Volume 137, Issue 4, Pages 672-684 (May 2009) Protein Architecture of the Human Kinetochore Microtubule Attachment Site  Xiaohu Wan, Ryan P. O'Quinn, Heather L. Pierce, Ajit P. Joglekar, Walt E. Gall, Jennifer G. DeLuca, Christopher W. Carroll, Song-Tao Liu, Tim J. Yen, Bruce F. McEwen, P. Todd Stukenberg, Arshad Desai, E.D. Salmon  Cell  Volume 137, Issue 4, Pages 672-684 (May 2009) DOI: 10.1016/j.cell.2009.03.035 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Delta Measurements for the Ndc80 Complex (A) Molecular structure of the tetrameric Ndc80 complex. (B) Images of metaphase HeLa cells fixed and stained for Hec1-9G3 (red) and anti-Spc24 (green). Inset: higher-magnification view of red/green sister kinetochore pair denoted by arrow. The scale bars represent 1.1 μm. (C) Schematic of a pair of sister kinetochores at metaphase with one kinetochore expanded to show Airy disk images of 9G3 and anti-Spc24 labels and Delta, the distance between their centroids along the inner-outer kinetochore axis. (D) Method for calculation of Delta that eliminates, locally, errors from lateral chromatic aberration in microscope optics. (E) Average Delta measurements for different label pairs along the Ndc80 complex. Cell 2009 137, 672-684DOI: (10.1016/j.cell.2009.03.035) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 Correlation of Delta Values with Centromere Stretch: The Ndc80 Complex Is Not Compliant and Maintains a Constant Shape (A) Schematic of sister kinetochore pairs in different mechanical states (top); predictions of Delta measurements for noncompliant (middle) and compliant (bottom) protein linkages. (B) Delta values of separations between Hec1-9G3 and antibody to Spc24 C terminus across the entire range of K-K distances. (C) Delta values for 9G3 anti-Hec1 labeling with a mixture of red/green fluorescent secondary antibodies are individually plotted versus K-K distance. For (B) and (C), measurements are insensitive to K-K separation. (D) Delta values of separations between Hec1-9G3 and GFP-CENP-A across the entire range of K-K distances show oscillation compliance. The whole data set of Delta values uncorrected for tilt are shown; average Deltas are within 1–2 nm of corrected values (Table S1). Cell 2009 137, 672-684DOI: (10.1016/j.cell.2009.03.035) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 Summary of Delta Measurements in Control and Taxol-Treated HeLa Cells Summary of Delta measurements for 19 epitopes in 16 kinetochore proteins in control cells (left) and taxol-treated cells (right). The scale (red) on the far right is set equal to zero at the position of the Hec1-9G3 centroid; positive values are outward (toward the spindle MTs), while negative values are inward (toward the centromeric chromatin). Color-coded boxes indicate complexes. Colored dotted lines indicate proposed “arms” of the structural kinetochore. Black dots indicate average Delta values corrected for tilt. Vertical lines indicate minimum and maximum Delta values measured during oscillations in centromere stretch—for most linkages that do not show significant compliance, the vertical lines do not extend beyond the symbol used to indicate the average. Cell 2009 137, 672-684DOI: (10.1016/j.cell.2009.03.035) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 Changes in Kinetochore Architecture Associated with Taxol Treatment (A) Images of a metaphase HeLa cell treated with 10 μM taxol and then fixed and stained for Hec1-9G3 (red) and anti-Spc24 (green). Inset: higher-magnification view of red/green sister kinetochore pair denoted by arrow. The scale bars represent 1.1 μm. (B) Graph of measured Delta value versus K-K distance for the Hec1 and Spc24 labels in untreated (red) and taxol-treated (green) cells. (C) Graph of measured Delta value versus K-K distance for the Hec1 label versus GFP-CENP-A (top) and antibodies to CENP-I (middle), and hKnl1 (bottom) in untreated (red) and taxol-treated (green) cells. The whole data set of Delta values uncorrected for tilt is shown; the average Deltas are within 1–2 nm of corrected values (Table S1). Cell 2009 137, 672-684DOI: (10.1016/j.cell.2009.03.035) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 A Two-Color Method for Locating the Plus Ends of kMTs Relative to the Hec1 Head in Cooled PtK2 Cells (A) Fluorescent image of PtK2 cells stably expressing GFP-α-tubulin cooled to 6°C, fixed, and stained with the Hec1-9G3 antibody and a red fluorescent secondary. The image shows a kinetochore fiber and its kinetochore in the same focal plane. At right is a magnified image of the boxed region, showing how line scans were drawn down centers of the fibers through the center of Hec1-9G3 fluorescence. (B) Sample line scan of (A) showing GFP-α-tubulin intensity (green) and Hec1-9G3 (red) fluorescent intensity along the line scan. (C) Plot of all normalized line scans (n = 92), with Hec1-9G3 centroid set to zero for each on the x axis and the error function (purple) that best fits the data set. Blue lines mark x and y positions of the 50% amplitude of the error function. Cell 2009 137, 672-684DOI: (10.1016/j.cell.2009.03.035) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 Centromere and Kinetochore Protein Architecture, Mechanics, Tension Sensing, and Force Generation (A and B) Schematics of the Ndc80 arm (A) and the hKnl1 arm (B) revealed by comparison of control and taxol-treated metaphase cells. (C) A tension/SAC activation-dependent intrakinetochore switch produced by an ∼12 nm translocation of the Ndc80 arm relative to the hKnl1 arm. This translocation is proposed to occur by rotation of Mis12 and hDsn1 subunits coupled to relaxation of a flexible filament-like linkage between the Ndc80 Arm and the inner kinetochore. (D–F) Models of the protein architecture of the KMN network of proteins within a kinetochore MT attachment site for depolymerizing ends (D), polymerizing ends (E), and taxol-stabilized ends (F). Cell 2009 137, 672-684DOI: (10.1016/j.cell.2009.03.035) Copyright © 2009 Elsevier Inc. Terms and Conditions