Electrophysiology of fluoride channels in the yeasts Saccharomyces cerevisiae and Candida albicans.

4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-01-20 DOI:10.1016/bs.mie.2024.01.005
Alberto Rivetta, Clifford Slayman
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引用次数: 0

Abstract

Tight regulation of molecules moving through the cell membrane is particularly important for free-living microorganisms because of their small cell volumes and frequent changes in the chemical composition of the extracellular environment. This is true for nutrients, but even more so for toxic molecules. Traditionally, the transport of these diverse molecules in microorganisms has been studied on cell populations rather than on single cells, mainly because of technical difficulties. The goal of this chapter is to make available a detailed method to prepare yeast spheroplasts to study the movement of fluoride ions across the plasma membrane of single cells by the patch-clamp technique. In this procedure, three steps are critical to achieve high resistance (GΩ) seals between the membrane and the glass electrode: (1) appropriate removal of the cell wall by enzymatic treatment; (2) balance between the osmotic strength of sealing solutions and cell membrane turgor; and (3) meticulous morphological inspection of spheroplasts suitable for gigaseal formation. We show now that this method, originally developed for Saccharomyces cerevisiae, can also be applied to Candida albicans, an opportunistic human pathogen.

酿酒酵母和白色念珠菌中氟化物通道的电生理学。
由于自由生活的微生物细胞体积小,细胞外环境的化学成分变化频繁,因此对通过细胞膜的分子进行严格调节尤为重要。营养物质如此,有毒分子更是如此。传统上,这些不同分子在微生物中的转运主要是通过细胞群而不是单细胞来研究的,这主要是由于技术上的困难。本章的目的是提供一种制备酵母球形体的详细方法,通过膜片钳技术研究氟离子在单细胞质膜上的移动。在此过程中,有三个步骤对实现膜和玻璃电极之间的高电阻(GΩ)密封至关重要:(1)通过酶处理适当去除细胞壁;(2)平衡密封溶液的渗透强度和细胞膜的张力;以及(3)对适合形成千亿pt老虎机的球形体进行细致的形态学检查。我们现在证明,这种最初为酿酒酵母开发的方法也可应用于白色念珠菌(一种机会性人类病原体)。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Methods in enzymology
Methods in enzymology 生物-生化研究方法
CiteScore
2.90
自引率
0.00%
发文量
308
审稿时长
3-6 weeks
期刊介绍: The critically acclaimed laboratory standard for almost 50 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Each volume is eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with over 500 volumes the series contains much material still relevant today and is truly an essential publication for researchers in all fields of life sciences, including microbiology, biochemistry, cancer research and genetics-just to name a few. Five of the 2013 Nobel Laureates have edited or contributed to volumes of MIE.
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