Conserved and divergent features of kinetochores and spindle microtubule ends from five species - PubMed (original) (raw)
Conserved and divergent features of kinetochores and spindle microtubule ends from five species
J Richard McIntosh et al. J Cell Biol. 2013.
Abstract
Interfaces between spindle microtubules and kinetochores were examined in diverse species by electron tomography and image analysis. Overall structures were conserved in a mammal, an alga, a nematode, and two kinds of yeasts; all lacked dense outer plates, and most kinetochore microtubule ends flared into curved protofilaments that were connected to chromatin by slender fibrils. Analyses of curvature on >8,500 protofilaments showed that all classes of spindle microtubules displayed some flaring protofilaments, including those growing in the anaphase interzone. Curved protofilaments on anaphase kinetochore microtubules were no more flared than their metaphase counterparts, but they were longer. Flaring protofilaments in budding yeasts were linked by fibrils to densities that resembled nucleosomes; these are probably the yeast kinetochores. Analogous densities in fission yeast were larger and less well-defined, but both yeasts showed ring- or partial ring-shaped structures girding their kinetochore microtubules. Flaring protofilaments linked to chromatin are well placed to exert force on chromosomes, assuring stable attachment and reliable anaphase segregation.
Figures
Figure 1.
Structural variety among the kinetochore–MT interfaces studied here. (A) Diagram of a mammalian chromosome (purple) and its kinetochore (K; orange disk) interacting with KMTs (dark green), which connect it with a spindle pole (yellow disk). KMT numbers are from McEwen et al. (1998a). Spindles also contain MTs that do not encounter kinetochores (non-KMTs; light green). (B) 4-nm slice from a tomogram of a kinetochore from a metaphase NRK cell. (C–F) Diagrams of the kinetochores analyzed in this paper. Images are colored as in A; the structures and numbers indicated are based on this paper,
Figs. S1–S4
.
Figure 2.
Tomographic slices of the spindles studied here. (A) 10-nm slice from a serial tomogram of a C. reinhardtii metaphase (CrM1, see
Fig. S1
for model). Chromosomes (CH), the nuclear envelope (NE), and an opening in the envelope (PO) through which MTs project toward the spindle pole are evident. (A′) 4-nm slice through a different metaphase spindle (CrM2 in Fig. S1). One MT ends near a chromosome (a KMT) but others continue. The arrow indicates a flared PF. (B) 10-nm slice from a serial tomogram of a one-cell embryo of C. elegans. Chromosomes (CH) are evident, and a centriole (arrow) marks the spindle pole. (B′) 4-nm slice through the chromosome–MT interface of a different C. elegans metaphase (CeM in
Fig. S2
). Chromatin, at left, is separated by a fibrous zone from the KMTs with flaring plus ends. No outer plate is seen. (C) 10-nm slice from a tomogram of a metaphase budding yeast (ScM1,
Fig. S3
); SPB, spindle pole body; NE, nuclear envelope. (C′ and C″) Two 4-nm slices from a prometaphase budding yeast (ScPM1); slender filaments connect the capped MT minus ends with the spindle pole. (D) 10-nm slice from a prometaphase fission yeast (SpPM in
Fig. S4 C
); SPB and the NE are evident, but chromosomes are not. (D′) A 4-nm slice from the same cell. Some MTs end in pairs where the texture of nucleoplasm is different (CH). Such MTs have been interpreted as KMTs. Bars: (A) 0.5 µm; (A′) 200 nm; (B) 1 µm; (B′) 100 nm; (C) 0.5 µm; (C′ and C″) 100 nm; (D) 0.5 µm; (D′) 100 nm.
Figure 3.
Galleries of spindle MT plus ends. (A) 4-nm slices that contain the ends of four algal metaphase KMTs. In all panels of this figure, arrows indicate flaring PFs and arrowheads demark fibrils that run from the PFs to nearby chromatin. (A′) Analogous images of non-KMTs. (B) 4-nm slices of four metaphase KMTs from C. elegans. (B′) Analogous images of non-MTs. (C) 4-nm slices from the plus ends of KMTs from S. cerevisiae. Here, no chromatin is evident, but there are small circular densities not far from each MT end. (C′) Budding yeast non-KMTs. Leftmost is the plus end of an “astral” MT that projected from the spindle pole body into the cytoplasm. The center and right images are from ipMTs in the anaphase interzone. (D and D′) 4-nm slices of KMTs and non-KMTs from fission yeast. The electron densities near the tips of the KMTs in D are suggestive of chromatin, but a well-formed chromosome is not seen. The non-KMTs of D′ are pole-distal ends of MTs that diverged widely from the spindle axis (two leftmost images) and from MTs that ended in the anaphase interzone (two rightmost images). Bars: (A) 100 nm; (A′) 75 nm; (B) 100 nm; (B′) 50 nm; (C and C′) 50 nm; (D and D′) 50 nm.
Figure 4.
Analysis of PF curvatures. (A) Tracings of all visible PFs from all the KMTs imaged in one algal metaphase spindle. The dark line displays the mean position of these PFs as a function of distance from the MT wall. Error bars indicate SEMs. (B) Average shapes of KMT PFs from PtK1 cells (data are from McIntosh et al. [2008] whenever PtK1 numbers are given) and the species described here, all in metaphase. (C) Mean local curvatures, measured as described in Materials and methods, based on circles fit to 10 adjacent points at each position along every PF (inset). KMT PFs from all organisms are compared, colors are as in B. (D) Diagram showing how PF slopes were determined for sorting. (E) Metaphase KMT PFs from a C. reinhardtii metaphase sorted by their slope in the region between the two green vertical lines (see Materials and methods). The lower cluster displays the “ram’s horns,” the upper cluster displays the “intermediate” PFs. There were no “short/blunt” or “extension” PFs in this cell. (F) Fractions of KMT PFs from each organism that fall into the four categories defined in Materials and methods. Comparisons include MTs polymerizing (P) or depolymerizing (D) in vitro, as characterized in McIntosh et al. (2008).
Figure 5.
Conserved and divergent features of PF curvatures. (A) Average shapes of KMT ram’s horn PFs from PtK1 and budding yeast compared with analogous PFs from MTs depolymerizing in vitro (D). (B) Mean local curvatures as a function of distance from the MT wall for all ram’s horn PFs from the KMTs of each species studied versus those from polymerizing (P-MTs) and depolymerizing MTs (D-MTs) in vitro. Colors are as in Fig. 4 B, both here and in C and G. (C) Analogous graph of the “intermediate” PFs from all species. (D) Comparison of mean local curvatures for intermediate PFs from nematode KMTs in metaphase and anaphase versus from non-KMTs. (E) Histograms of mean curvatures of intermediate PFs from KMTs 16–23 nm from the MT wall. (F) Local PF curvatures for three classes of MTs from S. pombe. Curvatures of KMTs and non-KMT PFs are similar, but ipMTs are different, both visually and in a test for significance (asterisks) using the unpaired Student’s t test with 95% confidence intervals. (G) Average local curvatures of all PF classes for non-KMTs compared with C. (H) Lengths of PFs versus fibrils in all species studied. For each new species, 100 fibrils were measured on each of two metaphase cells; the numbers of PFs studied are as in Table 1. Error bars indicate SEMs.
Figure 6.
Structures of averaged plus ends. The program PEET was used to align and average multiple KMT plus ends from each cell (number and kind of MTs plus cell name are given in black for each picture). Averaged structures were sampled with Slicer by cutting a lamina that contained the MT axis at an angle specified in degrees (white in each image). Orientations were selected to show fibrous connections between the averaged MT end and nearby chromatin (arrowheads). (A) Algal KMT shown with the mask used to occlude non-MT features during alignment. (A′) Leftmost is the slice of the reference volume used for MT alignment. Others panels show stages in averaging as more MTs are added. All images are slices at +1° (number in white). Arrowheads indicate a fibril that becomes more distinct with averaging. Bars, 100 nm. (B) Slices from the same averaged algal KMT at different angles. Arrowheads indicate apparent fibrils on all panels. (B′) Analogous images cut at angles shown in white from averaged KMTs from a different metaphase algal cell. (B″) Similar averages of MTs ending far from the chromatin in CrM1. No fibers are seen. (C) Averages from KMTs of one C. elegans metaphase (C) and one anaphase (C′). (C″) Averages of non-KMTs from blastomere spindles.
Figure 7.
Structures of averaged plus ends from yeast spindle MTs. (A) A slice from the single KMT (left) used as an alignment reference. No mask was used. Cell name and number of MTs averaged are shown in black. Slices are all at −43°. Strong development of image features is seen, including both a puck-shaped structure ∼55 nm from the MT tip and fibrils connecting this with flaring PFs. (B) Averages of KMTs from the S. cerevisiae cell named under each panel. Numbers of MTs are averaged at the top, the angle of the sampling slice is shown in white, and fibrils are indicated with arrowheads. Fibers associated with bending PFs are again visible, but not on an ipMT (B′). (C) Averages of KMTs from S. pombe. Fibers are again evident in averages (arrowheads), but they are oriented at diverse angles, perhaps because of the size of the fission yeast kinetochore. A mass of ill-defined shape ∼40 nm from the MT tip accompanies all KMTs (arrows) not ipMTs (C′). (D) PEET averages of particles from different regions of budding yeast nuclei. In each part of the figure, the left image is a slice cut through the average, where the right is a surface rendering. (D) Average of 100 particles far from the spindle, which we interpret as nucleosomes. (D′) Average of 64 particles near the plus ends of spindle MTs in a diploid prometaphase. These we interpret as kinetochores. (D″) Similar average of 58 particles from two haploid prometaphases. (D‴) Similar average from 68 particles from three haploid metaphases. Bars: (A–C) 50 nm; (D) 22 nm.
Figure 8.
Ring-shaped structures on KMTs in vivo. (A) MT polymerized in vitro from bovine tubulin, then mixed with recombinant Dam1 and negatively stained. (B) Negatively stained Dam1 rings. (B1) Unpublished image of the Dam1 ring (arrowhead) surrounding an MT (arrow), courtesy of E. Nogales (personal communication). (B2 and B3) Single (B2) and average of multiple (B3) negatively stained Dam1 rings in isolation (Wang et al. [2007], with permission from Nature Structural & Molecular Biology). (C) 4-nm slices cut from single KMTs and from averaged KMTs (labeled “Avg.,” then the number of MTs was averaged) from three budding yeast cells. Bottom panels are cross sections at the positions marked by black plusses in the top panels. The arrow indicates the MT wall in cross section; arrowheads indicate the ring and partial rings surrounding the MTs. (D) Comparable images for KMTs from fission yeast. (E) Localization of the S. pombe Dam1 complex, triply tagged with GFP, was accomplished with anti-GFP and a secondary anti-rabbit Fab coupled to 5 nm colloidal gold (arrowheads). The top image is a tomographic slice showing the stain and MT in the same slice. The bottom two images show a different tomogram; the top image slices the section’s surface where the antibody is bound (arrowheads), and the bottom image shows the MT in perfect alignment with the top image, so the position of the gold can be identified. Bars: (A) 100 nm; (B) 25 nm; (C and D) 50 nm; (E) 75 nm.
Figure 9.
Models for chromosome–KMT attachment. (A) Diagram of customary views about kinetochore–MT interaction: kinetochore plates are linked to KMTs via connections with the MT wall. Some models see the outer plate as a sleeve that can diffuse on the MT wall. (B) Diagram based on our results showing no plate-like organization and a majority of connections running from the chromosome to the PFs at the KMT end.
References
- Bajer A.S., Mole-Bajer J. 1972. Spindle dynamics and chromosome movements. International Review of Cytology, Supplement 3. Academic Press, New York: 271 pp
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