović K, Pajić T, Cvetić Antić T, Atlagić K, Krmpot A. Novel research project – BioPhysFun for advancement of characterization of Trichoderma as biological control agent. in Book of Abstracts: ICGEB Workshop: Trends in microbial solutions for sustainable agriculture; 2023 Sep13-15; Belgrade, Serbia. 2023;:84.
https://hdl.handle.net/21.15107/rcub_ibiss_6248 .

Todorović, Nataša, Živić, Miroslav, Stevanović, Katarina, Pajić, Tanja, Cvetić Antić, Tijana, Atlagić, Kristina, Krmpot, Aleksandar, "Novel research project – BioPhysFun for advancement of characterization of Trichoderma as biological control agent" in Book of Abstracts: ICGEB Workshop: Trends in microbial solutions for sustainable agriculture; 2023 Sep13-15; Belgrade, Serbia (2023):84,
https://hdl.handle.net/21.15107/rcub_ibiss_6248 .

TY  - CONF
AU  - Stevanović, Katarina
AU  - Pajić, Tanja
AU  - Krmpot, Aleksandar
AU  - Rabasović, Mihailo
AU  - Žižić, Milan
AU  - Živić, Miroslav
AU  - Todorović, Nataša
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6246
AB  - The success of patch-clamp giga-ohm seal formation on filamentous fungi Phycomyces blakesleeanus protoplasts was investigated to evaluate their usefulness in ion channel studies on filamentous fungi. Protoplasts were obtained by laser-mediated nanosurgery of the cell wall stained with Calcofluor White. To enable a successful seal formation, it is critical to prevent cell wall regeneration. Since wall integrity responses in fungi involve kinase-dependent pathways, we aimed to sufficiently reduce intracellular ATP availability using the respiratory inhibitor azide. The effect of azide on phosphate metabolites of Phycomyces blakesleeanus, as determined by 31P NMR spectroscopy, was a reduction in intracellular ATP accompanied by a decrease in long chain polyphosphates. Subsequently, all seal formation measurements were performed in the presence of azide, and protoplast viability was confirmed by cytoplasmic streaming. The success of seal formation depends on the size of the protoplasts, as larger protoplasts are more prone to successful seal formation. It was also found that the laser power used in nanosurgery could influence the success of seal formation, as higher values were associated with a lower success rate. The protoplasts released by laser nanosurgery produced by our newly developed method are a good model system for patch clamp on filamentous fungal membranes because their plasma membrane can easily form high-quality seals with the patch pipette.
PB  - Kragujevac: Institute for Information Technologies, University of Belgrade
C3  - Book of Proceedings: 2nd International Conference on Chemo and Bioinformatics ICCBIKG 2023; 2023 Sep 28-29; Kragujevac, Serbia
T1  - Patch clamp pipette giga seal forming success on the nanosurgery-obtained filamentous fungi protoplasts
DO  - 10.46793/ICCBI23.221S
SP  - 221
EP  - 224
ER  - 

@conference{
author = "Stevanović, Katarina and Pajić, Tanja and Krmpot, Aleksandar and Rabasović, Mihailo and Žižić, Milan and Živić, Miroslav and Todorović, Nataša",
year = "2023",
abstract = "The success of patch-clamp giga-ohm seal formation on filamentous fungi Phycomyces blakesleeanus protoplasts was investigated to evaluate their usefulness in ion channel studies on filamentous fungi. Protoplasts were obtained by laser-mediated nanosurgery of the cell wall stained with Calcofluor White. To enable a successful seal formation, it is critical to prevent cell wall regeneration. Since wall integrity responses in fungi involve kinase-dependent pathways, we aimed to sufficiently reduce intracellular ATP availability using the respiratory inhibitor azide. The effect of azide on phosphate metabolites of Phycomyces blakesleeanus, as determined by 31P NMR spectroscopy, was a reduction in intracellular ATP accompanied by a decrease in long chain polyphosphates. Subsequently, all seal formation measurements were performed in the presence of azide, and protoplast viability was confirmed by cytoplasmic streaming. The success of seal formation depends on the size of the protoplasts, as larger protoplasts are more prone to successful seal formation. It was also found that the laser power used in nanosurgery could influence the success of seal formation, as higher values were associated with a lower success rate. The protoplasts released by laser nanosurgery produced by our newly developed method are a good model system for patch clamp on filamentous fungal membranes because their plasma membrane can easily form high-quality seals with the patch pipette.",
publisher = "Kragujevac: Institute for Information Technologies, University of Belgrade",
journal = "Book of Proceedings: 2nd International Conference on Chemo and Bioinformatics ICCBIKG 2023; 2023 Sep 28-29; Kragujevac, Serbia",
title = "Patch clamp pipette giga seal forming success on the nanosurgery-obtained filamentous fungi protoplasts",
doi = "10.46793/ICCBI23.221S",
pages = "221-224"
}

Stevanović, K., Pajić, T., Krmpot, A., Rabasović, M., Žižić, M., Živić, M.,& Todorović, N.. (2023). Patch clamp pipette giga seal forming success on the nanosurgery-obtained filamentous fungi protoplasts. in Book of Proceedings: 2nd International Conference on Chemo and Bioinformatics ICCBIKG 2023; 2023 Sep 28-29; Kragujevac, Serbia
Kragujevac: Institute for Information Technologies, University of Belgrade., 221-224.
https://doi.org/10.46793/ICCBI23.221S

Stevanović K, Pajić T, Krmpot A, Rabasović M, Žižić M, Živić M, Todorović N. Patch clamp pipette giga seal forming success on the nanosurgery-obtained filamentous fungi protoplasts. in Book of Proceedings: 2nd International Conference on Chemo and Bioinformatics ICCBIKG 2023; 2023 Sep 28-29; Kragujevac, Serbia. 2023;:221-224.
doi:10.46793/ICCBI23.221S .

Stevanović, Katarina, Pajić, Tanja, Krmpot, Aleksandar, Rabasović, Mihailo, Žižić, Milan, Živić, Miroslav, Todorović, Nataša, "Patch clamp pipette giga seal forming success on the nanosurgery-obtained filamentous fungi protoplasts" in Book of Proceedings: 2nd International Conference on Chemo and Bioinformatics ICCBIKG 2023; 2023 Sep 28-29; Kragujevac, Serbia (2023):221-224,
https://doi.org/10.46793/ICCBI23.221S . .

TY  - CONF
AU  - Pajić, Tanja
AU  - Stevanović, Katarina
AU  - Todorović, Nataša
AU  - Lević, Steva
AU  - Savić Šević, Svetlana
AU  - Pantelić, Dejan
AU  - Živić, Miroslav
AU  - Rabasović, Mihailo D
AU  - Krmpot, Aleksandar J
PY  - 2023
UR  - https://www.eventclass.org/contxt_emim2023/online-program/session?s=PW36#e609
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6289
AB  - Introduction
Electrophysiology studies of ion channels, in live filamentous fungi by patch clamp method are notpossible due to presence of rigid chitinous cell wall that prevents patch clamp pipette to access theplasma membrane. We present laser nano-surgery of the fungal cell wall that enables patch clampelectrophysiology studies. Similar approaches as one-time reports utilizing nanosecond laser pulseslong time ago were not pursued further [1,2]. Here, we demonstrate reproducible method usingfemtosecond lasers accompanied by two-photon excitation fluorescence (TPEF) imaging of hyphae.
Methods
A wild-type strain of filamentous fungus
Phycomyces blakesleeanus
(Burgeff ) [NRRL 1555(-)] were grownon glass coverslips with hand-etched grid, coated with a thin layer of 50% collagen type I as animmobilizer. Home built nonlinear laser scanning microscope [3,4] utilizing Ti:Sa tunable fs laser wasused for TPEF imaging of hyphae and the cell surgery. The latter is enabled with the custom made add-on in soft ware. Coverslip with hyphae is transferred to another microscope setup for patch clamp,consisting of micromanipulators and precise electronics for pA current measurements. The surgicalincisions and released protoplasts were additionally imaged by scanning electron microscopy for whichtreated hyphae had to undergo critical point drying procedure.
Results/Discussion
Hyphae were stained by Calcofluor White and treated with an exocytosis inhibitor (brefeldin A) and arespiration inhibitor (sodium azide) to prevent cell wall regeneration. Since the cell wall and the plasmamembrane are in the close contact [4] hyphae were kept in hyperosmotic solution to retract thecytoplasm from the cell wall. Surgical spot-wise pattern was precisely positioned at TPEF image ofselected hypha at the place where the plasma membrane was retracted. The dwell time (1s) and thelaser power (4-15mW) were set with fixed repetition rate (76MHz), pulse duration (160fs) and laserwavelength (730nm). Upon the surgery, hyphae were gently deplasmolysed. A protoplast with plasmamembrane accessible for the patch clamp pipette was released through the surgical incision (Fig 1). The>GΩ seal resistance was achieved. Numerous ion channels are recorded in diff erent configurations (oncell, inside-out, whole cell and out-out) (Fig 2).
Conclusions
The whole process (cell surgery + patch clamping) is rather complex and specific steps have to be strictlyfollowed for high success rate and reproducibility. Also, chemicals concentrations, solutions osmolarity,timing and cutting parameters have to be kept in the specified narrow range. Obtained currentrecordings provide valuable information on fungal cell membrane ionic channels.
PB  - European Society for Molecular Imaging
C3  - European Molecular Imaging Meeting: 18th Annual Meeting of the European Society for Molecular Imaging: EMIM 2023; 2023 Mar 14-17; Saltzburg, Austria
T1  - Laser nano-surgery of fungal cell wall to enable patch clamping
SP  - 1095
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6289
ER  - 

@conference{
author = "Pajić, Tanja and Stevanović, Katarina and Todorović, Nataša and Lević, Steva and Savić Šević, Svetlana and Pantelić, Dejan and Živić, Miroslav and Rabasović, Mihailo D and Krmpot, Aleksandar J",
year = "2023",
abstract = "Introduction
Electrophysiology studies of ion channels, in live filamentous fungi by patch clamp method are notpossible due to presence of rigid chitinous cell wall that prevents patch clamp pipette to access theplasma membrane. We present laser nano-surgery of the fungal cell wall that enables patch clampelectrophysiology studies. Similar approaches as one-time reports utilizing nanosecond laser pulseslong time ago were not pursued further [1,2]. Here, we demonstrate reproducible method usingfemtosecond lasers accompanied by two-photon excitation fluorescence (TPEF) imaging of hyphae.
Methods
A wild-type strain of filamentous fungus
Phycomyces blakesleeanus
(Burgeff ) [NRRL 1555(-)] were grownon glass coverslips with hand-etched grid, coated with a thin layer of 50% collagen type I as animmobilizer. Home built nonlinear laser scanning microscope [3,4] utilizing Ti:Sa tunable fs laser wasused for TPEF imaging of hyphae and the cell surgery. The latter is enabled with the custom made add-on in soft ware. Coverslip with hyphae is transferred to another microscope setup for patch clamp,consisting of micromanipulators and precise electronics for pA current measurements. The surgicalincisions and released protoplasts were additionally imaged by scanning electron microscopy for whichtreated hyphae had to undergo critical point drying procedure.
Results/Discussion
Hyphae were stained by Calcofluor White and treated with an exocytosis inhibitor (brefeldin A) and arespiration inhibitor (sodium azide) to prevent cell wall regeneration. Since the cell wall and the plasmamembrane are in the close contact [4] hyphae were kept in hyperosmotic solution to retract thecytoplasm from the cell wall. Surgical spot-wise pattern was precisely positioned at TPEF image ofselected hypha at the place where the plasma membrane was retracted. The dwell time (1s) and thelaser power (4-15mW) were set with fixed repetition rate (76MHz), pulse duration (160fs) and laserwavelength (730nm). Upon the surgery, hyphae were gently deplasmolysed. A protoplast with plasmamembrane accessible for the patch clamp pipette was released through the surgical incision (Fig 1). The>GΩ seal resistance was achieved. Numerous ion channels are recorded in diff erent configurations (oncell, inside-out, whole cell and out-out) (Fig 2).
Conclusions
The whole process (cell surgery + patch clamping) is rather complex and specific steps have to be strictlyfollowed for high success rate and reproducibility. Also, chemicals concentrations, solutions osmolarity,timing and cutting parameters have to be kept in the specified narrow range. Obtained currentrecordings provide valuable information on fungal cell membrane ionic channels.",
publisher = "European Society for Molecular Imaging",
journal = "European Molecular Imaging Meeting: 18th Annual Meeting of the European Society for Molecular Imaging: EMIM 2023; 2023 Mar 14-17; Saltzburg, Austria",
title = "Laser nano-surgery of fungal cell wall to enable patch clamping",
pages = "1095",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6289"
}

Pajić, T., Stevanović, K., Todorović, N., Lević, S., Savić Šević, S., Pantelić, D., Živić, M., Rabasović, M. D.,& Krmpot, A. J.. (2023). Laser nano-surgery of fungal cell wall to enable patch clamping. in European Molecular Imaging Meeting: 18th Annual Meeting of the European Society for Molecular Imaging: EMIM 2023; 2023 Mar 14-17; Saltzburg, Austria
European Society for Molecular Imaging., 1095.
https://hdl.handle.net/21.15107/rcub_ibiss_6289

Pajić T, Stevanović K, Todorović N, Lević S, Savić Šević S, Pantelić D, Živić M, Rabasović MD, Krmpot AJ. Laser nano-surgery of fungal cell wall to enable patch clamping. in European Molecular Imaging Meeting: 18th Annual Meeting of the European Society for Molecular Imaging: EMIM 2023; 2023 Mar 14-17; Saltzburg, Austria. 2023;:1095.
https://hdl.handle.net/21.15107/rcub_ibiss_6289 .

Pajić, Tanja, Stevanović, Katarina, Todorović, Nataša, Lević, Steva, Savić Šević, Svetlana, Pantelić, Dejan, Živić, Miroslav, Rabasović, Mihailo D, Krmpot, Aleksandar J, "Laser nano-surgery of fungal cell wall to enable patch clamping" in European Molecular Imaging Meeting: 18th Annual Meeting of the European Society for Molecular Imaging: EMIM 2023; 2023 Mar 14-17; Saltzburg, Austria (2023):1095,
https://hdl.handle.net/21.15107/rcub_ibiss_6289 .

TY  - JOUR
AU  - Stevanović, Katarina S
AU  - Čepkenović, Bogdana
AU  - Križak, Strahinja
AU  - Pajić, Tanja
AU  - Todorović, Nataša
AU  - Živić, Miroslav Ž
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6088
AB  - Ion channels are vital components of filamentous fungi signaling in communication with their environment. We exploited the ability of the apical region of growing sporangiophores of Phycomyces blakesleeanus to form membrane-enveloped cytoplasmic droplets (CDs), to examine ion currents in the filamentous fungi native plasma membrane. In hypoosmotic conditions, the dominant current in the CDs is ORIC, an osmotically activated, anionic, outwardly rectified, fast inactivating instantaneous current that we have previously characterized. Here, we examined the effect of ATP on ORIC. We show that CDs contain active mitochondria, and that respiration inhibition by azide accelerates ORIC inactivation. ATP, added intracellularly, reduced ORIC run-down and shifted the voltage dependence of inactivation toward depolarized potentials, in a manner that did not require hydrolysis. Notably, ATP led to slowing down of ORIC inactivation, as evidenced by an increased time constant of inactivation, τin, and slower decline of τin during prolonged recordings. Flavonoids (genistein and quercetin) had the effect on ORIC opposite to ATP, acting as current inhibitors, possibly by disrupting the stabilizing effect of ATP on ORIC. The integration of osmotic sensing with ATP dependence of the anionic current, typical of vertebrate cells, is described here for the first time in filamentous fungi.
PB  - Springer Nature
T2  - Scientific Reports
T1  - ATP modulation of osmotically activated anionic current in the membrane of Phycomyces blakesleeanus sporangiophore
IS  - 1
VL  - 13
DO  - 10.1038/s41598-023-39021-9
SP  - 11897
ER  - 

@article{
author = "Stevanović, Katarina S and Čepkenović, Bogdana and Križak, Strahinja and Pajić, Tanja and Todorović, Nataša and Živić, Miroslav Ž",
year = "2023",
abstract = "Ion channels are vital components of filamentous fungi signaling in communication with their environment. We exploited the ability of the apical region of growing sporangiophores of Phycomyces blakesleeanus to form membrane-enveloped cytoplasmic droplets (CDs), to examine ion currents in the filamentous fungi native plasma membrane. In hypoosmotic conditions, the dominant current in the CDs is ORIC, an osmotically activated, anionic, outwardly rectified, fast inactivating instantaneous current that we have previously characterized. Here, we examined the effect of ATP on ORIC. We show that CDs contain active mitochondria, and that respiration inhibition by azide accelerates ORIC inactivation. ATP, added intracellularly, reduced ORIC run-down and shifted the voltage dependence of inactivation toward depolarized potentials, in a manner that did not require hydrolysis. Notably, ATP led to slowing down of ORIC inactivation, as evidenced by an increased time constant of inactivation, τin, and slower decline of τin during prolonged recordings. Flavonoids (genistein and quercetin) had the effect on ORIC opposite to ATP, acting as current inhibitors, possibly by disrupting the stabilizing effect of ATP on ORIC. The integration of osmotic sensing with ATP dependence of the anionic current, typical of vertebrate cells, is described here for the first time in filamentous fungi.",
publisher = "Springer Nature",
journal = "Scientific Reports",
title = "ATP modulation of osmotically activated anionic current in the membrane of Phycomyces blakesleeanus sporangiophore",
number = "1",
volume = "13",
doi = "10.1038/s41598-023-39021-9",
pages = "11897"
}

Stevanović, K. S., Čepkenović, B., Križak, S., Pajić, T., Todorović, N.,& Živić, M. Ž.. (2023). ATP modulation of osmotically activated anionic current in the membrane of Phycomyces blakesleeanus sporangiophore. in Scientific Reports
Springer Nature., 13(1), 11897.
https://doi.org/10.1038/s41598-023-39021-9

Stevanović KS, Čepkenović B, Križak S, Pajić T, Todorović N, Živić MŽ. ATP modulation of osmotically activated anionic current in the membrane of Phycomyces blakesleeanus sporangiophore. in Scientific Reports. 2023;13(1):11897.
doi:10.1038/s41598-023-39021-9 .

Stevanović, Katarina S, Čepkenović, Bogdana, Križak, Strahinja, Pajić, Tanja, Todorović, Nataša, Živić, Miroslav Ž, "ATP modulation of osmotically activated anionic current in the membrane of Phycomyces blakesleeanus sporangiophore" in Scientific Reports, 13, no. 1 (2023):11897,
https://doi.org/10.1038/s41598-023-39021-9 . .

TY  - JOUR
AU  - Stevanović, Katarina
AU  - Čepkenović, Bogdana
AU  - Križak, Strahinja
AU  - Živić, Miroslav
AU  - Todorović, Nataša
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5978
AB  - Studies of ion currents in filamentous fungi are a prerequisite for forming a complete
understanding of their physiology. Cytoplasmic droplets (CDs), obtained from sporangiophores of
Phycomyces blakesleeanus, are a model system that enables the characterization of ion currents in
the native membrane, including the currents mediated by the channels not yet molecularly identified.
Osmotically activated anionic current with outward rectification (ORIC) is a dominant current in
the membrane of cytoplasmic droplets under the conditions of hypoosmotic stimulation. We have
previously reported remarkable functional similarities of ORIC with the vertebrate volume regulated
anionic current (VRAC), such as dose-dependent activation by osmotic difference, ion selectivity
sequence, and time and voltage dependent profile of the current. Using the patch clamp method on
the CD membrane, we further resolve VRAC-like ORIC characteristics in this paper. We examine
the inhibition by extracellular ATP and carbenoxolone, the permeation of glutamate in presence of
chloride, selectivity for nitrates, and activation by GTP, and we show its single channel behavior
in excised membrane. We propose that ORIC is a functional counterpart of vertebrate VRAC in
filamentous fungi, possibly with a similar essential role in anion efflux during cell volume regulation.
PB  - Basel: MDPI
T2  - Journal of Fungi
T1  - Osmotically activated anion current of Phycomyces blakesleeanus-filamentous fungi counterpart to vertebrate volume regulated anion current
IS  - 6
VL  - 9
DO  - 10.3390/jof9060637
SP  - 637
ER  - 

@article{
author = "Stevanović, Katarina and Čepkenović, Bogdana and Križak, Strahinja and Živić, Miroslav and Todorović, Nataša",
year = "2023",
abstract = "Studies of ion currents in filamentous fungi are a prerequisite for forming a complete
understanding of their physiology. Cytoplasmic droplets (CDs), obtained from sporangiophores of
Phycomyces blakesleeanus, are a model system that enables the characterization of ion currents in
the native membrane, including the currents mediated by the channels not yet molecularly identified.
Osmotically activated anionic current with outward rectification (ORIC) is a dominant current in
the membrane of cytoplasmic droplets under the conditions of hypoosmotic stimulation. We have
previously reported remarkable functional similarities of ORIC with the vertebrate volume regulated
anionic current (VRAC), such as dose-dependent activation by osmotic difference, ion selectivity
sequence, and time and voltage dependent profile of the current. Using the patch clamp method on
the CD membrane, we further resolve VRAC-like ORIC characteristics in this paper. We examine
the inhibition by extracellular ATP and carbenoxolone, the permeation of glutamate in presence of
chloride, selectivity for nitrates, and activation by GTP, and we show its single channel behavior
in excised membrane. We propose that ORIC is a functional counterpart of vertebrate VRAC in
filamentous fungi, possibly with a similar essential role in anion efflux during cell volume regulation.",
publisher = "Basel: MDPI",
journal = "Journal of Fungi",
title = "Osmotically activated anion current of Phycomyces blakesleeanus-filamentous fungi counterpart to vertebrate volume regulated anion current",
number = "6",
volume = "9",
doi = "10.3390/jof9060637",
pages = "637"
}

Stevanović, K., Čepkenović, B., Križak, S., Živić, M.,& Todorović, N.. (2023). Osmotically activated anion current of Phycomyces blakesleeanus-filamentous fungi counterpart to vertebrate volume regulated anion current. in Journal of Fungi
Basel: MDPI., 9(6), 637.
https://doi.org/10.3390/jof9060637

Stevanović K, Čepkenović B, Križak S, Živić M, Todorović N. Osmotically activated anion current of Phycomyces blakesleeanus-filamentous fungi counterpart to vertebrate volume regulated anion current. in Journal of Fungi. 2023;9(6):637.
doi:10.3390/jof9060637 .

Stevanović, Katarina, Čepkenović, Bogdana, Križak, Strahinja, Živić, Miroslav, Todorović, Nataša, "Osmotically activated anion current of Phycomyces blakesleeanus-filamentous fungi counterpart to vertebrate volume regulated anion current" in Journal of Fungi, 9, no. 6 (2023):637,
https://doi.org/10.3390/jof9060637 . .

TY  - CONF
AU  - Stevanović, Katarina
AU  - Pajić, Tanja
AU  - Živić, Miroslav
AU  - Krmpot, Aleksandar
AU  - Rabasović, Mihailo
AU  - Todorović, Nataša
PY  - 2022
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5742
AB  - Prisustvo i aktivni karakter ćelijskog zida koji prekriva plazma membranu filamentoznih gljiva su do sad bili nesavladive prepreke da joj se pristupi elektrofiziološkim metodama na način koji bi omogućio kvalitetno registrovanje jonskih struja. Nedavno, mi smo razvili i optimizovali protokol koji omogućava kvalitetno snimanje struja, metodom nametnute voltaže na deliću membrane, sa plazma membrane protoplasta gljive oslobođenih od zida subćelijskom mikrohirurgijom korišćenjem femtosekundnog Ti:Sa lasera.1,2 Među registrovanim plazmamembranskim strujama protoplasta gljive dominantne su struje nošene različitim anjonskim vrstama. Na osnovu potencijala reverzije u asimetričnim jonskim uslovima identifikovani tipovi jonskih struja su, u najvećem broju slučajeva, bili slabo (42%) ili izrazito (35%) selektivni za hlor u prisustvu K, NO3, glutamatnog jona i visokih koncentracija Ca+2. Među nekoliko različitih struja slabije selektivnosti za hlor, daleko najzastupljenija je struja, konduktivnosti 21 ± 1 pS, prisutna u 42% svih kontakata registracije. Analiza aktivnosti pojedinačnih kanala ove, najzastupljenije, struje pokazuje da verovatnoća otvorenosti kanala slabo raste sa depolarizacijom membrane (Po(-80) = 0,3; Po(50) = 0,45) i opada na više depolarisanim vrednostima (Po(80) = 0,2). Učestalost paketića aktivnosti je najveća na hiperpolarisanim potencijalima (f(-80 mV) > 200 Hz) i dvostruko manja na depolarisanim (f(80 mV) < 100 Hz).
AB  - Присуство и активни карактер ћелијског зида који прекрива плазма мембрану
филаментозних гљива су до сад били несавладиве препреке да јој се приступи
електрофизиолошким методама на начин који би омогућио квалитетно
регистровање јонских струја. Недавно, ми смо развили и оптимизовали протокол
који омогућава квалитетно снимање струја, методом наметнуте волтаже на делићу
мембране, са плазма мембране протопласта гљиве ослобођених од зида
субћелијском микрохирургијом коришћењем фемтосекундног Ti:Sa ласера.1,2 Међу
регистрованим плазмамембранским струјама протопласта гљиве доминантне су
струје ношене различитим анјонским врстама. На основу потенцијала реверзије у
асиметричним јонским условима идентификовани типови јонских струја су, у
највећем броју случајева, били слабо (42%) или изразито (35%) селективни за хлор
у присуству K, NO3, глутаматног јона и високих концентрација Ca+2. Међу
неколико различитих струја слабије селективности за хлор, далеко најзаступљенија
је струја, кондуктивности 21 ± 1 pS, присутна у 42% свих контаката регистрације.
Анализа активности појединачних канала ове, најзаступљеније, струје показује да
вероватноћа отворености канала слабо расте са деполаризацијом мембране (Po(-80) =
0,3; Po(50) = 0,45) и опада на више деполарисаним вредностима (Po(80) = 0,2).
Учесталост пакетића активности је највећа на хиперполарисаним потенцијалима
(f(-80 mV) > 200 Hz) и двоструко мања на деполарисаним (f(80 mV) < 100 Hz).
PB  - Belgrade: Serbian Biological Society
C3  - Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia
T1  - Dominantne struje plazma membrane gljive Phycomyces blakesleeanus registrovane sa protoplasta dobijenih mikrohirurgijom ćelijskog zida femtosekundnim laserom
T1  - Доминантне струје плазма мембране гљиве Phycomyces blakesleeanus регистроване са протопласта добијених микрохирургијом ћелијског зида фемтосекундним ласером
SP  - 24
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5742
ER  - 

@conference{
author = "Stevanović, Katarina and Pajić, Tanja and Živić, Miroslav and Krmpot, Aleksandar and Rabasović, Mihailo and Todorović, Nataša",
year = "2022",
abstract = "Prisustvo i aktivni karakter ćelijskog zida koji prekriva plazma membranu filamentoznih gljiva su do sad bili nesavladive prepreke da joj se pristupi elektrofiziološkim metodama na način koji bi omogućio kvalitetno registrovanje jonskih struja. Nedavno, mi smo razvili i optimizovali protokol koji omogućava kvalitetno snimanje struja, metodom nametnute voltaže na deliću membrane, sa plazma membrane protoplasta gljive oslobođenih od zida subćelijskom mikrohirurgijom korišćenjem femtosekundnog Ti:Sa lasera.1,2 Među registrovanim plazmamembranskim strujama protoplasta gljive dominantne su struje nošene različitim anjonskim vrstama. Na osnovu potencijala reverzije u asimetričnim jonskim uslovima identifikovani tipovi jonskih struja su, u najvećem broju slučajeva, bili slabo (42%) ili izrazito (35%) selektivni za hlor u prisustvu K, NO3, glutamatnog jona i visokih koncentracija Ca+2. Među nekoliko različitih struja slabije selektivnosti za hlor, daleko najzastupljenija je struja, konduktivnosti 21 ± 1 pS, prisutna u 42% svih kontakata registracije. Analiza aktivnosti pojedinačnih kanala ove, najzastupljenije, struje pokazuje da verovatnoća otvorenosti kanala slabo raste sa depolarizacijom membrane (Po(-80) = 0,3; Po(50) = 0,45) i opada na više depolarisanim vrednostima (Po(80) = 0,2). Učestalost paketića aktivnosti je najveća na hiperpolarisanim potencijalima (f(-80 mV) > 200 Hz) i dvostruko manja na depolarisanim (f(80 mV) < 100 Hz)., Присуство и активни карактер ћелијског зида који прекрива плазма мембрану
филаментозних гљива су до сад били несавладиве препреке да јој се приступи
електрофизиолошким методама на начин који би омогућио квалитетно
регистровање јонских струја. Недавно, ми смо развили и оптимизовали протокол
који омогућава квалитетно снимање струја, методом наметнуте волтаже на делићу
мембране, са плазма мембране протопласта гљиве ослобођених од зида
субћелијском микрохирургијом коришћењем фемтосекундног Ti:Sa ласера.1,2 Међу
регистрованим плазмамембранским струјама протопласта гљиве доминантне су
струје ношене различитим анјонским врстама. На основу потенцијала реверзије у
асиметричним јонским условима идентификовани типови јонских струја су, у
највећем броју случајева, били слабо (42%) или изразито (35%) селективни за хлор
у присуству K, NO3, глутаматног јона и високих концентрација Ca+2. Међу
неколико различитих струја слабије селективности за хлор, далеко најзаступљенија
је струја, кондуктивности 21 ± 1 pS, присутна у 42% свих контаката регистрације.
Анализа активности појединачних канала ове, најзаступљеније, струје показује да
вероватноћа отворености канала слабо расте са деполаризацијом мембране (Po(-80) =
0,3; Po(50) = 0,45) и опада на више деполарисаним вредностима (Po(80) = 0,2).
Учесталост пакетића активности је највећа на хиперполарисаним потенцијалима
(f(-80 mV) > 200 Hz) и двоструко мања на деполарисаним (f(80 mV) < 100 Hz).",
publisher = "Belgrade: Serbian Biological Society",
journal = "Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia",
title = "Dominantne struje plazma membrane gljive Phycomyces blakesleeanus registrovane sa protoplasta dobijenih mikrohirurgijom ćelijskog zida femtosekundnim laserom, Доминантне струје плазма мембране гљиве Phycomyces blakesleeanus регистроване са протопласта добијених микрохирургијом ћелијског зида фемтосекундним ласером",
pages = "24",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5742"
}

Stevanović, K., Pajić, T., Živić, M., Krmpot, A., Rabasović, M.,& Todorović, N.. (2022). Dominantne struje plazma membrane gljive Phycomyces blakesleeanus registrovane sa protoplasta dobijenih mikrohirurgijom ćelijskog zida femtosekundnim laserom. in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia
Belgrade: Serbian Biological Society., 24.
https://hdl.handle.net/21.15107/rcub_ibiss_5742

Stevanović K, Pajić T, Živić M, Krmpot A, Rabasović M, Todorović N. Dominantne struje plazma membrane gljive Phycomyces blakesleeanus registrovane sa protoplasta dobijenih mikrohirurgijom ćelijskog zida femtosekundnim laserom. in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia. 2022;:24.
https://hdl.handle.net/21.15107/rcub_ibiss_5742 .

Stevanović, Katarina, Pajić, Tanja, Živić, Miroslav, Krmpot, Aleksandar, Rabasović, Mihailo, Todorović, Nataša, "Dominantne struje plazma membrane gljive Phycomyces blakesleeanus registrovane sa protoplasta dobijenih mikrohirurgijom ćelijskog zida femtosekundnim laserom" in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia (2022):24,
https://hdl.handle.net/21.15107/rcub_ibiss_5742 .

TY  - CONF
AU  - Stevanović, Katarina
AU  - Živić, Miroslav
AU  - Todorović, Nataša
PY  - 2022
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5741
AB  - Izlazno-rektifikujuća inaktivirajuća struja (IRIS) je dominantna anjonska struja u uslovima osmotskog stresa na membrani poreklom iz sporangiofora gljive Phycomyces blakesleeanus.1 Jedna od njenih upečatljivih karakteristika je obrazac inaktivacije na depolarišućim potencijalima.1 Sa gubitkom adenozin trifosfata (ATF) sa unutarćelijske strane tokom dijalize pipetnim rastvorom, inaktivacija se vremenom ubrzava i dovodi do nepovratnog pada amplitude ukupne struje, dok se sa ATF-om prisutnim u pipetnom rastvoru amplituda struje i konstanta inaktivacije τ održavaju u vremenu.2 Naši najskoriji rezultati ukazuju da je, u prisustvu unutarćelijskog ATF-a oporavak od inaktivacije izazvane depolarizacijom voltažno- i vremenski-zavisan: (1) izlaganje hiperpolarizaciji je neophodno za dostizanje oporavka ≥ 90%; (2) procenat oporavka pri minimalnom vremenu odmora raste sa dubinom hiperpolarizacije; (3) brzina eksponencijalnog pada amplitude pri skraćenju vremena oporavka opada sa dubinom hiperpolarizacije. U uslovima smanjene koncentracije ATF-a koja je postignuta preinkubacijom natrijum-azidom, već u prvom minutu po ulasku u konfiguraciju „cela ćelija“ uočena je ubrzana inaktivacija.2 Dalja analiza voltažne-zavisnosti τ inaktivacije IRIS u različitim uslovima jasno pokazuje pomeraj ka manjim vrednostima τ sa opadanjem očekivane koncentracije ATF-a dok je voltažna zavisnost neznatno izmenjena. IRIS je identifikovana i u istrgnutoj konfiguraciji („spolja spolja“), pri čemu je obrazac oporavka od inaktivacije poslužio kao dodatna potvrda da su registrovana pojedinačna otvaranja upravo kanali u osnovi IRIS.
AB  - Излазно-ректификујућа инактивирајућа струја (ИРИС) је доминантна анјонска
струја у условима осмотског стреса на мембрани пореклом из спорангиофора
гљиве Phycomyces blakesleeanus.1 Једна од њених упечатљивих карактеристика је
образац инактивације на деполаришућим потенцијалима.1 Са губитком аденозин
трифосфата (АТФ) са унутарћелијске стране током дијализе пипетним раствором,
инактивација се временом убрзава и доводи до неповратног пада амплитуде укупне
струје, док се са АТФ-ом присутним у пипетном раствору амплитуда струје и
константа инактивације τ одржавају у времену.2 Наши најскорији резултати указују
да је, у присуству унутарћелијског АТФ-а опоравак од инактивације изазване
деполаризацијом волтажно- и временски-зависан: (1) излагање хиперполаризацији
је неопходно за достизање опоравка ≥ 90%; (2) проценат опоравка при минималном
времену одмора расте са дубином хиперполаризације; (3) брзина експоненцијалног
пада амплитуде при скраћењу времена опоравка опада са дубином
хиперполаризације. У условима смањене концентрације АТФ-а која је постигнута
преинкубацијом натријум-азидом, већ у првом минуту по уласку у конфигурацију
„цела ћелија“ уочена је убрзана инактивација.2 Даља анализа волтажне-зависности
τ инактивације ИРИС у различитим условима јасно показује померај ка мањим
вредностима τ са опадањем очекиване концентрације АТФ-а док је волтажна
зависност незнатно измењена. ИРИС је идентификована и у истргнутој
конфигурацији („споља споља“), при чему је образац опоравка од инактивације
послужио као додатна потврда да су регистрована појединачна отварања управо
канали у основи ИРИС.
PB  - Belgrade: Serbian Biological Society
C3  - Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia
T1  - Biofizički profil procesa inaktivacije izlazno-rektifikujuće inaktivirajuće struje gljive Phycomyces blakesleeanus
T1  - Биофизички профил процеса инактивације излазно-ректификујуће инактивирајуће струје гљиве Phycomyces blakesleeanus
SP  - 23
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5741
ER  - 

@conference{
author = "Stevanović, Katarina and Živić, Miroslav and Todorović, Nataša",
year = "2022",
abstract = "Izlazno-rektifikujuća inaktivirajuća struja (IRIS) je dominantna anjonska struja u uslovima osmotskog stresa na membrani poreklom iz sporangiofora gljive Phycomyces blakesleeanus.1 Jedna od njenih upečatljivih karakteristika je obrazac inaktivacije na depolarišućim potencijalima.1 Sa gubitkom adenozin trifosfata (ATF) sa unutarćelijske strane tokom dijalize pipetnim rastvorom, inaktivacija se vremenom ubrzava i dovodi do nepovratnog pada amplitude ukupne struje, dok se sa ATF-om prisutnim u pipetnom rastvoru amplituda struje i konstanta inaktivacije τ održavaju u vremenu.2 Naši najskoriji rezultati ukazuju da je, u prisustvu unutarćelijskog ATF-a oporavak od inaktivacije izazvane depolarizacijom voltažno- i vremenski-zavisan: (1) izlaganje hiperpolarizaciji je neophodno za dostizanje oporavka ≥ 90%; (2) procenat oporavka pri minimalnom vremenu odmora raste sa dubinom hiperpolarizacije; (3) brzina eksponencijalnog pada amplitude pri skraćenju vremena oporavka opada sa dubinom hiperpolarizacije. U uslovima smanjene koncentracije ATF-a koja je postignuta preinkubacijom natrijum-azidom, već u prvom minutu po ulasku u konfiguraciju „cela ćelija“ uočena je ubrzana inaktivacija.2 Dalja analiza voltažne-zavisnosti τ inaktivacije IRIS u različitim uslovima jasno pokazuje pomeraj ka manjim vrednostima τ sa opadanjem očekivane koncentracije ATF-a dok je voltažna zavisnost neznatno izmenjena. IRIS je identifikovana i u istrgnutoj konfiguraciji („spolja spolja“), pri čemu je obrazac oporavka od inaktivacije poslužio kao dodatna potvrda da su registrovana pojedinačna otvaranja upravo kanali u osnovi IRIS., Излазно-ректификујућа инактивирајућа струја (ИРИС) је доминантна анјонска
струја у условима осмотског стреса на мембрани пореклом из спорангиофора
гљиве Phycomyces blakesleeanus.1 Једна од њених упечатљивих карактеристика је
образац инактивације на деполаришућим потенцијалима.1 Са губитком аденозин
трифосфата (АТФ) са унутарћелијске стране током дијализе пипетним раствором,
инактивација се временом убрзава и доводи до неповратног пада амплитуде укупне
струје, док се са АТФ-ом присутним у пипетном раствору амплитуда струје и
константа инактивације τ одржавају у времену.2 Наши најскорији резултати указују
да је, у присуству унутарћелијског АТФ-а опоравак од инактивације изазване
деполаризацијом волтажно- и временски-зависан: (1) излагање хиперполаризацији
је неопходно за достизање опоравка ≥ 90%; (2) проценат опоравка при минималном
времену одмора расте са дубином хиперполаризације; (3) брзина експоненцијалног
пада амплитуде при скраћењу времена опоравка опада са дубином
хиперполаризације. У условима смањене концентрације АТФ-а која је постигнута
преинкубацијом натријум-азидом, већ у првом минуту по уласку у конфигурацију
„цела ћелија“ уочена је убрзана инактивација.2 Даља анализа волтажне-зависности
τ инактивације ИРИС у различитим условима јасно показује померај ка мањим
вредностима τ са опадањем очекиване концентрације АТФ-а док је волтажна
зависност незнатно измењена. ИРИС је идентификована и у истргнутој
конфигурацији („споља споља“), при чему је образац опоравка од инактивације
послужио као додатна потврда да су регистрована појединачна отварања управо
канали у основи ИРИС.",
publisher = "Belgrade: Serbian Biological Society",
journal = "Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia",
title = "Biofizički profil procesa inaktivacije izlazno-rektifikujuće inaktivirajuće struje gljive Phycomyces blakesleeanus, Биофизички профил процеса инактивације излазно-ректификујуће инактивирајуће струје гљиве Phycomyces blakesleeanus",
pages = "23",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5741"
}

Stevanović, K., Živić, M.,& Todorović, N.. (2022). Biofizički profil procesa inaktivacije izlazno-rektifikujuće inaktivirajuće struje gljive Phycomyces blakesleeanus. in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia
Belgrade: Serbian Biological Society., 23.
https://hdl.handle.net/21.15107/rcub_ibiss_5741

Stevanović K, Živić M, Todorović N. Biofizički profil procesa inaktivacije izlazno-rektifikujuće inaktivirajuće struje gljive Phycomyces blakesleeanus. in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia. 2022;:23.
https://hdl.handle.net/21.15107/rcub_ibiss_5741 .

Stevanović, Katarina, Živić, Miroslav, Todorović, Nataša, "Biofizički profil procesa inaktivacije izlazno-rektifikujuće inaktivirajuće struje gljive Phycomyces blakesleeanus" in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia (2022):23,
https://hdl.handle.net/21.15107/rcub_ibiss_5741 .

TY  - CONF
AU  - Pajić, Tanja
AU  - Krmpot, Aleksandar J.
AU  - Todorović, Nataša
AU  - Živić, Miroslav
AU  - Stevanović, Katarina
AU  - Rabasović, Mihailo D.
PY  - 2021
UR  - https://iupab2020.sbbq.org.br/
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/4719
AB  - Nonlinear laser scanning microscopy (NLSM), is an advanced optical technique that utilizes ultrashort laser pulses 
for structural and functional imaging, as well as laser manipulation of live organisms and cells. Two modalities of 
NLSM, two photon excitation fluorescence (TPEF) and third harmonic generation (THG) were applied for in vivo and 
label-free study of oxidative and lipid metabolism of individual cells of filamentous fungus Phycomyces 
blakesleeanus. Cell membranes and lipid droplets (LDs) are major sources of THG signal. TPEF allows us to 
determine the redox ratio (reflecting metabolic activity of cells) of the metabolic cofactors FAD and NAD(P)H 
autofluorescence. In addition, slight modifications of the experimental setup, mostly on software, enabled utilization 
of femtosecond laser pulses for precise cell microsurgery of hyphal cell wall. The optimized microsurgery procedure 
we than utilized to obtain protoplasts suitable for patch-clamp electrophysiological recording. Cell surgery of 
filamentous fungus Phycomyces blakesleeanus, were performed by ultrafast Ti:Sa laser (160 fs pulses). The same 
laser was used for in vivo autoTPEF imaging of NAD(P)H and FAD at different wavelengths. For in vivo THG imaging 
of label-free hyphae, we used 1040 nm, 200 fs pulses from Yb KGW laser. In vivo and label-free application of THG 
imaging enabled, accurately and reliably, detection of changes in distribution, total number, and size of LDs in control 
and treatment group of cells. Two-photon microscopy made it possible to obtain a redox ratio using autofluorescences 
of NAD(P)H and FAD in the same regions of live hyphae. The cell microsurgery procedure has been optimized and 
developed, which enabled the subsequent registration of currents on otherwise unaccessible membrane.
PB  - São Paulo: Sociedade Brasileira de Bioquímica e Biologia Molecular
C3  - 20th IUPAB Congress, 45th SBBf Congress and 50th Annual Meeting of SBBq
T1  - In vivo metabolic imaging and micromanipulation of individual filamentous fungus cells using different nonlinear laser scanning microscopy modalities
SP  - 358
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_4719
ER  - 

@conference{
author = "Pajić, Tanja and Krmpot, Aleksandar J. and Todorović, Nataša and Živić, Miroslav and Stevanović, Katarina and Rabasović, Mihailo D.",
year = "2021",
abstract = "Nonlinear laser scanning microscopy (NLSM), is an advanced optical technique that utilizes ultrashort laser pulses 
for structural and functional imaging, as well as laser manipulation of live organisms and cells. Two modalities of 
NLSM, two photon excitation fluorescence (TPEF) and third harmonic generation (THG) were applied for in vivo and 
label-free study of oxidative and lipid metabolism of individual cells of filamentous fungus Phycomyces 
blakesleeanus. Cell membranes and lipid droplets (LDs) are major sources of THG signal. TPEF allows us to 
determine the redox ratio (reflecting metabolic activity of cells) of the metabolic cofactors FAD and NAD(P)H 
autofluorescence. In addition, slight modifications of the experimental setup, mostly on software, enabled utilization 
of femtosecond laser pulses for precise cell microsurgery of hyphal cell wall. The optimized microsurgery procedure 
we than utilized to obtain protoplasts suitable for patch-clamp electrophysiological recording. Cell surgery of 
filamentous fungus Phycomyces blakesleeanus, were performed by ultrafast Ti:Sa laser (160 fs pulses). The same 
laser was used for in vivo autoTPEF imaging of NAD(P)H and FAD at different wavelengths. For in vivo THG imaging 
of label-free hyphae, we used 1040 nm, 200 fs pulses from Yb KGW laser. In vivo and label-free application of THG 
imaging enabled, accurately and reliably, detection of changes in distribution, total number, and size of LDs in control 
and treatment group of cells. Two-photon microscopy made it possible to obtain a redox ratio using autofluorescences 
of NAD(P)H and FAD in the same regions of live hyphae. The cell microsurgery procedure has been optimized and 
developed, which enabled the subsequent registration of currents on otherwise unaccessible membrane.",
publisher = "São Paulo: Sociedade Brasileira de Bioquímica e Biologia Molecular",
journal = "20th IUPAB Congress, 45th SBBf Congress and 50th Annual Meeting of SBBq",
title = "In vivo metabolic imaging and micromanipulation of individual filamentous fungus cells using different nonlinear laser scanning microscopy modalities",
pages = "358",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_4719"
}

Pajić, T., Krmpot, A. J., Todorović, N., Živić, M., Stevanović, K.,& Rabasović, M. D.. (2021). In vivo metabolic imaging and micromanipulation of individual filamentous fungus cells using different nonlinear laser scanning microscopy modalities. in 20th IUPAB Congress, 45th SBBf Congress and 50th Annual Meeting of SBBq
São Paulo: Sociedade Brasileira de Bioquímica e Biologia Molecular., 358.
https://hdl.handle.net/21.15107/rcub_ibiss_4719

Pajić T, Krmpot AJ, Todorović N, Živić M, Stevanović K, Rabasović MD. In vivo metabolic imaging and micromanipulation of individual filamentous fungus cells using different nonlinear laser scanning microscopy modalities. in 20th IUPAB Congress, 45th SBBf Congress and 50th Annual Meeting of SBBq. 2021;:358.
https://hdl.handle.net/21.15107/rcub_ibiss_4719 .

Pajić, Tanja, Krmpot, Aleksandar J., Todorović, Nataša, Živić, Miroslav, Stevanović, Katarina, Rabasović, Mihailo D., "In vivo metabolic imaging and micromanipulation of individual filamentous fungus cells using different nonlinear laser scanning microscopy modalities" in 20th IUPAB Congress, 45th SBBf Congress and 50th Annual Meeting of SBBq (2021):358,
https://hdl.handle.net/21.15107/rcub_ibiss_4719 .

TY  - CONF
AU  - Stevanović, Katarina
AU  - Živić, Miroslav
AU  - Todorović, Nataša
PY  - 2021
UR  - https://iupab2020.sbbq.org.br/
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/4710
AB  - Outwardly rectifying, inactivating current (ORIC), recorded in cytoplasmatic droplets of P.blakesleeanus is 
characterized by a mild outward rectification, selectivity for anions, and a characteristic inactivation on 
depolarizing potentials. In absence of ATP, ORIC has a fast rundown with increasing speed of inactivation, while both processes are delayed by ATPpip (for at least 8min) and the non-hydrolysable AM-PCP (minimum of 5min). Extracellularly applied, ATP has a blocking effect. Flavonoid querzetin leads to a gradual loss of current despite the presence of intracellular ATP. Our research group has shown azide to be the potent respiration inhibitor of P. blakesleeanus, leaving the cells viable but significantly depleted from ATP. We are presenting data recorded in whole cell configuration, showing that: 1.ORIC can be activated despite the depletion of ATP in the cytoplasmatic droplet. However, its inactivation profile is significantly different comparing to normal conditions with or without ATP in the pipette in the first minute of recording, with reduced τ on lower depolarizing potentials. The fraction of current blocked by querzetin with AM-PCP in pipette solution is greater comparing to experiments with ATPpip, even with the 5-fold increase of AM-PCPpip concentration. The time required for current to recover from depolarizing steps is shorter at more hyperpolarized potentials. We have also started to look for the first insights into ORIC single channel activity in out-out configuration, and recorded, upon stimulation of ORIC in whole cell, and patch excision, the step-like pattern of inactivation that strongly resembles one of ORIC. Based on current data, we can conclude that ATP has a significant role in the inactivation process, with a more potent effect comparing to AM-PCP, whether by the means of greater affinity for the binding site or an additional mechanism involving kinase activity.
PB  - São Paulo: Sociedade Brasileira de Bioquímica e Biologia Molecular
C3  - 20th IUPAB Congress, 45th SBBf Congress and 50th Annual Meeting of SBBq
T1  - Inactivation properties of ORIC, VRAC-like current of filamentous fungus P. blakesleeanus: the role of ATP and the first glimpse of the single channel behavior
SP  - 143
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_4710
ER  - 

@conference{
author = "Stevanović, Katarina and Živić, Miroslav and Todorović, Nataša",
year = "2021",
abstract = "Outwardly rectifying, inactivating current (ORIC), recorded in cytoplasmatic droplets of P.blakesleeanus is 
characterized by a mild outward rectification, selectivity for anions, and a characteristic inactivation on 
depolarizing potentials. In absence of ATP, ORIC has a fast rundown with increasing speed of inactivation, while both processes are delayed by ATPpip (for at least 8min) and the non-hydrolysable AM-PCP (minimum of 5min). Extracellularly applied, ATP has a blocking effect. Flavonoid querzetin leads to a gradual loss of current despite the presence of intracellular ATP. Our research group has shown azide to be the potent respiration inhibitor of P. blakesleeanus, leaving the cells viable but significantly depleted from ATP. We are presenting data recorded in whole cell configuration, showing that: 1.ORIC can be activated despite the depletion of ATP in the cytoplasmatic droplet. However, its inactivation profile is significantly different comparing to normal conditions with or without ATP in the pipette in the first minute of recording, with reduced τ on lower depolarizing potentials. The fraction of current blocked by querzetin with AM-PCP in pipette solution is greater comparing to experiments with ATPpip, even with the 5-fold increase of AM-PCPpip concentration. The time required for current to recover from depolarizing steps is shorter at more hyperpolarized potentials. We have also started to look for the first insights into ORIC single channel activity in out-out configuration, and recorded, upon stimulation of ORIC in whole cell, and patch excision, the step-like pattern of inactivation that strongly resembles one of ORIC. Based on current data, we can conclude that ATP has a significant role in the inactivation process, with a more potent effect comparing to AM-PCP, whether by the means of greater affinity for the binding site or an additional mechanism involving kinase activity.",
publisher = "São Paulo: Sociedade Brasileira de Bioquímica e Biologia Molecular",
journal = "20th IUPAB Congress, 45th SBBf Congress and 50th Annual Meeting of SBBq",
title = "Inactivation properties of ORIC, VRAC-like current of filamentous fungus P. blakesleeanus: the role of ATP and the first glimpse of the single channel behavior",
pages = "143",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_4710"
}

Stevanović, K., Živić, M.,& Todorović, N.. (2021). Inactivation properties of ORIC, VRAC-like current of filamentous fungus P. blakesleeanus: the role of ATP and the first glimpse of the single channel behavior. in 20th IUPAB Congress, 45th SBBf Congress and 50th Annual Meeting of SBBq
São Paulo: Sociedade Brasileira de Bioquímica e Biologia Molecular., 143.
https://hdl.handle.net/21.15107/rcub_ibiss_4710

Stevanović K, Živić M, Todorović N. Inactivation properties of ORIC, VRAC-like current of filamentous fungus P. blakesleeanus: the role of ATP and the first glimpse of the single channel behavior. in 20th IUPAB Congress, 45th SBBf Congress and 50th Annual Meeting of SBBq. 2021;:143.
https://hdl.handle.net/21.15107/rcub_ibiss_4710 .

Stevanović, Katarina, Živić, Miroslav, Todorović, Nataša, "Inactivation properties of ORIC, VRAC-like current of filamentous fungus P. blakesleeanus: the role of ATP and the first glimpse of the single channel behavior" in 20th IUPAB Congress, 45th SBBf Congress and 50th Annual Meeting of SBBq (2021):143,
https://hdl.handle.net/21.15107/rcub_ibiss_4710 .

TY  - CONF
AU  - Živić, Miroslav
AU  - Stevanović, Katarina
AU  - Pajić, Tanja
AU  - Rabasović, Mihailo D.
AU  - Krmpot, Aleksandar J.
AU  - Todorović, Nataša
PY  - 2021
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/4713
AB  - Electrophysiology of cell membrane ion channels in filamentous fungi, unlike that in yeast, plant and 
animal cells is still in its infancy. The only two reports on single channel patch clamp recordings from 
native cell membrane of filamentous fungi were made on protoplasts obtained after cell wall 
microsurgery with UV pulsed laser more than 20 years ago. Both pioneering papers reported 
fairly high success rates of obtaining gigaohm contacts, but neither was followed by more studies. 
Advanced imaging techniques that enable more controllable surgery process and utilizing the 
femtosecond pulses, after optimizing the protocol, could result in minimally damaging cell wall 
microsurgery. The end result would be reproducibly high quality membrane of “de-walled” protoplasts. 
The membrane quality, property that is of utmost importance for application of patch clamp method, is 
a term describing not only mechanical integrity and cleanliness of the membrane, but the physiological 
fitness of the cell as well, as cells about to enter apoptosis or necrosis, or that were subjected to oxidative 
stress do not have it. We are presenting here, to the best of our knowledge, the first electrophysiological snapshot obtained on filamentous fungi protoplast after cell wall removal by a femtosecond laser microsurgery. Utilization 
of the Ti:Sa femtosecond laser with optimizations of the cell wall microsurgery protocol explained in this conference resulted in protoplasts that were prone to form contacts of high electrical resistance (GΩ) with a patch pipette. Ti:Sa laser operating at 730 nm (76 MHz, 160 fs pulse duration) combined with homemade nonlinear laser scanning microscope, physiological 40x 1.0 NA objective was employed for microsurgery and imaging. Standard patch-clamp set up was used for electrophysiology. In single channel recordings from more than 30 patches, 11 different channel types were distinguished, based on the reversal potential in asymmetric ionic conditions and on the conductance. By far, the most frequent types of conductance were anionic. We have found four groups of ion channel currents, based 
on ion selectivity: 1. Unselective anion currents (not discriminating between chloride and glutamate) 44% of all recorded currents. 2. Anionic currents selective for chloride (carried exclusively or mostly by chloride) 35%. 3. Organic acid permeable anionic currents (discriminating for glutamate over chloride) 17%; 4. Calcium cationic current was recorded once. The range of conductance size (g) was variable, with unselective anionic currents encompassing the
smallest (5 pS) and largest (160 pS) recorded conductances. Calcium conductance was small (6 pS), while organic acid conductances and Cl-selective conductances had similar ranges (10-60 pS). Most of the conductances displayed linear current-voltage relationships.
PB  - Belgrade : Institute of Physics
C3  - VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop
T1  - First glance at a multitude of ion currents on filamentous fungus P. blakesleeanus protoplasts obtained by femtosecond laser microsurgery
SP  - 111
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_4713
ER  - 

@conference{
author = "Živić, Miroslav and Stevanović, Katarina and Pajić, Tanja and Rabasović, Mihailo D. and Krmpot, Aleksandar J. and Todorović, Nataša",
year = "2021",
abstract = "Electrophysiology of cell membrane ion channels in filamentous fungi, unlike that in yeast, plant and 
animal cells is still in its infancy. The only two reports on single channel patch clamp recordings from 
native cell membrane of filamentous fungi were made on protoplasts obtained after cell wall 
microsurgery with UV pulsed laser more than 20 years ago. Both pioneering papers reported 
fairly high success rates of obtaining gigaohm contacts, but neither was followed by more studies. 
Advanced imaging techniques that enable more controllable surgery process and utilizing the 
femtosecond pulses, after optimizing the protocol, could result in minimally damaging cell wall 
microsurgery. The end result would be reproducibly high quality membrane of “de-walled” protoplasts. 
The membrane quality, property that is of utmost importance for application of patch clamp method, is 
a term describing not only mechanical integrity and cleanliness of the membrane, but the physiological 
fitness of the cell as well, as cells about to enter apoptosis or necrosis, or that were subjected to oxidative 
stress do not have it. We are presenting here, to the best of our knowledge, the first electrophysiological snapshot obtained on filamentous fungi protoplast after cell wall removal by a femtosecond laser microsurgery. Utilization 
of the Ti:Sa femtosecond laser with optimizations of the cell wall microsurgery protocol explained in this conference resulted in protoplasts that were prone to form contacts of high electrical resistance (GΩ) with a patch pipette. Ti:Sa laser operating at 730 nm (76 MHz, 160 fs pulse duration) combined with homemade nonlinear laser scanning microscope, physiological 40x 1.0 NA objective was employed for microsurgery and imaging. Standard patch-clamp set up was used for electrophysiology. In single channel recordings from more than 30 patches, 11 different channel types were distinguished, based on the reversal potential in asymmetric ionic conditions and on the conductance. By far, the most frequent types of conductance were anionic. We have found four groups of ion channel currents, based 
on ion selectivity: 1. Unselective anion currents (not discriminating between chloride and glutamate) 44% of all recorded currents. 2. Anionic currents selective for chloride (carried exclusively or mostly by chloride) 35%. 3. Organic acid permeable anionic currents (discriminating for glutamate over chloride) 17%; 4. Calcium cationic current was recorded once. The range of conductance size (g) was variable, with unselective anionic currents encompassing the
smallest (5 pS) and largest (160 pS) recorded conductances. Calcium conductance was small (6 pS), while organic acid conductances and Cl-selective conductances had similar ranges (10-60 pS). Most of the conductances displayed linear current-voltage relationships.",
publisher = "Belgrade : Institute of Physics",
journal = "VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop",
title = "First glance at a multitude of ion currents on filamentous fungus P. blakesleeanus protoplasts obtained by femtosecond laser microsurgery",
pages = "111",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_4713"
}

Živić, M., Stevanović, K., Pajić, T., Rabasović, M. D., Krmpot, A. J.,& Todorović, N.. (2021). First glance at a multitude of ion currents on filamentous fungus P. blakesleeanus protoplasts obtained by femtosecond laser microsurgery. in VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop
Belgrade : Institute of Physics., 111.
https://hdl.handle.net/21.15107/rcub_ibiss_4713

Živić M, Stevanović K, Pajić T, Rabasović MD, Krmpot AJ, Todorović N. First glance at a multitude of ion currents on filamentous fungus P. blakesleeanus protoplasts obtained by femtosecond laser microsurgery. in VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop. 2021;:111.
https://hdl.handle.net/21.15107/rcub_ibiss_4713 .

Živić, Miroslav, Stevanović, Katarina, Pajić, Tanja, Rabasović, Mihailo D., Krmpot, Aleksandar J., Todorović, Nataša, "First glance at a multitude of ion currents on filamentous fungus P. blakesleeanus protoplasts obtained by femtosecond laser microsurgery" in VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop (2021):111,
https://hdl.handle.net/21.15107/rcub_ibiss_4713 .

TY  - CONF
AU  - Stevanović, Katarina
AU  - Pajić, Tanja
AU  - Todorović, Nataša
AU  - Krmpot, Aleksandar J.
AU  - Živić, Miroslav
AU  - Rabasović, Mihailo D.
PY  - 2021
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/4714
AB  - We have developed an advanced protocol for laser cell surgery to obtain protoplasts of filamentous 
fungi, suitable for investigation of ion channels, relying on few attempts already made in the past. Among obtained protoplasts, 32% were shown to be “patchable”, meaning that formation of the 
gigaseal by the micropipette was possible. Protoplasts were produced by first making an incision on the 
cell wall of plasmolysed hyphae by a tightly focused femtosecond laser beam. Cell surgery 
is followed by a reduction of solution osmolarity to promote extrusion of protoplast (or more often, a 
part of it) through the cut. The two key points- cell surgery parameters and the proper sequence of the 
solutions used – were subjected to variations to gain insight into parameters that contribute to protoplast 
production and stability. The proper selection of the pipette size and shape with respect to the protoplast 
size was also of great importance. Cell surgery and hyphae imaging was performed by a nonlinear laser scanning microscope. Ti:Sa laser was operating at 730nm, with 76MHz repetition rate and 160fs pulse duration. Water dipping objective lens (40 x 1.0, Zeiss W Plan-Apochromat) on the upright non-linear microscope system made possible to perform laser surgery and patch clamp on two separate systems, while working in the same microscopic chamber. Prior to microsurgery, two-photon excitation fluorescence was used to scan the hyphae cell wall stained with Calcofluor white dye, using the same wavelength as for the surgery. Two-step plasmolysis, with increased concentration of calcium in the more hyperosmotic solution was both efficient for microsurgery to be performed and had a stabilizing effect on protoplasts. Subtle deplasmolysis prior to the patch clamping effective enough to stimulate protoplasts to exit, without 
making the membrane overstretched to interact with the pipette was employed. Optimized concentration 
and type of chemical agens for inhibition of the cell wall production was continuously present in all 
solutions, as an indispensable factor for success.
PB  - Belgrade : Institute of Physics
C3  - VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop
T1  - Laser microsurgery of filamentous fungi: The latest protocol enabling patch-clamp amenable protoplasts
SP  - 104
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_4714
ER  - 

@conference{
author = "Stevanović, Katarina and Pajić, Tanja and Todorović, Nataša and Krmpot, Aleksandar J. and Živić, Miroslav and Rabasović, Mihailo D.",
year = "2021",
abstract = "We have developed an advanced protocol for laser cell surgery to obtain protoplasts of filamentous 
fungi, suitable for investigation of ion channels, relying on few attempts already made in the past. Among obtained protoplasts, 32% were shown to be “patchable”, meaning that formation of the 
gigaseal by the micropipette was possible. Protoplasts were produced by first making an incision on the 
cell wall of plasmolysed hyphae by a tightly focused femtosecond laser beam. Cell surgery 
is followed by a reduction of solution osmolarity to promote extrusion of protoplast (or more often, a 
part of it) through the cut. The two key points- cell surgery parameters and the proper sequence of the 
solutions used – were subjected to variations to gain insight into parameters that contribute to protoplast 
production and stability. The proper selection of the pipette size and shape with respect to the protoplast 
size was also of great importance. Cell surgery and hyphae imaging was performed by a nonlinear laser scanning microscope. Ti:Sa laser was operating at 730nm, with 76MHz repetition rate and 160fs pulse duration. Water dipping objective lens (40 x 1.0, Zeiss W Plan-Apochromat) on the upright non-linear microscope system made possible to perform laser surgery and patch clamp on two separate systems, while working in the same microscopic chamber. Prior to microsurgery, two-photon excitation fluorescence was used to scan the hyphae cell wall stained with Calcofluor white dye, using the same wavelength as for the surgery. Two-step plasmolysis, with increased concentration of calcium in the more hyperosmotic solution was both efficient for microsurgery to be performed and had a stabilizing effect on protoplasts. Subtle deplasmolysis prior to the patch clamping effective enough to stimulate protoplasts to exit, without 
making the membrane overstretched to interact with the pipette was employed. Optimized concentration 
and type of chemical agens for inhibition of the cell wall production was continuously present in all 
solutions, as an indispensable factor for success.",
publisher = "Belgrade : Institute of Physics",
journal = "VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop",
title = "Laser microsurgery of filamentous fungi: The latest protocol enabling patch-clamp amenable protoplasts",
pages = "104",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_4714"
}

Stevanović, K., Pajić, T., Todorović, N., Krmpot, A. J., Živić, M.,& Rabasović, M. D.. (2021). Laser microsurgery of filamentous fungi: The latest protocol enabling patch-clamp amenable protoplasts. in VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop
Belgrade : Institute of Physics., 104.
https://hdl.handle.net/21.15107/rcub_ibiss_4714

Stevanović K, Pajić T, Todorović N, Krmpot AJ, Živić M, Rabasović MD. Laser microsurgery of filamentous fungi: The latest protocol enabling patch-clamp amenable protoplasts. in VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop. 2021;:104.
https://hdl.handle.net/21.15107/rcub_ibiss_4714 .

Stevanović, Katarina, Pajić, Tanja, Todorović, Nataša, Krmpot, Aleksandar J., Živić, Miroslav, Rabasović, Mihailo D., "Laser microsurgery of filamentous fungi: The latest protocol enabling patch-clamp amenable protoplasts" in VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop (2021):104,
https://hdl.handle.net/21.15107/rcub_ibiss_4714 .

TY  - CONF
AU  - Stevanović, Katarina
AU  - Križak, Strahinja
AU  - Todorović, Nataša
AU  - Živić, Miroslav
PY  - 2018
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6375
AB  - Jonski kanali filamentoznih gljiva su još uvek u velikoj meri neistraženi, uglavnom usled prisustva brzo regenerišućeg ćelijskog zida. Naš model sistem citoplazmatskih kapi dobijenih iz sporangiofora gljive Phycomyces blakesleeanus, odnosno ćelijske membrane koja još uvek nije pokrivena ćelijskim zidom, nam omogućava da registrujemo struje metodom nametnute voltaže na deliću membrane. U hipoosmotskim uslovima, pored izlazno ispravljene anjonske struje (ORIC), u familiji struja sa cele membrane su uočljive i ulazno ispravljene anjonske struje koje su propustljive za glukonat. Ovde će biti opisane po prvi put. U hipoosmotskoj sredini (kada je prisutan i ORIC), sa hlorom simetrično zamenjenim glukonatom, glukonat propustljiva struja se javlja u 50% eksperimenata (n=10). U izoosmotskim uslovima, kada ORIC nije aktivan, ulazna struja je 2.5 puta veća kada je simetrični glukonat provodni jon nego kada je prisutan hlor (n=11, prisutna u 72% zapisa). Fit voltažne zavisnosti struje Bolcmanovom funkcijom pokazuje jasno ulazno ispravljanje, sa vrednošću nalektrisanja vratnica -0.55±0.10 (p<0.001). Upadljiva osobina glukonat provodljive struje je spora deaktivacija i odložena aktivacija (na potencijalu -150 mV struja se aktivira posle 137±36 ms (n=8)). Snimanje aktivnosti pojedinačnih kanala u asimetričnim anjonskim uslovima (glukonat/hlor) pokazalo je da u membrani citoplazmatske kapi postoje najmanje tri različita jonska kanala (u 3 od 30 eksperimenata) koji su propustljiviji za glukonat nego za hlor sa sledećim karakteristikama: provodljivost (u pS) 83, 60 i 75; potencijal reverzije (mV) -13,13, 9,42, -5,28; propustljivost za glukonat/propustljivost za hlor 1.80, 1.53, 1.27.
PB  - Belgrade: Serbian Biological Society
C3  - Drugi kongres biologa Srbije; 2018 Sep 25-30; Kladovo, Srbija
T1  - Ulazno ispravljena anjonska struja u membrani citoplazmatskih kapi iz gljive Phycomyces blakesleeanus
SP  - 34
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6375
ER  - 

@conference{
author = "Stevanović, Katarina and Križak, Strahinja and Todorović, Nataša and Živić, Miroslav",
year = "2018",
abstract = "Jonski kanali filamentoznih gljiva su još uvek u velikoj meri neistraženi, uglavnom usled prisustva brzo regenerišućeg ćelijskog zida. Naš model sistem citoplazmatskih kapi dobijenih iz sporangiofora gljive Phycomyces blakesleeanus, odnosno ćelijske membrane koja još uvek nije pokrivena ćelijskim zidom, nam omogućava da registrujemo struje metodom nametnute voltaže na deliću membrane. U hipoosmotskim uslovima, pored izlazno ispravljene anjonske struje (ORIC), u familiji struja sa cele membrane su uočljive i ulazno ispravljene anjonske struje koje su propustljive za glukonat. Ovde će biti opisane po prvi put. U hipoosmotskoj sredini (kada je prisutan i ORIC), sa hlorom simetrično zamenjenim glukonatom, glukonat propustljiva struja se javlja u 50% eksperimenata (n=10). U izoosmotskim uslovima, kada ORIC nije aktivan, ulazna struja je 2.5 puta veća kada je simetrični glukonat provodni jon nego kada je prisutan hlor (n=11, prisutna u 72% zapisa). Fit voltažne zavisnosti struje Bolcmanovom funkcijom pokazuje jasno ulazno ispravljanje, sa vrednošću nalektrisanja vratnica -0.55±0.10 (p<0.001). Upadljiva osobina glukonat provodljive struje je spora deaktivacija i odložena aktivacija (na potencijalu -150 mV struja se aktivira posle 137±36 ms (n=8)). Snimanje aktivnosti pojedinačnih kanala u asimetričnim anjonskim uslovima (glukonat/hlor) pokazalo je da u membrani citoplazmatske kapi postoje najmanje tri različita jonska kanala (u 3 od 30 eksperimenata) koji su propustljiviji za glukonat nego za hlor sa sledećim karakteristikama: provodljivost (u pS) 83, 60 i 75; potencijal reverzije (mV) -13,13, 9,42, -5,28; propustljivost za glukonat/propustljivost za hlor 1.80, 1.53, 1.27.",
publisher = "Belgrade: Serbian Biological Society",
journal = "Drugi kongres biologa Srbije; 2018 Sep 25-30; Kladovo, Srbija",
title = "Ulazno ispravljena anjonska struja u membrani citoplazmatskih kapi iz gljive Phycomyces blakesleeanus",
pages = "34",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6375"
}

Stevanović, K., Križak, S., Todorović, N.,& Živić, M.. (2018). Ulazno ispravljena anjonska struja u membrani citoplazmatskih kapi iz gljive Phycomyces blakesleeanus. in Drugi kongres biologa Srbije; 2018 Sep 25-30; Kladovo, Srbija
Belgrade: Serbian Biological Society., 34.
https://hdl.handle.net/21.15107/rcub_ibiss_6375

Stevanović K, Križak S, Todorović N, Živić M. Ulazno ispravljena anjonska struja u membrani citoplazmatskih kapi iz gljive Phycomyces blakesleeanus. in Drugi kongres biologa Srbije; 2018 Sep 25-30; Kladovo, Srbija. 2018;:34.
https://hdl.handle.net/21.15107/rcub_ibiss_6375 .

Stevanović, Katarina, Križak, Strahinja, Todorović, Nataša, Živić, Miroslav, "Ulazno ispravljena anjonska struja u membrani citoplazmatskih kapi iz gljive Phycomyces blakesleeanus" in Drugi kongres biologa Srbije; 2018 Sep 25-30; Kladovo, Srbija (2018):34,
https://hdl.handle.net/21.15107/rcub_ibiss_6375 .