Organization of the smallest eukaryotic spindle

Abstract

In metazoans, plants, and fungi, the spindle checkpoint delays mitosis until each chromosome is attached to one or more of its own kinetochore microtubules (kMTs). Some unicellular eukaryotes, however, have been reported to have fewer kMTs than chromosomes [1-5]. If this is the case, it is unclear how the spindle checkpoint could be satisfied. In the vast majority of the previous studies, mitotic cells were chemically fixed at room temperature, but this does not always preserve dynamic and/or small structures like spindle MTs and kinetochores [6]. Indeed, later higher-resolution studies have reversed some earlier claims [7-11]. Here we show that in Ostreococcus tauri (the smallest eukaryote known), mitosis does involve fewer spindle microtubules than chromosomes. O. tauri cultures were enriched for mitotic cells, high-pressure frozen, and then imaged in 3D both in plastic and in a near-native ("frozen-hydrated") state through electron tomography. Mitotic cells have a distinctive intranuclear heterochromatin-free "spindle tunnel" with approximately four short and occasionally one long, incomplete (unclosed) microtubule at each end of the spindle tunnel. Because other aspects of O. tauri's spindle checkpoint seem typical, these data suggest that O. tauri's 20 chromosomes are physically linked and segregated as just one or a small number of groups.

Figure 1. O. tauri has only a few spindle microtubules

(A–D) Tomographic slices (19 nm thick) through merged serial-section tomograms of MG132-treated, high-pressure frozen, freeze-substituted O. tauri cells. The entire nuclei and spindles are reconstructed in the merged tomogram. The cells in columns A-C were sectioned nearly transverse to the spindle tunnel, while the cell in column D nearly longitudinal. Rows 1 –3 show tomographic slices through upper, middle, and lower plastic sections of the nucleus. Row 4 shows enlargements of the areas boxed in white in Rows 1 or 2, with small rotations out of plane to enhance the visibility of the spindle MTs. Arrows in row 4 indicate example MTs. Row 5 shows 3-D segmentations of the chromatin boundary (blue), spindle tunnel (arrowheads), and MTs (green). Note that the tunnel-spanning portion of long MTs have incomplete walls and are modeled as thin rods instead of tubes. The cell in (B) is shown in greater detail in Movie S1.

Figure 2. O. tauri spindle microtubules are short and mostly incomplete

(A) Tomographic slices (20 nm thick) through cryotomograms of vitreously sectioned MG132-treated cells. (B) Enlarged view of the spindle MT clusters boxed in (A). The right subpanel shows an longitudinal slice of the MT indicated by the arrowhead, taken through the center of the MT barrel (left cell) or along three protofilaments (right cell). (C, D) Segmentations of the spindle MTs in (B), colored randomly for clarity. The apparent "holes" in the MT walls are likely artifacts of the low signal-to-noise.

Figure 3. Example microtubules from cryosectioned cells

Tomographic slices (10 or 20 nm thick) through many MTs taken perpendicular (upper subpanels) and parallel (lower subpanels) to the MT axis. MTs 14 and 26 are complete with 13-protofilaments at one end (top subpanel) but not the other (lower subpanels with black border). In contrast to cytoplasmic MTs, which have long complete walls, most of the intranuclear (spindle) MTs are short and C-shaped along most of their length. MTs 10, 19, and 26 had one conical and one open end; MTs 14 and 20 have two open ends. Some MTs were only ~50 nm long (MTs 10, 19, and 20), which would consist of just a few α-β tubulin dimers (8-nm long). O. tauri spindle MTs are in general short with C-shaped profiles along most of their length, which is in agreement with the short MTs observed in the plastic-sectioned cells. See Table S2 for additional example MTs.

Figure 4. Organization of the O. tauri spindle

Scale model of the O. tauri spindle in the context of the chromatin. A small bundle of incomplete MTs (green) reside at each pole, and at most one long incomplete MT extends deep into the nucleus from each pole. Heterochromatin (blue) forms a torus-like structure with a central channel that we call the spindle tunnel. Individual chromosomes could not be resolved. The nuclear envelope (not shown) has openings at both spindle poles.