Laser nano-surgery of fungal cell wall to enable patch clamping
2023
Аутори:
Pajić, TanjaStevanović, Katarina
Todorović, Nataša
Lević, Steva
Savić Šević, Svetlana
Pantelić, Dejan
Živić, Miroslav
Rabasović, Mihailo D
Krmpot, Aleksandar J
Тип документа:
Конференцијски прилог (Објављена верзија)
,
© 2023 by the European Society for Molecular Imaging
Метаподаци
Приказ свих података о документуАпстракт:
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spot-wise pattern was precisely positioned at TPEF image ofselected hypha at the place where the plasma membrane was retracted.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.
Кључне речи:
laser nanosurgery; patch clamp; electrophysiology; two-photon imaging; cell surgeryФинансирање / пројекти:
- HEMMAGINERO - Hemoglobin-Based Spectroscopy and Nonlinear Imaging of Erythrocytes and Their Membranes As Emerging Diagnostic Tool (RS-ScienceFundRS-Promis-6066079)
- Qatar National Research Fund (project #NPRP12S-0205-190047)
- Министарство науке, технолошког развоја и иновација Републике Србије, институционално финансирање - 200178 (Универзитет у Београду, Биолошки факултет) (RS-MESTD-inst-2020-200178)
- Министарство науке, технолошког развоја и иновација Републике Србије, институционално финансирање - 200116 (Универзитет у Београду, Пољопривредни факултет) (RS-MESTD-inst-2020-200116)
- Министарство науке, технолошког развоја и иновација Републике Србије, институционално финансирање - 200007 (Универзитет у Београду, Институт за биолошка истраживања 'Синиша Станковић') (RS-MESTD-inst-2020-200007)
У:
- 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; 2023. p. 1095.