Tanja Pajić, Katarina Stevanović, Nataša V. Todorović, Aleksandar J. Krmpot, Miroslav Živić, Svetlana Savić-Šević, Steva M. Lević, Marina Stanić, Dejan Pantelić, Brana Jelenković, Mihailo D. Rabasović
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引用次数: 0
摘要
研究丝状真菌的膜生理学是了解其与环境相互作用的关键,也是开发针对致病病原体的新治疗策略的关键。然而,由于僵硬的壳质细胞壁,用微米大小的贴片钳移液管记录 pA 电流一直无法触及它们的质膜。在这里,我们首次报道了对丝状真菌细胞壁的飞秒红外激光纳米手术,从而实现了对从菌丝中释放出来的原生质体的贴片钳测量。研究人员开发了一种可重复且高度精确(衍射限制、亚微米分辨率)的方法,用于获取有活力的释放原生质体。原生质体从纳米手术在细胞壁上造成的切口中释放出来,这些切口来自菌丝的不同区域。获得的原生质体的质膜与记录吸管形成了紧密的高阻(GΩ)接触。整个纳米手术过程和膜片钳技术可在 1 小时内完成。与以往使用异源表达通道进行的研究相比,该技术为鉴定新的离子电流和研究丝状真菌原生质体在原生环境中的离子通道特性提供了机会。
In vivo femtosecond laser nanosurgery of the cell wall enabling patch-clamp measurements on filamentous fungi
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.
期刊介绍:
Microsystems & Nanoengineering is a comprehensive online journal that focuses on the field of Micro and Nano Electro Mechanical Systems (MEMS and NEMS). It provides a platform for researchers to share their original research findings and review articles in this area. The journal covers a wide range of topics, from fundamental research to practical applications. Published by Springer Nature, in collaboration with the Aerospace Information Research Institute, Chinese Academy of Sciences, and with the support of the State Key Laboratory of Transducer Technology, it is an esteemed publication in the field. As an open access journal, it offers free access to its content, allowing readers from around the world to benefit from the latest developments in MEMS and NEMS.