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The influence of the cellular milieu, a complex and crowded solvent, is often neglected when biomolecular structure and function are studied in vitro. To mimic the cellular environment, crowding effects are commonly induced in vitro using artificial crowding agents like Ficoll or dextran. However, it is unclear if such effects are also observed in cellulo. Diverging results on protein stability in living cells point out the need for new quantitative methods to investigate the contributions of excluded volume and nonspecific interactions to the cellular crowding effect. We show how new crowding sensitive probes may be utilized to directly investigate crowding effects in living cells. Moreover, we discuss processes where crowding effects could play a crucial role in molecular cell biology.

Biomolecules evolve and function in densely crowded and highly heterogeneous cellular environments. Such conditions are often mimicked in the test tube by the addition of artificial macromolecular crowding agents. Still, it is unclear if such cosolutes indeed reflect the physicochemical properties of the cellular environment as the in-cell crowding effect has not yet been quantified. We have developed a macromolecular crowding sensor based on a FRET-labeled polymer to probe the macromolecular crowding effect inside single living cells. Surprisingly, we find that excluded-volume effects, although observed in the presence of artificial crowding agents, do not lead to a compression of the sensor in the cell. The average conformation of the sensor is similar to that in aqueous buffer solution and cell lysate. However, the in-cell crowding effect is distributed heterogeneously and changes significantly upon cell stress. We present a tool to systematically study the in-cell crowding effect as a modulator of biomolecular reactions. [ABSTRACT FROM AUTHOR]

Biomolecular dynamics and stability are predominantly investigated in vitro and extrapolated to explain function in the living cell. We present fast relaxation imaging (FreI), which combines fluorescence microscopy and temperature jumps to probe biomolecular dynamics and stability inside a single living cell with high spatiotemporal resolution. We demonstrated the method by measuring the reversible fast folding kinetics as well as folding thermodynamics of a fluorescence resonance energy transfer (FRET) probe-labeled phosphoglycerate kinase construct in two human cell lines. Comparison with in vitro experiments at 23–49 °C showed that the cell environment influences protein stability and folding rate. FReI should also be applicable to the study of protein-protein interactions and heat-shock responses as well as to comparative studies of cell populations or whole organisms. [ABSTRACT FROM AUTHOR]

The cover page of the journal "Angewandte Chemie International Edition" is presented.