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A sensor for quantication of macromolecular crowding in living cells
npg 201 5 Nature America, Inc. All rights reserved.
Arnold J Boersma1,2, Inge S Zuhorn3 & Bert Poolman1,2
Macromolecular crowding in cells inuences processes such as folding, association and diffusion of proteins and polynucleic acids. Direct spatiotemporal readout of crowding would be a powerful approach for unraveling the structure of the cytoplasm and determining the impact of excluded volume on protein function in living cells. Here, we introduce a genetically encodable uorescence resonance energy transfer (FRET) sensor for quantifying macromolecular crowding and discuss our application of the sensor in bacterial and mammalian cells.
The cell is highly crowded with biomacromolecules, and the excluded volume influences processes such as diffusion, folding, conformation, and aggregation or association of proteins and poly-nucleic acids1. The values reported for the total macromolecular content range from 50 to 400 mg ml1 (refs. 2,3), depending on the type of cell. The crowding is estimated from the total biopolymer volume fraction3 or inferred from the diffusion coefficient of a protein4. Estimates of the total biopolymer volume fraction of a cell might not reflect the actual crowding because biopolymers associate, aggregate and change shape as a result of crowding, which in turn alters the volume available. Such estimates also do not resolve local crowding differences. Determination of the diffusion coefficient reveals insights into, for example, the organization and structure of the cytosol and the response of cells to osmotic stress or energy depletion5,6. However, diffusion is influenced not only by crowding but also by chemical interactions with cellular components7, by viscosity and by confinement imposed by, for example, nucleoid or membrane invaginations8. Hence, a probe for direct quantification of macromolecular crowding would be a useful addition to the toolbox for studying the physicochemical nature of the cytoplasm.
Here, we present a FRET-based sensor for direct determination of the crowding in a living cell. Genetically encoded FRET-based sensors are widely applied in vivo as, for example, small-molecule or force sensors9,10. We designed our new sensor to contain a conformationally flexible domain that adopts more condensed conformations when placed in a crowded environment, similar to
1Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, the Netherlands. 2Zernike Institute for Advanced Materials, University of...