Abstract
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.
Highlights
Traditional techniques in biochemistry and molecular biology are usually performed on a population ensemble average level
The stoichiometry of each fluorescent spot was identified by comparing its fluorescent intensity with that corresponding to a single green fluorescent protein (GFP) molecule
Consistent with previously published data and observations of aquaporins in other eukaryotes, Fps1 seems to be present as tetramers, which are organized in higher stoichiometry spots (Fig. 1C)
Summary
Traditional techniques in biochemistry and molecular biology are usually performed on a population ensemble average level. Such approaches “smooth” the noise by averaging the observations from anomalous outlying units. Ensemble averaging masks important effects of subpopulations [1,2,3], such as drug resistant bacteria or cancer cells [4,5,6]. Novel localization-based super-resolved microscopy techniques allow tracking individual molecules of the same type (e.g. FliM protein of Escherichia coli [8]) or different types (e.g. correlating separate motions of proteins and lipids in the same bacteria cell [11]) to uncover “hidden” subpopulations, determining precise biological functions [9]
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