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Figure 5 | Theoretical Biology and Medical Modelling

Figure 5

From: Can visco-elastic phase separation, macromolecular crowding and colloidal physics explain nuclear organisation?

Figure 5

Macromolecular crowding plays a role in chromatin condensation. Hela cells were exposed to different concentrations of PBS and Ficoll 400. Following 10 minutes of exposure to the experimental conditions, cells were fixed with 2% paraformaldehyde, stained with 20 nM Topro 3 for 5 min, and then images were collected. Panels A to N show representative images for each condition. Autocorrelation analysis was performed as follows: intensity profiles were measured across the nucleus, taking care to avoid nucleoli. These measurements were used to compute correlation by self-reference, for a series of lag distances (unit used was 40 nm) from each point n≥ 50, and the average and standard deviation were plotted. The autocorrelation plots show the variation of correlation with the distance. Ecologists have used this type of analysis extensively, as it gives information about spatial patterns. Panel A: chromatin distribution in a non-permeabilised cell. Panels B to J: representative images illustrating cells permeabilised with 0.1% Triton and incubated for 10 minutes with buffers of different ionic strength and with different concentrations of macromolecular crowding agents. Incubation with PBS alone is sufficient to decondense chromatin (B, E), and this decondensation increases with lowering PBS concentrations (H, K and N, Q). The addition of Ficoll was able to maintain chromatin condensation and even to hyper-condense chromatin (C, D, I, J, O, P). However, to achieve conditions similar to unperturbed cells, a combination of both ionic strength and macromolecular crowding is required. Panels E, F, G, K, L, M, Q, R, S show the autocorrelation analysis of images from nuclei treated with the varying conditions represented in the picture panels. The correlogram plots show measurements from chromatin of control nuclei (grey) and nuclei exposed to the different experimental conditions (black). Values above or below control correlogram, represents hyper-condensation or decondensation respectively. This analysis is sensitive enough to detect small variations, and may be used as a quantitative test for chromatin structure. The experimental conditions that best mimic the nuclear milieu are an ionic strength of 50% PBS and 5% Ficoll 400, as shown by the overlap of both correlograms (Panels I and L). Panels U and V show the chromatin distribution in nuclei incubated with 2 M NaCl. Most of the DNA is extruded from the nuclear interior (U) but when the same experiment was performed in presence of 10 % Ficoll (V) the chromatin integrity was maintained. Panels X and Y illustrate the chromatin distribution in nuclei treated with SDS (1%), which completely destroys nuclear integrity and massively decondenses the chromatin (X). Addition of 10% Ficoll (Y) was able to preserve chromatin integrity. Bar 5 μm.

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