Chromatin in a marine picoeukaryote is a disordered assemblage of nucleosomes

Abstract

Chromatin organization is central to many conserved biological processes, but it is generally unknown how the underlying nucleosomes are arranged in situ. Here, we have used electron cryotomography to study chromatin in the picoplankton Ostreococcus tauri, the smallest known free-living eukaryote. By visualizing the nucleosome densities directly, we find that O. tauri chromosomes do not arrange into discrete, compact bodies or any other higher level of order. In contrast to the textbook 30-nm fiber model, O. tauri chromatin resembles a disordered assemblage of nucleosomes akin to the polymer melt model. This disorganized nucleosome arrangement has important implications for potentially conserved functions in tiny eukaryotes such as the clustering of nonhomologous chromosomes at the kinetochore during mitosis and the independent regulation of closely positioned adjacent genes.

Fig. 1

O. tauri chromatin is not organized as 30-nm fibers. a Tomographic slice, 60 nm thick, through a cryosection of a mitotic O. tauri cell. The intranuclear spindle microtubules are not located in this slice. The nucleus (Nuc), chloroplast (Chl), mitochondrion (Mito), a granule (gr), and gold fiducials (Au) are labeled. A position including either chromatin (blue) or cytoplasmic ribosomes (red) was selected for Fourier analysis. The semiperiodic horizontal structures (most visible in the left side of the chloroplast) are crevasses from cryomicrotomy. b A 10-nm-thick tomographic slice corresponding to the black/white box in a, enlarged 3-fold and rotated 90° counterclockwise. A cytoplasmic ribosome is indicated by the arrow and an intranuclear nucleosome-sized density is indicated by the arrowhead. c Rotationally averaged amplitudes (log scale, arbitrary units) of the Fourier transform of the two color-coded positions boxed in a. Arrows point to the ~30-nm (1/30 nm⁻¹) (left) and 10-nm (1/10 nm⁻¹) (right) spatial frequencies. A 10-nm-thick tomographic slice corresponding to the blue box in a is shown, enlarged 3-fold, either d unmodified or e with a Gaussian-shaped bandpass filter centered at 10 nm (1/10 nm⁻¹). The arrowhead points to an example nucleosome-sized density. Note that image compression artifacts make the chromatin densities look smaller than in the original, uncompressed image

Fig. 2

Some nucleosomes appear to form small clusters. Tomographic slices (10 nm) showing examples of small nucleosome clusters (indicated by arrows) in an interphase cell (a–d) and a mitotic cell (e–h). Both the bandpass-filtered images (a, e) and the original lowpass-filtered images (b, f) are shown. The template-matching result for the 10-nm-thick volume is indicated by green circles overlaid on the tomographic densities (c, g) and alone (d, h). The diameter of the circle is related to the nucleosome’s “z” position within the subvolume: the largest circles denote nucleosomes centered within the subvolume, while the smallest circles denote nucleosomes centered above or below the subvolume. Some densities appear to be smaller than the nominal ~10-nm nucleosome diameter; these smaller densities may be the “tops” or “bottoms” of nucleosomes that are just within the 10-nm-thick volume. They may also result from the noise in the cryotomogram that produces some false-positive hits. The presence of some false-positives and false-negatives does not affect, however, the main conclusion that there are no discernable higher-order structures present

Fig. 3

O. tauri nucleosomes do not undergo large-scale reorganization in mitosis. Tomographic slices (1.3 nm thick) through the nucleus of an interphase (a–e) and a mitotic (f–j) O. tauri cell. To enhance the visualization of nucleosome-sized densities in such thin tomographic slices, the tomograms were bandpass-filtered as in Fig. 1d. Template-matching results showing 0.66× (b, g), 1× (c, h), and 1.5× (d, i) the nominal number of hits are shown as green circles superposed on the tomographic densities. Only those hits that are centered in the particular tomographic slice are circled; hits in slices “above” and “below” are not circled for clarity. Many of the ~10-nm densities are nucleosomes centered just below or above the current slice and therefore are not circled; in contrast, see Fig. 2. e, j 3D model of the centers of masses of all the nucleosome-like densities. Each sphere is ~8 nm wide

Fig. 4

Spindle microtubules reside in a nucleosome-depleted zone. a, b Tomographic slices (10 nm) of two mitotic O. tauri cells, taken at the spindle tunnel. The chromatin (Chr), mitochondrion (Mito), and chloroplast (Chl) are indicated. Each tomogram was rotated to a view along the longitudinal axis of the spindle microtubules (arrowhead), one of which is incomplete (b). As a result of the image rotation, crevasses (arrows) are visible in the right-hand side of b. Subvolumes containing the spindle microtubule(s) and the spindle tunnel are boxed and enlarged in c and d, corresponding to the cells (a) and (b), respectively. The boundary surrounding the nucleosome-depleted zone is delineated by the blue dotted line

Fig. 5

O. tauri chromatin is disorganized. Cartoon models of nucleosomes (blue disks) in O. tauri chromatin in a interphase and b mitosis. In textbook models (c), chromatin is universally presented using the 30-nm fiber; modeled from Scheffer et al. (2011). d Hypothetical model of O. tauri polymer melt chromatin in a mitotic cell, viewed along the spindle axis. Canonical nucleosomes (light blue spheres) and centromeric nucleosomes from nonhomologous chromosomes (multicolored spheres) are positioned around the spindle. Kinetochore protein complexes (lilac rods) connect the centromere to the spindle microtubules (green rings)