Excluded‐Volume Effects in Living Cells

Abstract

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.

Macromolecular Crowding In Vitro , In Vivo , and In Between

“…Technical advances in fluorescence microscopy have provided the ability to measure a probe signal as a function of location within an intact cell 55, 56, 63 . Experiments conducted using this technique have shown clearly that the equilibrium thermal stability and kinetics of conformational change of a given fluorescently labeled probe can vary substantially within a single cell 64, 65 , and between cells of the same type 39 .…”

Design and Properties of Genetically Encoded Probes for Sensing Macromolecular Crowding

“…Other sensors have been developed, including a synthetic sensor based on polyethylene glycol that is compressed by macromolecular crowding (15), and a genetically encoded sensor that is based on protein-induced destabilization of an impaired YFP (16). The polyethylene glycol 10,000 (PEG)-based sensor may function via a similar mechanism to our sensor, whereas the mechanism behind the destabilization of the YFP sensor is not yet clear.…”

Molecular simulations of cellular processes

“…The third challenge, here defined, shall try to overcome the limitations of the previous steps. Specifically, the force field and reaction scheme of a cell model, parameterized through the two-body approximation in challenge 2, shall be refined by using a large number of data measured under minimally perturbing conditions for the cell (Xia et al 2013;Di Rienzo et al 2014;Gnutt et al 2015;Luchinat and Banci 2017;Liu et al 2017) (Fig. 2c).…”

“…The limitations of the assumption of inert crowders are now becoming evident, especially in the cell milieu, where promiscuous and attractive interactions are frequent (Robinson et al 2012;Sarkar et al 2013;Gnutt et al 2015;Smith et al 2016;Pérez Santero et al 2016;Minton 2017;Rotta et al 2017). The stabilizing effect arising from the crowder excluded volume has been observed to be counteracted in several cases where weakly interacting biological crowders were employed (Zhou et al 2008;Peixoto et al 2015;Gnutt et al 2015;Smith et al 2016;Rothe et al 2016;Starzyk et al 2016).…”

“…The stabilizing effect arising from the crowder excluded volume has been observed to be counteracted in several cases where weakly interacting biological crowders were employed (Zhou et al 2008;Peixoto et al 2015;Gnutt et al 2015;Smith et al 2016;Rothe et al 2016;Starzyk et al 2016). However, the largest effect of favorable interactions is experienced during diffusion (Saxton 1996;Putzel et al 2014;Peixoto et al 2015;Bucciarelli et al 2016;Smith et al 2016).…”