Mol Biol Cell
January 1, 2016;
Heterogeneous architecture of vertebrate kinetochores revealed by three-dimensional superresolution fluorescence microscopy.
is often depicted as having a disk-like architecture in which the outer layer of proteins, which engage microtubules and control checkpoint signaling, are built on a static inner layer directly linked to CENP-A chromatin. Here, applying three-dimensional (3D) structural illumination microscopy (SIM) and stochastic optical reconstruction microscopy (STORM) to Xenopus egg
extracts and tissue
culture cells, we report various distribution patterns of inner and outer kinetochore
proteins. In egg
extracts, a configuration in which outer kinetochore
proteins surround the periphery of CENP-A chromatin is common, forming an ∼200-nm ring-like organization that may engage a bundle of microtubule
ends. Similar rings are observed in Xenopus tissue
culture cells at a lower frequency but are enriched in conditions in which the spindle
is disorganized. Although rings are rare in human cells, the distribution of both inner and outer kinetochore
proteins elongates in the absence of microtubule
attachment in a manner dependent on Aurora B. We propose a model in which the 3D organization of both the outer and inner kinetochore
regions respond to the progression from lateral
to end-on microtubule
attachments by coalescing into a tight disk from less uniform distributions early in prometaphase.
Mol Biol Cell
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FIGURE 1:. Superresolution imaging of kinetochore proteins in Xenopus egg extracts. (A) Bipolar spindle assembled in Xenopus egg extract, imaged using conventional wide-field deconvolution microscopy. Scale bar, 1 μm. (B) Comparison of images of the same spindle shown in A produced using conventional deconvolution (left) and SIM (middle). Scale bars, 1 μm. Higher-magnification images of single kinetochores shown with intensity scaling from 0 to the maximum value in each image; scale bars, 0.2 μm. Line scans corresponding to the dotted yellow line in each are shown below. (C) Kinetochores from samples as in A and B imaged using direct STORM. (D) Bub1 (magenta) and CENP-A (green) on a metaphase spindle. Images were produced using conventional deconvolution (left) and SIM (right). Scale bars, 1 μm. (E) Intensity plots for the yellow dotted lines in D in which values are normalized separately to 100 for the maximum intensity in each channel.
FIGURE 2:. Systematic imaging of kinetochore proteins in Xenopus egg extracts. (A) Schematic of Xenopus kinetochore components emphasizing their location relative to the centromere (inner) and microtubule (outer). (B) SIM images of pairs of sister kinetochores, indicated as inner and outer kinetochore components (green), costained with Bub1 or BubR1 (magenta). Scale bars, 0.2 μm. (C) Line scans of kinetochores with ring-shaped BubR1 stained as in B. Top, overlays; bottom, average intensity. Average BubR1 line scan from Zwint-stained kinetochores. (D) Ring diameters calculated by interpolating individual line scans from C using cubic splines and finding the distances between the two intensity peaks. Each point represents an independent diameter measurement of one kinetochore; mean and SD are shown in black. Mann–Whitney test: ***p < 0.0001; ns, p = 0.4 (n = 30, 48, 48, and 90 for Zwint, Ndc80, CENP-E, and BubR1, respectively). (E) Kinetochores stained for Ndc80 and imaged with STORM (left; scale bar, 0.2 μm); individual (middle) and average (right) line scans generated for ring-shaped kinetochores.
FIGURE 3:. Kinetochore rings may engage a hollow bundle of microtubules but do not depend on microtubules. (A, B) Spindles assembled in the presence of Alexa 488–labeled EB1 and incubated on ice for 2 min (scale bar, 1 μm). Pairs of sister kinetochores are shown at higher magnification (scale bars, 0.2 μm), with locations indicated by white boxes. (C, D) Mitotic chromosomes assembled in extract treated with 33 mM nocodazole to depolymerize all microtubules (scale bar, 1 μm). Pairs of sister kinetochores are shown at higher magnification (scale bars, 0.2 μm), with locations indicated by arrowheads. All images are maximum-intensity projections of SIM data. (E) Lines scans of ring-shaped Ndc80 signals in nocodazole-treated extract. Top, overlays; bottom, average intensity.
FIGURE 4:. Diverse kinetochore configurations, including rings, are also seen in Xenopus cells. (A, B) Xenopus A6 kidney cells were synchronized using RO-3306, released into medium containing MG132, and processed for immunofluorescence and classified based on spindle morphology (scale bars, 1 μm). Single kinetochores and adjacent microtubules are shown at higher magnification (scale bars, 0.2 μm). Locations of single kinetochores are indicated by numbered yellow arrowheads. (C) The 1-μm line scans of individual kinetochores were overlaid (left) or averaged (right) to illustrate the difference in the ring-like kinetochore configurations seen in disorganized and monopolar spindles. (D) Quantification of kinetochore shapes in Xenopus extract and A6 cells. (E) Xenopus A6 cells were treated with monastrol and processed for immunofluorescence (scale bar, 1 μm). Locations of higher-magnification images of kinetochores are indicated by numbered yellow boxes. (F, G) Higher-magnification images of CENP-A and Bub1 staining in sister kinetochores (scale bars, 0.2 μm); line scan indicated by the dotted yellow line in G. All images are maximum-intensity projections of SIM data.
FIGURE 5:. Centromere and core kinetochore configurations are both sensitive to microtubule attachment status. (A) hTERT-RPE1 cells in prometaphase and metaphase. Locations of higher- magnification images of sister kinetochore pairs (scale bars, 0.2 μm) indicated by boxes. (B) Quantification of kinetochore shapes in human cells and Xenopus (C–E) hTERT-RPE1 cells arrested in mitosis for 4 h in the presence or absence of nocodazole and ZM447439 (scale bars, 1 μm). Locations of higher-magnification images of sister kinetochore pairs (scale bars, 0.2 μm) indicated by boxes. Quantification of CENP-C foci lengths from nuclei in C is shown in D.
Ando, CENP-A, -B, and -C chromatin complex that contains the I-type alpha-satellite array constitutes the prekinetochore in HeLa cells. 2002, Pubmed