Stability of cytoplasmic nanoviscosity during cell cycle of HeLa cells synchronized with Aphidicolin

Nanoviscosity of the cytoplasm is a key factor affecting diffusion of biomolecules and – as a consequence – rates of biochemical reactions in a cell. Nanoviscosity is an outcome of variable chemical and structural factors, which can temporarily change with cell-cycle associated changes of intracellular architecture. Thus, the question arises, whether rates of biochemical reactions depend on the point of cell cycle. In this paper we address this topic by constant observation of nanoviscosity of HeLa cells cytoplasm during S, G2 and G1 phases after Aphidicolin synchronization. For this purpose we measured diffusion rates of EGFP molecules using fluorescence correlation spectroscopy (FCS). To our surprise, a counter-intuitive stability of cytoplasmic viscosity was observed during the cell cycle. Our results hint at possible existence of robust mechanism maintaining stable physiological viscosity of the cytoplasm, despite huge structural changes during cell cycle.


FCS of EGFP in cytoplasm of HeLa cells
EGFP freely diffuses in cytoplasm of HeLa cells (see reference 20), and thus FCS autocorrelation curve recorded for its motion can be fitted with one-component 3D diffusion model with triplets (Equation SI.1): (SI.1) where N stands for the average number of fluorescent probes inside the focal volume, τD is the average time of diffusion of a probe across the focal volume, κ is the aspect ratio of the focal volume (measured during calibration), ptrip is fraction of triplet states and τtrip is a decay time of a triplet state.
Each autocorrelation curve was fitted with N, τD, ptrip and τtrip as free parameters. Example fit is presented in Figure

Residuals
Control experiment was performed to compare inter-and intracellular variances of diffusion coefficients of EGFP measured by FCS in the cytoplasm of HeLa cells. To reveal cytoplasmic heterogeneity, FCS was performed at 8 different spots, picked randomly in the cytoplasmic area of the cell. For each cell, variance and average of the results was calculated. Variance for diffusion coefficients measured for different cells varied between 12 and 52% of a mean values for given cells (average variance was 42%). For comparison, variance of average diffusion coefficients of the cells considered in this experiment was 128%.
Variance of the diffusion coefficients for the 0.5h time windows considered in the cell cycle experiment varied between 17 and 670%, with the average of 129%. The data was presented in Table SI.1.

Changes in cytoplasmic viscosity upon osmotic shock
Cells were exposed to osmotic shock to measure levels of cytoplasmic viscosity that are possible to achieve in HeLa cells for EGFP protein. For this purpose Influx hypertonic cell culture medium was used (Influx kit, Sigma-Algrich). The stock hypertonic medium had osmolarity of 900 mOsmol/L, while basic cell culture medium is 300 mOsmol/L. A series of dilutions of the stock solution in cell culture medium resulted in media of 400 mOsmol/L, 500 mOsmol/L, 600 mOsmol/L, 700 mOsmol/L and 800 mOsmol/L. EGFP diffusion coefficients could have been measured with FCS for the osmolarity of up to 600 mOsmol/L. The results are presented in Figure SI.2.

Figure SI.2.
Diffusion coefficients of EGFP in the cytoplasm of HeLa cells exposed to media of different osmolarity.

Contribution of EGFP to overall cytoplasmic crowding
The cell line used in experiments was HeLa-EGFP inducible line, where EGFP is expressed under tetracycline inducible suCMV promoter. For FCS measurements it is required to have ~1 fluorescent molecule per focal volume. Thus, amount of EGFP produced solely as a result of suCMV promoter leakage was sufficient for our experiments and no induction was applied to enhance EGFP expression.
All FCS measurements were performed in the regime of 1-10 EGFP molecules per focal volume. It corresponds to total number of ~70 000 EGFP copies per cell. According to reference 24 total number of copies of proteins natively present in cytosol is ~640 000. Thus, EGFP expressed additionally is 0.01% of total number of cytosolic proteins. We believe, that contribution of such a rare crowder can be neglected in overall crowding.
Data used for calculation are presented in Table SI.2.