2nd International Conference on Chemo and Bioinformatics ICCBIKG 2023 (221-224)
АУТОР(И) / AUTHOR(S): Katarina Stevanović, Tanja Pajić, Aleksandar Krmpot, Mihailo Rabasović, Milan Žižić, Miroslav Živić, Nataša V. Todorović
Е-АДРЕСА / E-MAIL: katarina.stevanovic@bio.bg.ac.rs
DOI: 10.46793/ICCBI23.221S
САЖЕТАК / 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.
КЉУЧНЕ РЕЧИ / KEYWORDS:
Phycomyces, azide, laser surgery
ЛИТЕРАТУРА / REFERENCES:
- A. Yoshimi, K. Miyazawa, et al. Cell Wall Integrity and Its Industrial Applications in Filamentous Fungi. J. Fungi. 8 (2022) 435.
- T. Pajić, K. Stevanović, N.V. Todorović, et al. Laser nano-surgery of fungal cell wall to enable patch clamping.18th European Molecular Imaging Meeting, Saltzburg, 2023. PW36#1095.
- M. Zivić, J. Zakrzewska, et al. 31P NMR study of polyphosphate levels during different growth phases of Phycomyces blakesleeanus. Antonie Van Leeuwenhoek 91(2) (2007)169-177.
- T. Pajić, N.V. Todorović, M. Živić, et al. Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi. Sci Rep. 12(1) (2022) 18760.