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.

Keywords: FRET; biophysics; biosensors; excluded-volume effect; macromolecular crowding.

Figure 1

a) Illustration of the labeled PEG sensor in different concentrations of crowding agent (PEG, 10 kDa). Scale bar: 30 Å. b–e) FRET efficiencies plotted as a function of increased cosolute concentration in DPBS buffer for: b) Ficoll 70 and sucrose, c) polyethylene glycol (10 kDa), d) BSA, and e) Xenopus laevis oocyte lysate. Error bars represent mean ±s.d.

Figure 2

a) Representative donor (Atto488) images of cytosolic and nuclear injected cells. Further, false-colored images of FRET efficiencies calculated for each pixel are shown. Scale bar: 20 μm. b) Comparison of the mean FRET efficiencies in HeLa cytosol (n=17), HeLa nuclei (n=11), 10 mg mL⁻¹ DNA solution (n=3), and diluted buffer (n=3). One-way ANOVA with multiple comparisons and a Tukey’s post-test were used to determine statistical differences. *P<0.05, **P<0.01, ***P<0.001. The data are reported as mean ±s.d. c) FRET efficiency histogram of the cytosol and nucleus.

Figure 3

a) Snapshots at three different times during the hypertonic shock are shown: before the hypertonic shock (0 s), immediately after addition of 500 mm NaCl (10 s) and after the hypertonic shock (20 s). Images of the calculated FRET efficiency are shown. Scale bar: 20 μm. b) Histograms of the FRET efficiencies at the three different time points illustrate the subcellular heterogeneity. c) Cell-averaged FRET efficiencies plotted against time. The addition of NaCl is indicated by the red arrow. Error bars represent mean ±s.d.