Three-dimensional ultrastructure of Saccharomyces cerevisiae meiotic spindles - PubMed (original) (raw)

Three-dimensional ultrastructure of Saccharomyces cerevisiae meiotic spindles

Mark Winey et al. Mol Biol Cell. 2005 Mar.

Abstract

Meiotic chromosome segregation leads to the production of haploid germ cells. During meiosis I (MI), the paired homologous chromosomes are separated. Meiosis II (MII) segregation leads to the separation of paired sister chromatids. In the budding yeast Saccharomyces cerevisiae, both of these divisions take place in a single nucleus, giving rise to the four-spored ascus. We have modeled the microtubules in 20 MI and 15 MII spindles by using reconstruction from electron micrographs of serially sectioned meiotic cells. Meiotic spindles contain more microtubules than their mitotic counterparts, with the highest number in MI spindles. It is possible to differentiate between MI versus MII spindles based on microtubule numbers and organization. Similar to mitotic spindles, kinetochores in either MI or MII are attached by a single microtubule. The models indicate that the kinetochores of paired homologous chromosomes in MI or sister chromatids in MII are separated at metaphase, similar to mitotic cells. Examination of both MI and MII spindles reveals that anaphase A likely occurs in addition to anaphase B and that these movements are concurrent. This analysis offers a structural basis for considering meiotic segregation in yeast and for the analysis of mutants defective in this process.

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Figures

Figure 1.

Electron micrographs of HPF/FS prepared meiotic yeast cells. MI (A and B) and MII (C and D) spindles are shown in serial longitudinal sections. Arrows indicate SPBs (A and C), and the enhanced outer plaque on the SPB of a MII spindle is indicated in C. Images of spindle cross sections (E and F) show typical microtubule cross sections (E, arrow) and show more oblique microtubule profiles (F, arrow) that had to be traced by hand. Bar, 0.2 μm.

Figure 2.

Meiotic spindle length distribution from fluorescent microscopy (FM) images of cells with GFP-tagged Tub1p compared with those from 3-D reconstruction from EMs. The spindle lengths were determined as described in Materials and Methods and are reported in 1.5-μm bins. The spindle lengths are reported separately for MI (A) and MII (B) and are reported as percent of total number of spindles. The number of spindles in the data set includes 41 MI spindles and 56 MII spindles identified by immunofluorescence microscopy, and 20 MI spindles and 15 MII spindles identified by electron microscopy (see Table 1).

Figure 3.

General description of the microtubules in MI (○) and MII (×) spindles derived from the 35 meiotic spindles (see Table 1) as described in Materials and Methods. (A) The total number of microtubules versus spindle length. (B) The microtubule total polymer versus spindle length. (C) Mean microtubule length versus spindle length. Spindles of similar length were grouped together. Error bars represent 74% confidence interval.

Figure 4.

Representative meiotic spindle models (models MSI-9 and MSII-11; see Table 1). (A) The 3-D model of the microtubules in the MI spindle MSI-9. The supplemental data includes this image in a movie where the microtubules are color coded, allowing one to roll the model around the spindle axis. The supplemental data contains similar movies for all of the models and movies of parts of the EM data sets used to derive the models. (B) The 3-D model of the microtubules in the MII spindle MSII-11. The position of a spindle cross section (for 1 serial section of 50 nm) shown in F and G is indicated by a box (∼40 sections made up the spindle). This spindle model is deconstructed into putative kinetochore microtubules (C) and the core spindle microtubules (D) as determined in Figure 6 (also see text). (E) A microtubule overlap graph showing the length of each microtubule from one SPB or the other with two microtubules at the bottom whose polarity cannot be determined (“continuous microtubules”; see text). The microtubule overlap graphs for all spindle models are in the supplemental data. (F and G) Representation of the positions of microtubules in a single cross-section of spindle MSII-11 (position shown in B) where the polarity of the microtubules is indicated by a circle for microtubules from one SPB, a triangle for microtubules from the other SPB, and squares for microtubules that are continuous. (F) Neighbor density analysis used in Figure 7 is done by drawing concentric circles (gray) of user-defined radii (20 nm) around each microtubule and recording the number of microtubules in each ring created by the circles. (G) The outermost microtubules are used to define a polygon whose area is the cross sectional area for this section of the spindle. These areas are multiplied by section thickness to determine a volume, and the volumes are summed for all of the sections yielding the spindle volume (see Figure 5).

Figure 5.

Spindle volumes distinguish MI (○) from MII (×) spindles. Spindle volumes were determined as described in Figure 4 and volumes of spindles of similar length were grouped together. Average spindle volumes are plotted versus spindle length. Error bars represent 74% confidence interval.

Figure 6.

Putative kinetochore microtubules seem to be the shortest microtubules and to have the least amount of pairing between microtubules in the spindle models. Histograms of the number of microtubules per spindle sorted by the ratio of microtubule length to spindle length for MI spindles (A) and MII (B) spindles normalizes microtubule lengths across all of the spindle models and reveals two classes of microtubules, where the relatively short microtubules (≤0.5 of spindle length) are thought to be kinetochore microtubules (see text). Histograms of the number of microtubules per spindle sorted by microtubule lengths for MI spindles (C) and MII spindles (D). The shaded areas indicate microtubules with significant pairing (lengths of ≥0.2 μm in MI spindles and of >0.3 μm in MII spindles) with microtubules from the opposing SPB. Here again, the shorter microtubules without significant pairing (unshaded) seem to be kinetochore microtubules and number very near 32 per spindle (1 per kinetochore; see text).

Figure 7.

Antiparallel microtubules in the middle of MII spindles show a preferred packing distance. Neighbor density analysis (see Figure 4F) of spindle midzone microtubules was examined in three size classes of MII spindles (1.47–1.62 μm, A and B; 1.7–2.0, C and D; >2.0 μm, E and F) and plotted as histograms showing the density of microtubules (_x_-axis) at given distances from the reference microtubule (_y_-axis, the reference microtubule is the one in the center in the Figure 4F schematic). The data for each microtubule in the models being used as the reference microtubule is summed. No significant preferred packing was detected between microtubules from the same SPB (parallel) in the shorter spindles (A and C), but a peak of ∼35 nm was seen in the longest spindles (E). A preferred distance of ∼40 nm is observed between microtubules from opposite SPBs (antiparallel) at all spindle lengths (B, D, and F). This distance of 40 nm is particularly apparent in longer spindle lengths (D and F). Such organization is not apparent in MI spindles (see text; our unpublished data).

Figure 8.

Apparent kinetochore microtubules diminish in length as MII proceeds indicating anaphase A movements. Plotting the mean length of noncore spindle microtubules in different spindle length classes versus spindle length shows that these microtubules shorten during MII (×), but not during MI (○). This analysis can be biased by a population of stable noncore microtubules, or by the loss (below detection) of microtubules from the spindle models (see text). Error bars represent 74% confidence interval.

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Three-dimensional ultrastructure of Saccharomyces cerevisiae meiotic spindles - PubMed (original) (raw)