Figure S1: Male Human Fibroblast Nucleus (G0): Deconvolution 3D Microscopy, CT Classification, and 3D Reconstruction
Images selected from a stack of 20 light-optical serial sections. Sections from a fibroblast nucleus following chromosome painting with 24-color M-FISH were recorded by wide-field epifluorescence microscopy. Images from left to right show optical sections obtained from the bottom to the top of the nucleus.
(A) RGB images without deconvolution.
(B) RGB images after deconvolution.
(C) False color images after classification.
(D) Still shot from Video S1
, which shows the simulation of CT expansion in a fibroblast model nucleus according to the SCD model (compare with ).
(766 KB JPG).
Figure S2: Positions of IGCs of Each CT in G0 Human Fibroblast Nuclei
Each point represents the 2D projection of the ICG of an individual CT. The x- and y-half-axes of the ellipses shown represent the mean x- and y-half-axes of all the 54 nuclei evaluated. Each nucleus was rotated until its long axis fitted the abscissa of the coordinate system. The inner ellipsoid lines were drawn to facilitate the comparison of the ICG locations from different CTs.
(1.5 MB JPG).
Figure S3: Prometaphase Rosettes: Positions of IGCs of Each Chromosome
2D projections of the IGCs determined for all chromosome types (1–22, X, and Y) present in 28 prometaphase rosettes of male diploid human fibroblasts (compare to Figure S2
(1.2 MB JPG).
Figure S4: Cumulative Normalized 3D Radial Distances in Human Fibroblast Nuclei (G0)
Cumulative frequency of normalized 3D CN–CT distances for all chromosome types (1–22, X, and Y) measured in 54 G0 human fibroblast nuclei of the 2D projections shown in Figure S2
. The origin on the abscissa corresponds to the CN, and “1” corresponds to the maximum 3D distance from CN to any site of the nuclear periphery. Distances were sorted into ten classes representing ten concentric 3D nuclear shells. To define these shells, normalized 3D radii were subdivided into ten equal segments.
(919 KB JPG).
Figure S5: Prometaphase Rosette: Deconvolution 3D Microscopy, PC Classification, and 3D Reconstruction
Prometaphase rosette from a male fibroblast following chromosome painting with the 24-color M-FISH protocol.
(A–C) Typical sections selected from a stack of 20 light-optical serial sections obtained by wide-field epifluorescence microscopy (A) before deconvolution, (B) after deconvolution, and (C) after classification.
(D) 3D reconstruction from the entire stack of deconvoluted light-optical images.
(E) Mid-plane section recorded in eight channels. Note that the Cy7 channel was not analyzable in this experiment. As a consequence, Chromosomes 5 and 19 were both labeled solely by Spectrum Green, while Chromosomes 8 and 20 were labeled solely by Cy3. Despite these limitations, differences in size and labeling intensity allowed the classification of these PCs.
(876 KB JPG).
Figure S6: Cumulative Normalized 3D Radial Distances in Human Fibroblast Prometaphase Rosettes
Cumulative frequency plot of normalized 3D CR–PC distances for all chromosome types (1–22, X, and Y) measured in 28 rosettes of the 2D projections shown in Figure S3
. The origin on the abscissa corresponds to the CR, and “1” corresponds to the maximum 3D distance from CR to any site of the rosette periphery. Distances were sorted into ten classes as in Figure S4
(922 KB JPG).
Figure S7: Mean Normalized 3D CN–CT/CR–PC Distances and Standard Deviation of All Chromosome Types in Human Male Fibroblasts
Left: normalized mean radial 3D CN–CT distances from the 54 evaluated 3D fibroblast nuclei. Right: normalized mean radial 3D CR–PC distances of the 28 evaluated 3D prometaphase rosettes. SD, standard deviation.
(424 KB JPG).
Figure S8: Comparison of Radial CT Positions in Quiescent and Proliferating Fibroblasts
(A–C) Left: for quality control chromosome paint probes were hybridized to metaphase spreads from phytohemagglutinin-stimulated human lymphocytes. For example, shown are metaphase spreads after two-color 3D FISH with the following pairs of chromosome paint probes: (A) HSA 18 (red) and HSA 19 (green); (B) HSA 17 (green) and HSA Y (red); and (C) HSA 1 (red) and HSA 20 (green). Painted metaphase chromosomes show a rather homogeneous coverage, except for the centromere region, which remained unstained because of the signal suppression with Cot-1 DNA. Right: maximum intensity projections of confocal image stacks from selected nuclei in quiescent (G0) and proliferating (early S-phase) human male fibroblasts demonstrate the variability of CT positioning. CT colors are the same as described for metaphase chromosomes.
(D) To compare the voxel-based analysis of 3D radial CT 18 and CT 19 distributions in G0 and S-phase fibroblasts (see A and D) with an IGC-based 3D analysis, we allocated CT 18 IGCs and CT 19 IGCs to one of five concentric nuclear shells with equal volume. Shell 1 corresponds to the most peripheral shell. The data was analyzed in this way to compare with in Bridger et al [27
], except that 3D distances were used here. In both quiescent and S-phase nuclei a higher fraction of CT 19 IGCs was found in the two central shells 4 and 5 than for CT 18 IGCs. In contrast, in S-phase nuclei the fraction of CT 18 IGCs was larger in the more peripheral shells 1–3. In quiescent nuclei only shells 2 and 3 revealed a higher fraction of IGCs from HSA 18 CTs, while the fraction of IGCs from HSA 19 CTs was slightly higher in the most peripheral shell, shell 1. The results are consistent with a voxel-based approach (see ).
(E) Comparison of the median and the mean 3D CN–CT distances obtained for HSA 18 CTs and HSA 19 CTs in G0 and S-phase nuclei. A plus sign indicates a slight shift of CT 18 IGCs towards the nuclear periphery in early S-phase fibroblast nuclei compared to G0 nuclei, whereas a minus sign indicates a shift to the interior of the CT 19 IGCs. However the measured shifts were statistically not significant (ns).
(F) Median and mean normalized 3D CN–CT 18 distances were not significantly different from CN–CT 19 distances in G0 fibroblast nuclei, but showed a marginal difference (* p < 0.05) in early S-phase nuclei.
(1.3 MB JPG).
Figure S9: G0 Nuclei: Mean Angular Separation between 3D IGCs of Homologous CTs and the CN
CT–CN–CT angles were measured between the IGCs of homologous CTs and the center of the nucleus (CN) in 54 G0 fibroblast nuclei. Sample sizes (n) indicate the number of nuclei in which CT–CN–CT angles could be measured for a given pair of homologous CTs. The experimental distribution did not deviate from a normal distribution (p > 0.05; one-tailed K-S test of goodness of fit). With few exceptions pairwise comparisons of the mean angular separation between a pair of homologous CTs with the respective mean angle distribution in 60 random point distribution model nuclei did not show a significant difference (p > 0.05; two-tailed K-S test). Significant differences (p < 0.05) are indicated by an asterisk. The comparison of mean angular separation of homologous CTs with statistically placed homologous CTs in 50 SCD model nuclei also did not reveal significant differences except for 4-CN–4 and 20-CN–20 angles, which were smaller in the experiment than in the model (**p < 0.01).
(503 KB JPG).
Figure S10: Prometaphase Rosettes: Mean Angular Separation between 3D IGCs of Homologs and the CR
PC–CR–PC angles between the IGCs of homologous PCs and the rosette center (CR) measured in 28 prometaphase rosettes. No significant difference was detected in comparison with angular separations found in the random point distribution model (p > 0.05; two-tailed K-S test).
(426 KB JPG).
Figure S11: Significance Levels for Pairwise Comparisons between Heterologous 3D CT–CN–CT Angles in 54 G0 Fibroblast Nuclei
Significance levels were determined by the two-tailed K-S test. Green, not significant, p > 0.05; yellow, p < 0.05; red, p < 0.01. Minus/plus signs in a colored field indicate that the chromosome pair given on the left shows a significantly shorter/greater mean radial distance than the chromosome pair presented at the top.
(A) Large chromosomes: HSAs 1–5.
(B) Small, acrocentric chromosomes: HSAs 13, 14, 15, 21, and 22.
(C) Other small chromosomes: HSAs 16–20.
(292 KB JPG).
Figure S12: Arrangements of HSA 7 and HSA 8 CTs in 50 Fibroblast Nuclei
Nuclei of G0 fibroblasts were subjected to 3D FISH with painting probes for HSA 7 and HSA 8, labeled with dUTP-Cy3 (green) and dUTP-FITC (red), respectively. Maximum intensity projections of confocal image stacks from 50 scanned nuclei are shown to demonstrate the variability of proximity patterns (for quantitative measurements see Figure S13
(870 KB JPG).
Figure S13: Quantitative Evaluation of the Angular Separation of Homologous and Heterologous Pairs of HSA 7 and HSA 8 CTs in 50 Fibroblast Nuclei
Upper left: Ellipses represent the normalized 2D shape of the nuclei and show 2D projections of the radial IGC locations of HSA 7 and HSA 8 CTs. Upper right: The IGCs of CT 7 and CT 8 pairs, respectively, were rotated around the nuclear center until one IGC lay on the positive abscissa (closed circles). Open circles show the IGC position of the corresponding homolog. Below are the mean 3D CT–CN–CT angles between homologous and heterologous HSA 7 and HSA 8 CTs, their ranges, and their standard deviations. Angles between homologous and heterologous pairs showed a normal distribution. Comparisons of experimental data with mean 3D angle distributions in the random point distribution model or SCD model did not show a significant difference (p > 0.05; two-tailed K-S test).
(328 KB JPG).
Video S1: Model Nucleus: CT Simulation
The video shows the simulation of CT expansion in a fibroblast model nucleus according to the SCD model (compare with ).
(567 KB MPG).