Fig. 1: Single-nucleosome imaging in mitotic HeLa cells. | Nature Communications

Fig. 1: Single-nucleosome imaging in mitotic HeLa cells.

From: Single-nucleosome imaging unveils that condensins and nucleosome–nucleosome interactions differentially constrain chromatin to organize mitotic chromosomes

Fig. 1

a Western blot of H2B-Halo expression in HeLa cells using the anti-HaloTag and anti-H2B antibody. Lane 1, parental HeLa cells; lane 2, H2B-Halo expressed HeLa cells. b HeLa cells expressing H2B-Halo labeled with HaloTag TMR ligand (center). (Left) DAPI-stained DNA. (Right) Merged image (DNA, cyan; H2B-Halo, magenta). Representative images of interphase nuclei (top) and mitotic chromosome spreads (bottom). Note that the TMR pattern is similar to DNA staining on interphase nuclei. c Enlarged image of the mitotic chromosomes in b (marked with the dot rectangle) showing a more uniform TMR labeling than DAPI, as depicted in the line plot below (DNA, cyan; TMR, magenta). A.U. means arbitrary unit. d A small fraction of H2B-Halo in mitotic chromosomes was sparsely labeled with the HaloTag fluorescent ligand (red). The labeled nucleosome movements can be tracked at super-resolution. e Scheme of oblique illumination microscopy. The illumination laser (green) excites fluorescent nucleosomes within a thin optical layer (red) of the mitotic cells to reduce background. f Single-nucleosome (H2B-Halo-TMR) images of interphase chromatin (left) or metaphase chromosomes (right) in a live HeLa cell after background subtraction. Also see Supplementary movies 1 and 2. g Two representative trajectories of nucleosome of interphase chromatin (left) or metaphase chromosomes (right). h MSD plot (±SD among cells) of TMR-labeled nucleosomes in live interphase (black, n = 20 cells), metaphase (magenta, n = 17 cells), apoptotic (blue, n = 19 cells), and FA-fixed (gray, n = 13 cells) HeLa cells from 0.05 to 0.5 s. MSD exponents from Supplementary Fig. 1f and asymmetry coefficient (AC) values from Supplementary Fig. 2b are also shown. The Kolmogorov-Smirnov test (two-sided) was used. ***, P < 0.0005, for interphase versus metaphase (P = 6.0 × 10−8), metaphase versus apoptosis (P = 4.2 × 10−9), and apoptosis versus FA-fixed (P = 1.2 × 10−4). i Schematic for angle-distribution analysis60,74. j Schematic for AC. See Methods for details. AC shows deviation from a homogeneous distribution and is negative for angular distributions, where pulling back force is dominant.

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