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Correlating single-molecule characteristics of the yeast aquaglyceroporin Fps1 with environmental perturbations directly in living cells
Methods ( IF 4.8 ) Pub Date : 2020-05-06 , DOI: 10.1016/j.ymeth.2020.05.003
Sviatlana Shashkova 1 , Mikael Andersson 2 , Stefan Hohmann 3 , Mark C Leake 1
Affiliation  

Membrane proteins play key roles at the interface between the cell and its environment by mediating selective import and export of molecules via plasma membrane channels. Despite a multitude of studies on transmembrane channels, understanding of their dynamics directly within living systems is limited. To address this, we correlated molecular scale information from living cells with real time changes to their microenvironment. We employed super-resolved millisecond fluorescence microscopy with a single-molecule sensitivity, to track labelled molecules of interest in real time. We use as example the aquaglyceroporin Fps1 in the yeast Saccharomyces cerevisiae to dissect and correlate its stoichiometry and molecular turnover kinetics with various extracellular conditions. We show that Fps1 resides in multi tetrameric clusters while hyperosmotic and oxidative stress conditions cause Fps1 reorganization. Moreover, we demonstrate that rapid exposure to hydrogen peroxide causes Fps1 degradation. In this way we shed new light on aspects of architecture and dynamics of glycerol-permeable plasma membrane channels.



中文翻译:

将酵母水甘油蛋白 Fps1 的单分子特征与活细胞中的环境扰动直接相关联

膜蛋白通过介导分子通过质膜通道的选择性输入和输出,在细胞与其环境之间的界面上发挥关键作用。尽管对跨膜通道进行了大量研究,但对其直接在生命系统内的动态的理解是有限的。为了解决这个问题,我们将来自活细胞的分子尺度信息与其微环境的实时变化相关联。我们采用具有单分子灵敏度的超分辨率毫秒荧光显微镜来实时跟踪感兴趣的标记分子。我们以酵母酿酒酵母中的水甘油孔蛋白 Fps1 为例剖析其化学计量学和分子转换动力学并将其与各种细胞外条件相关联。我们表明 Fps1 位于多四聚体簇中,而高渗和氧化应激条件会导致 Fps1 重组。此外,我们证明快速接触过氧化氢会导致 Fps1 降解。通过这种方式,我们对甘油可渗透质膜通道的结构和动力学方面有了新的认识。

更新日期:2020-05-06
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