In vivo femtosecond laser nanosurgery of cell wall enables patch-clamp on filamentous fungi

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 micron sized patch-clamp pipette for pA currents recordings due to the rigid chitinous cell wall. We report here the first femtosecond IR laser nanosurgery of the cell wall of filamentous fungi, enabling patch-clamp measurements on protoplasts released from hyphae. A reproducible and highly precise (diffraction limited, sub-micron resolution) method for obtaining viable released protoplasts was developed. Protoplast release from the nanosurgery-generated incisions in cell wall was achieved from different regions of the hyphae. Plasma membrane of obtained protoplasts formed tight and high-resistance (GΩ) contacts with the recording pipette. The entire nanosurgical procedure followed by patch-clamp technique can 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 ion channels in protoplasts of filamentous fungi in their native environment.