Quantitative assessment of the spatial crowding heterogeneity in cellular fluids

Mammalian cells are crowded with macromolecules, supramolecular complexes, and organelles, all of which equip intracellular fluids, e.g., the cytoplasm, with a dynamic and spatially heterogeneous occupied volume fraction. Diffusion in such fluids has been reported to be heterogeneous, i.e., even individual single-particle trajectories feature spatiotemporally varying transport characteristics. Complementing diffusion-based experiments, we have used here an imaging approach to assess the spatial heterogeneity of the nucleoplasm and the cytoplasm in living interphase cells. As a result, we find that the cytoplasm is more crowded and more heterogeneous than the nucleoplasm on several length scales. This phenomenon even persists in dividing cells, where the mitotic spindle region and its periphery form a contiguous fluid but remain nucleoplasmlike and cytoplasmlike, respectively.

Figure 1

Representative fluorescence images of HeLa cells expressing free EGFP (a) in interphase ($750\times 750$ pixels, scale bar: $10\mu \mathrm{m}$), and (b) in metaphase ($300\times 300$ pixels, scale bar: $4\mu \mathrm{m}$). Indicated rectangles ($150\times 40$ and $180\times 20$ pixels, respectively) were used for an averaged fluorescence profile $F\left(x\right)$ along the horizontal direction $x$. (c) In interphase, the fluorescence profile $F\left(x\right)$ shows a roughly 30% higher mean in the nucleus than the cytoplasm (indicated by dashed lines). (d) A smaller but still significant increase in the fluorescence (about 15%) is seen in metaphase when crossing the mitotic spindle region. (e) An elevated fluorescence in the nucleus (red) as compared to the cytoplasm (black) is also seen in the probability distribution function of voxel-wise fluorescence values $p\left(F\right)$. (f) A similar result is found when comparing $p\left(F\right)$ from the mitotic spindle region (red) versus its periphery (black).

Figure 2

The probability distribution function of voxel-wise fluorescence values $p\left(F\right)$ obtained from a homogeneous dye solution (black) is broader than a Poissonian with the same mean value (blue). The overdispersion can be modeled by a customized mapping that preserves the mean but increases the variance of fluorescence values (see main text for details), eventually yielding a very good fit to the data (red dashed line). Inset: a linear relation between the mean fluorescence $\langle F\rangle$ and the variance ${\sigma }^2\left(F\right)$ is observed for homogeneous samples. Varying the dye concentration and illumination intensity at fixed detector settings (photomultplier voltage: 800 V) does not change the prefactor (black circles and filled red squares). Yet, changing detector settings (to 900 V) increases the prefactor (filled black circles), leading to larger variances at the same mean. Please note the scale factor at both plots.

Figure 3

The PDF of fluorescence ratios $p\left(r\right)$ obtained for cell ensembles in interphase (90 cells, gray histogram) and in metaphase (116 cells, red-hatched histogram) highlight an increased mean fluorescence in the nucleus (spindle) versus the surrounding cytoplasm (periphery). For each interphase cell, the ratio $r$ of the mean fluorescence in the nucleus and in the cytoplasm was used; in metaphase cells the ratio employed the mean fluorescence in the spindle region as compared to its periphery. Treatment of interphase cells with nocodazole did not significantly change the ratio $r$, therefore, these data were included into the ensemble of interphase cells. Dashed blue lines are Gaussians with the mean and variance given by the experimental data, highlighting a significant deviation of the mean ratio $\langle r\rangle$ from unity.

Figure 4

(a) The PDF of the compartment-specific regional fluorescence heterogeneity $\eta \left(F_{\mathrm{ROI}}\right)$, which is used as a proxy for the heterogeneity of the regional accessible volume fraction $\eta \left(\phi \right)$ on intermediate length scales, shows marked differences between cytoplasm and nucleoplasm. (b) In contrast, little to no differences are visible for the crowding heterogeneity in the spindle region and its periphery in an ensemble of metaphase cells. Dashed vertical lines indicate the significance limit. See also main text for discussion.

Figure 5

The PDF of the voxel-wise heterogeneity $\eta \left(F\right)$ [see Eq. (5) and main text for details] indicates a similar heterogeneity of the accessible volume fraction for (a) cytoplasm and nucleoplasm, and for (b) the spindle region and its periphery.