Three-dimensional structure of kinetochore-fibers in human mitotic spindles
- Experimental Center, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Germany
- Department of Physics, Harvard University, United States
- Department of Visual and Data-Centric Computing, Zuse Institute Berlin, Germany
- Department of Molecular and Cellular Biology, Harvard University, United States
- John A. Paulson School of Engineering and Applied Sciences, Harvard University, United States
- Center for Computational Biology, Flatiron Institute, United States
Figures
Figure 1
Models of k-fiber organization in mammalian mitosis.
(A) Direct connection with KMTs (red lines) spanning the distance between the kinetochore and the spindle pole. Chromosomes are shown in blue with kinetochores in red. The mother (m) and the …
Figure 2 with 10 supplements
Three-dimensional reconstruction of metaphase spindles by large-scale electron tomography.
(A) Tomographic slice showing a HeLa cell (spindle #1) in metaphase. The chromosomes (ch) and the spindle poles (p) are indicated. (B) Three-dimensional reconstruction of the same spindle as shown …
Figure 2—figure supplement 1
Illustration of metaphase in HeLa cells.
(A–B) Tomographic slices through spindle #3 illustrating the morphology of HeLa cells. Spindle poles (p) with a centriole, chromosomes (ch), and mitochondria (m) are indicated. The boxed regions …
Figure 2—figure supplement 2
Correction of sample collapse caused during data acquisition by electron tomography.
(A) Schematic illustration of correction for a sample collapse in Z. Left panel shows a stack of semi-thick sections after ultramicrotomy (total height, Z1). The mid panel illustrates a sample …
Figure 2—figure supplement 3
Analysis of pole-to-pole and sister k-fiber-to-sister k-fiber distances.
(A) Schematic illustration of the pole-to-pole distance. The 3D distance between the mother centrioles (m, marked in gray) of the two opposing spindle poles (P1 and P2) is measured. Chromosomes …
Figure 2—figure supplement 4
Morphology of KMT plus ends.
(A) Schematic illustration of a KMT (red tube) with different viewing planes (colored in green, blue, orange, and purple). (B–D) Tomographic slices of KMT plus ends visualized at different viewing …
Figure 2—video 1
Generation of a 3D model from joined serial electron tomograms displaying spindle #1.
Series of stitched tomograms and corresponding 3D model of spindle #1. The stacking of serial tomograms to increase the tomographic volume is illustrated. MTs are shown as white lines, and …
Figure 2—video 2
Generation of a 3D model from joined serial electron tomograms displaying spindle #2.
Series of stitched tomograms and corresponding 3D model of spindle #2. The stacking of serial tomograms to increase the tomographic volume is illustrated. MTs are shown as white lines, and …
Figure 2—video 3
Generation of a 3D model from joined serial electron tomograms displaying spindle #3.
Series of stitched tomograms and corresponding 3D model of spindle #3. The stacking of serial tomograms to increase the tomographic volume is illustrated. MTs are shown as white lines, and …
Figure 2—video 4
Organization of KMTs in spindle #1.
Three-dimensional model of spindle #1. Non-KMTs are shown in yellow, KMTs in red. Chromosomes are illustrated in blue and centrioles in gray. This video corresponds to Figure 2F. Scale bar, 1 µm.
Figure 2—video 5
Organization of KMTs in spindle #2.
Three-dimensional model of spindle #2. Non-KMTs are shown in yellow, KMTs in red. Chromosomes are illustrated in blue and centrioles in gray. This video corresponds to Figure 2G. Scale bar, 1.5 µm.
Figure 2—video 6
Organization of KMTs in spindle #3.
Three-dimensional model of spindle #3. Non-KMTs are shown in yellow, KMTs in red. Chromosomes are illustrated in blue and centrioles in gray. This video corresponds to Figure 2H. Scale bar, 1.5 µm.
Figure 3 with 9 supplements
Morphology of k-fibers and number of KMTs associated per kinetochore.
(A) Examples of individual sister k-fibers extracted from the full 3D reconstruction of metaphase spindle #1. The numbering of these examples (corresponding to the supplementary videos) is given in …
Figure 3—figure supplement 1
MT segmentation and stitching across consecutive serial sections.
(A) Schematic illustration of MT segmentation across stacked consecutive serial sections. Each section is represented as a gray area. MTs are shown as red lines and their ends as red circles. …
Figure 3—figure supplement 2
Correlation of k-fiber circumference and number of attached KMTs.
(A) Graph showing the number of KMTs per kinetochore for each data set and the average number for all data sets (error bars are mean ±STD). (B) Schematic illustration of the indirect measurement of …
Figure 3—figure supplement 3
Positioning of k-fibers in the mitotic spindle.
(A) Schematic illustration of the k-fiber positions on the X/Z projection of the metaphase plate. Kinetochores are indicated as blue circles. The fitted ellipse (center marked by a black dot) …
Figure 3—video 1
3D reconstruction of a single k-fiber pair.
Three-dimensional model of selected sister k-fibers in spindle #1. The KMTs are shown as red lines, KMT ends as white spheres and the centrioles as gray cylinders. This video corresponds to Figure 3A…
Figure 3—video 2
3D reconstruction of a single k-fiber pair.
Three-dimensional model of selected sister k-fibers in spindle #1. The KMTs are shown as red lines, KMT ends as white spheres and the centrioles as gray cylinders. This video corresponds to Figure 3A…
Figure 3—video 3
3D reconstruction of a single k-fiber pair.
Three-dimensional model of selected sister k-fibers in spindle #1. The KMTs are shown as red lines, KMT ends as white spheres and the centrioles as gray cylinders. This video corresponds to Figure 3A…
Figure 3—video 4
3D reconstruction of a single k-fiber pair.
Three-dimensional model of selected sister k-fibers in spindle #1. The KMTs are shown as red lines, KMT ends as white spheres and the centrioles as gray cylinders. This video corresponds to Figure 3A…
Figure 3—video 5
3D reconstruction of a single k-fiber pair.
Three-dimensional model of selected sister k-fibers in spindle #1. The KMTs are shown as red lines, KMT ends as white spheres and the centrioles as gray cylinders. This video corresponds to Figure 3A…
Figure 3—video 6
3D reconstruction of a single k-fiber pair.
Three-dimensional model of selected sister k-fibers in spindle #1. The KMTs are shown as red lines, KMT ends as white spheres and the centrioles as gray cylinders. This video corresponds to Figure 3A…
Figure 4 with 3 supplements
Analysis of MT length distribution.
(A) Histogram showing the length distribution of KMTs from all data sets (n=2579). The dashed line indicates the average length of KMTs. (B) Histogram showing the length distribution of non-KMTs …
Figure 4—figure supplement 1
Length distribution of KMTs and non-KMTs.
(A) Beeswarm plot showing the length distribution of KMTs in the individual data sets. The average of all data sets is also given (error bars are mean ±STD). (B) Beeswarm plot showing the length …
Figure 4—figure supplement 2
KMT length distribution based on the k-fiber position in the spindle.
(A) Box plot showing the length distribution of KMTs in individual k-fibers as observed in spindle #1 (n=98). The k-fibers are displayed and color-coded according to their position within the …
Figure 4—figure supplement 3
KMT length distribution based on the position of individual k-fibers within the spindle.
Beeswarm plot showing the length distribution of KMTs and the average of all data sets based on the k-fiber position in the spindle. The plots show the mean indicated by black horizontal bars. The …
Figure 5 with 4 supplements
Analysis of MT minus ends.
(A) Measurement of MT minus-end positioning. A KMT (red line) with its ends (red circles) and a non-KMT (yellow line) with its ends (yellow circles) are shown. The distance of both the KMT and the …
Figure 5—figure supplement 1
Normalization of minus-end positioning on the pole-to-kinetochore axis.
(A) Three-dimensional model of the kinetochores visualized in spindle #1. For each kinetochore pair, each kinetochore is assigned to the closest spindle pole (p1, left pole, kinetochores in light …
Figure 5—figure supplement 2
Minus-end distribution of KMTs and non-KMTs.
(A) Beeswarm plot showing the distribution of KMT minus-end distances to the center of the mother centrioles. The MT-centrosome interaction area is indicated in gray (error bars are mean ±STD). (B) …
Figure 5—figure supplement 3
Analysis of KMT minus ends reaching the pole.
Plot showing the number of KMT minus ends per k-fiber detected within the MT-centrosome interaction area. The individual data sets and the average numbers for all data sets are given (error bars are …
Figure 5—figure supplement 4
KMT minus-end distribution based on the k-fiber position in the spindle.
(A) Plot showing the number of KMTs in each k-fiber associated with the spindle poles for the three positional regions in the mitotic spindle for all data sets. The mean values are indicated. (B) …
Figure 6 with 4 supplements
Global and local tortuosity of KMTs.
(A) Schematic illustration of tortuosity (T) as given for a straight line, a quarter of a circle, and a half of a circle. (B) Schematic illustration of global tortuosity (Tg) of KMTs given by the …
Figure 6—figure supplement 1
Global tortuosity of KMTs based on the k-fiber position in the spindle.
(A) Histogram showing the number of central KMTs plotted against the global tortuosity (n=338). (B) Histogram showing intermediate KMTs (n=541). (C) Histogram showing peripheral KMTs (n=1700). The …
Figure 6—video 1
Analysis of k-fiber global tortuosity in spindle #1.
Three-dimensional model of all KMTs of spindle #1 with indicated global tortuosity. KMTs are color-coded as indicated in Figure 6D. KMT ends are shown as white spheres, centrioles as gray cylinders. …
Figure 6—video 2
Analysis of k-fiber global tortuosity in spindle #2.
Three-dimensional model of all KMTs of spindle #2 with indicated global tortuosity. KMTs are color-coded as indicated in Figure 6D. KMT ends are shown as white spheres, centrioles as gray cylinders. …
Figure 6—video 3
Analysis of k-fiber global tortuosity in spindle #3.
Three-dimensional model of all KMTs of spindle #3 with indicated global tortuosity. KMTs are color-coded as indicated in Figure 6D. KMT ends are shown as white spheres, centrioles gray cylinders. …
Figure 7 with 1 supplement
Shape of k-fibers.
(A) Schematic illustration of the analysis of polygonal areas as obtained from k-fiber cross-sections. KMTs are shown as lines (red), KMT ends as spheres (light red). Cross-sections of the given …
Figure 7—figure supplement 1
Schematic illustration of the analysis of k-fiber area and KMT density.
(A) Selection of cross-sections along a k-fiber (KMTs, red lines; KMT ends, light red spheres) every 500 nm (upper row - blue squares; lower row - blue lines). (B) Assignment of a median position …
Figure 8 with 6 supplements
Association of KMTs with the MT network.
(A) Graph showing the number of KMT minus ends associated with KMT lattices within 35 nm of interaction (n=2579). Numbers of KMT minus ends are normalized by the density of surrounding MTs and …
Figure 8—figure supplement 1
Association of KMT minus ends with other MT lattices.
(A) Bar plot showing the percentage of all KMT minus ends associated with a MT lattice depending on defined distances of interaction (25–100 nm; n=2579; error bars are mean ±STD). (B) …
Figure 8—figure supplement 2
Association of KMT minus ends with KMT and non-KMT lattices at an interaction distance of 35 nm.
(A) Visualization of KMTs and non-KMTs obtained from the ASGA 3D-Viewer (https://cfci.shinyapps.io/ASGA_3DViewer/) showing a k-fiber pair from spindle #1 (ASGA k-fiber ID: pole1_08). This 3D model …
Figure 8—figure supplement 3
Association of KMTs with other MTs.
(A) Graph showing the number of KMT minus ends associated with KMT lattices within a distance of interaction of 35 nm (n=2579). The number of KMT minus ends normalized by the spindle density is …
Figure 8—figure supplement 4
Association of KMT lattices with other MT minus ends at an interaction distance of 35 nm.
(A) Visualization of KMTs and non-KMTs obtained from the ASGA 3D-Viewer (https://cfci.shinyapps.io/ASGA_3DViewer) showing a k-fiber pair from spindle #1 (ASGA k-fiber ID: pole1_08 / pole2_08). This …
Figure 8—video 1
Associations of KMT minus ends with MT lattices in spindle #1.
Three-dimensional model of all KMTs from spindle #1. KMT minus ends interacting with other MTs at association distances up to 35 nm are shown. KMT minus ends interacting with either KMT (red lines) …
Figure 8—video 2
Association of KMT minus end with MT lattices.
Three-dimensional model of KMTs from spindle #1 (ASGA k-fiber ID: pole1_08). The association of KMT minus-end with other MTs is shown. The distance of interaction is 35 nm. KMT minus-end association …
Figure 9 with 4 supplements
Positions of MT-MT associations.
(A) Schematic illustration showing the mapping of the number of MT-MT associations on the pole-to-pole axis (P1, position = 0; P2, position = 1). The number of associations is measured in defined …
Figure 9—figure supplement 1
Association of KMT lattices with other MT lattices at an interaction distance of 35 nm.
(A) Visualization of KMTs and non-KMTs obtained from the ASGA 3D-Viewer (https://cfci.shinyapps.io/ASGA_3DViewer/) showing a k-fiber pair from spindle #1 (ASGA k-fiber ID: pole1_08). This 3D model …
Figure 9—figure supplement 2
Association of KMT lattices with MT minus ends.
(A) Frequency of the number of associations of KMTs with other MTs in the spindle (n=2579). Plots are shown for five different interaction distances (insert). (B) Frequency of the number of …
Figure 9—video 1
Associations of KMT lattices with other MTs.
Three-dimensional model of all KMTs in spindle #1. KMT lattices associated with other MT lattices at are shown. The distances of interaction is 35 nm. KMTs are shown as gray lines with color-coded …
Figure 9—video 2
Associations of non-KMTs with other MTs.
Three-dimensional model of all non-KMTs in spindle #1. Non-KMT lattices associated with other MT lattices are shown. The distances of interaction is 35 nm. Non-KMTs are shown as gray lines with …
Figure 10
Model of a k-fiber showing a semi-direct connection between a kinetochore and a spindle pole.
(A–B) Three-dimensional views of a selected 3D-reconstructed k-fiber with an overlay area drawn around KMTs using the alpha shape method. The KMTs are shown as red lines and the ends are marked with …
Tables
Table 1
Characterization of the 3D-reconstructed metaphase spindles in HeLa cells.
| Data set | Spindle pole distance [µm] | Inter-kinetochore distance [µm]* | No. of MTs in the tomographic volume | No. of kinetochores | No. of KMTs | No. of non-KMTs | No. of k-fibers |
|---|---|---|---|---|---|---|---|
| Spindle #1 | 7.16 | 1.08 ± 0.20 (n=43) | 4884 | 92 | 797 (16.3%) | 4087 (83.7%) | 92 |
| Spindle #2 | 10.39 | 1.24 ± 0.21 (n=50) | 8047 | 110 | 1,102 (13.7%) | 6945 (86.3%) | 110 |
| Spindle #3 | 9.48 | 1.03 ± 0.27 (n=40) | 5904 | 90 | 680 (11.5%) | 5224 (88.5%) | 90 |
-
*
Numbers are given as mean ± STD.
Table 2
Tomographic data sets as used throughout this study.
| Data set | Original data set | Montage (X/Y) | No. of serial sections[300 nm each] | Estimated tomographic volume [µm3] | Data set size [Gb] |
|---|---|---|---|---|---|
| Spindle #1 | T_0475 | 2 × 3 | 22 | 598 | 46.5 |
| Spindle #2 | T_0479 | 2 × 3 | 29 | 996 | 77.9 |
| Spindle #3 | T_0494 | 2 × 3 | 35 | 904 | 71.9 |
Table 3
Quantitative analysis of KMTs and non-KMTs.
| Data set | Length of KMTs [µm]* | Length of non- KMTs [µm]* | No. of KMTs per kinetochore* | No. of KMTs in the MT-centrosome interaction area* | Mean KMT minus-end distance to poles [µm] | % of KMTs associated with poles | % of non-KMTs associated with poles |
|---|---|---|---|---|---|---|---|
| Spindle #1 | 3.59 (±1.57) | 2.13 (±1.67) | 8.04 (±1.86) | 5.0 (±1.8) | 1.72 | 61.2 | 44.3 |
| Spindle #2 | 3.82 (±1.97) | 1.95 (±1.60) | 9.75 (±2.18) | 3.1 (±2.3) | 2.87 | 31.5 | 28.6 |
| Spindle #3 | 4.27 (±1.93) | 2.07 (±1.93) | 7.49 (±1.91) | 4.1 (±2.0) | 2.12 | 54.2 | 41.9 |
-
*
Numbers are given as mean ±STD.
Table 4
Quantitative analysis of k-fiber organization.
| Data set | KMT density at the kinetochore [KMT/µm2]* | KMT-KMT distance at the kinetochore [nm]* | Global tortuosity of KMTs* | % of curved KMTs | Area of k-fibers [µm2]* | % of KMTs in a k-fibers* |
|---|---|---|---|---|---|---|
| Spindle #1 | 122 (±62) | 67 (±20) | 1.11 (±0.11) | 39.8 | 0.08 (±0.1) | 64 (±27) |
| Spindle #2 | 99 (±45) | 78 (±23) | 1.07 (±0.07) | 28.4 | 0.09 (±0.11) | 70 (±25) |
| Spindle #3 | 117 (±72) | 76 (±23) | 1.13 (±0.13) | 47.1 | 0.12 (±0.24) | 59 (±29) |
-
*
Numbers are given as mean ±STD.
Table 5
Quantitative analysis of k-fiber positioning in the spindle.
| Region | Length of KMTs [µm]* | No. of KMTs per kinetochore* | No. of KMTs at MT-centrosome interaction area† | Mean KMT minus-end distance to poles [µm]* | No. of KMTs associated with poles* | Global tortuosity of KMTs* |
|---|---|---|---|---|---|---|
| Central | 3.5 (±1.7) | 8.2 (±2.4) | 162 (~48%) | 2.0 (±1.3) | 4.3 (±2.3) | 1.08 (±0.08) |
| Intermediate | 3.6 (±1.7) | 8.6 (±2.1) | 266 (~49%) | 2.1 (±1.3) | 4.6 (±1.9) | 1.11 (±0.12) |
| Peripheral | 3.9 (±2.0) | 8.6 (±2.4) | 730 (~45%) | 2.5 (±1.6) | 4.1 (±2.0) | 1.10 (±0.10) |
-
*
Numbers are given as mean ±STD.
-
†
Number and percentage of KMTs is shown.
Table 6
Analysis of the association of KMT minus ends with other neighboring KMT lattices.
| Data set | Analysis | Interaction distances [nm] | ||||||
|---|---|---|---|---|---|---|---|---|
| 25 | 30 | 35 | 45 | 50 | 75 | 100 | ||
| Spindle #1 | No. of KMTs | 37 | 68 | 112 | 204 | 238 | 306 | 330 |
| % of KMTs | 4.9 | 9.1 | 15.0 | 27.3 | 32.0 | 40.9 | 44.1 | |
| Spindle #2 | No. of KMTs | 20 | 37 | 68 | 142 | 177 | 266 | 290 |
| % of KMTs | 1.9 | 3.5 | 6.3 | 13.2 | 16.5 | 24.8 | 27.1 | |
| Spindle #3 | No. of KMTs | 13 | 27 | 66 | 116 | 135 | 199 | 218 |
| % of KMTs | 1.9 | 4.0 | 9.8 | 17.2 | 20.0 | 29.5 | 32.3 | |
Table 7
Analysis of the association of KMT minus ends with neighboring non-KMT lattices.
| Data set | Analysis | Interaction distances [nm] | ||||||
|---|---|---|---|---|---|---|---|---|
| 25 | 30 | 35 | 45 | 50 | 75 | 100 | ||
| Spindle #1 | No. of KMTs | 37 | 82 | 132 | 217 | 248 | 353 | 384 |
| % of KMTs | 4.9 | 11.0 | 17.6 | 29.0 | 33.2 | 47.2 | 51.3 | |
| Spindle #2 | No. of KMTs | 245 | 313 | 353 | 469 | 525 | 677 | 732 |
| % of KMTs | 22.9 | 29.2 | 33.0 | 43.8 | 49.0 | 63.2 | 68.3 | |
| Spindle #3 | No. of KMTs | 28 | 64 | 107 | 198 | 230 | 355 | 410 |
| % of KMTs | 4.2 | 9.5 | 15.9 | 29.4 | 34.1 | 52.7 | 60.8 | |
Table 8
Analysis of the association of KMT lattices with other neighboring KMT minus ends.
| Data set | Analysis | Interaction distances [nm] | ||||||
|---|---|---|---|---|---|---|---|---|
| 25 | 30 | 35 | 45 | 50 | 75 | 100 | ||
| Spindle #1 | No. of KMTs | 39 | 71 | 117 | 210 | 236 | 336 | 403 |
| % of KMTs | 5% | 10% | 15% | 28% | 31% | 45% | 54% | |
| Spindle #2 | No. of KMTs | 24 | 46 | 86 | 179 | 237 | 401 | 470 |
| % of KMTs | 2% | 4% | 8% | 17% | 22% | 37% | 43% | |
| Spindle #3 | No. of KMTs | 14 | 27 | 61 | 127 | 148 | 227 | 284 |
| % of KMTs | 2% | 4% | 9% | 19% | 22% | 34% | 43% | |
Table 9
Analysis of the association of KMT lattices with other neighboring non-KMT minus ends.
| Data set | Analysis | Interaction distances [nm] | ||||||
|---|---|---|---|---|---|---|---|---|
| 25 | 30 | 35 | 45 | 50 | 75 | 100 | ||
| Spindle #1 | No. of KMTs | 81 | 151 | 223 | 362 | 415 | 534 | 577 |
| % of KMTs | 11% | 20% | 30% | 48% | 55% | 71% | 77% | |
| Spindle #2 | No. of KMTs | 51 | 100 | 173 | 351 | 433 | 640 | 717 |
| % of KMTs | 5% | 9% | 16% | 33% | 40% | 59% | 67% | |
| Spindle #3 | No. of KMTs | 34 | 93 | 176 | 301 | 348 | 471 | 507 |
| % of KMTs | 5% | 14% | 26% | 44% | 51% | 69% | 75% | |
Table 10
Average number of associations of KMTs and non-KMTs with MT lattices.
| Data set | MT type | Interaction distances [nm] | ||||
|---|---|---|---|---|---|---|
| 25 | 30 | 35 | 45 | 50 | ||
| Spindle #1* | KMTs | 4.8 (±1.5) | 8.6 (±2.5) | 12.4 (±3.4) | 18.8 (±4.9) | 1.3 (±5.5) |
| Non-KMTs | 4.4 (±1.5) | 7.0 (±2.6) | 9.6 (±3.7) | 13.0 (±5.4) | 16.2 (±6.2) | |
| Spindle #2* | KMTs | 4.2 (±1.3) | 5.8 (±1.8) | 8.0 (±2.6) | 13.3 (±4.1) | 16.0 (±4.8) |
| Non-KMTs | 3.2 (±0.9) | 4.0 (±1.3) | 5.2 (±1.8) | 8.2 (±3.1) | 9.8 (±3.7) | |
| Spindle #3* | KMTs | 4.2 (±1.2) | 8.0 (±2.3) | 12.4 (±3.4) | 18.6 (±4.8) | 21.2 (±5.4) |
| Non-KMTs | 3.6 (±1.2) | 5.2 (±2.3) | 8.0 (±3.3) | 11.6 (±4.7) | 13.2 (±5.3) | |
| All spindles* | KMTs | 4.4 (±1.3) | 7.4 (±2.3) | 10.6 (±3.2) | 16.4 (±4.7) | 19.0 (±5.3) |
| Non-KMTs | 3.6 (±1.2) | 5.4 (±2.2) | 7.4 (±3.1) | 10.8 (±4.5) | 12.4 (±5.1) | |
-
*
Numbers are given as mean ±STD.
Table 11
Average length of associations of KMTs and non-KMTs with MT lattices.
| Data set | MT type | Interaction distances [nm] | ||||
|---|---|---|---|---|---|---|
| 25 | 30 | 35 | 45 | 50 | ||
| Spindle #* | KMTs | 81.3 (±88.8) | 119.7 (±151.1) | 163.9 (±207.5) | 241.3 (±301.9) | 271.0 (±335.9) |
| Non-KMTs | 58.3 (±54.1) | 78.3 (±79.9) | 107.7 (±116.9) | 165.1 (±195.1) | 187.9 (±227.0) | |
| Spindle #2* | KMTs | 69.5 (±69.9) | 93.2 (±107.0) | 124.3 (±146.0) | 207.8 (±252.3) | 252.2 (±314,9) |
| Non-KMTs | 59.2 (±53.2) | 73.2 (±71.9) | 92.3 (±97.3) | 145.8 (±170.5) | 175.1 (±213.0) | |
| Spindle #3* | KMTs | 66.1 (±63.4) | 97.3 (±117.8) | 143.2 (±191.7) | 231.3 (±321.2) | 263.4 (±362.2) |
| Non-KMTs | 54.3 (±51.7) | 74.5 (±86.4) | 104.6 (±133.7) | 165.6 (±218.7) | 191.3 (±252.0) | |
| All spindles* | KMTs | 73.0 (±76.2) | 104.6 (±129.2) | 145.1 (±186.0) | 225.6 (±292.3) | 261.9 (±336.8) |
| Non-KMTs | 57.2 (±53.1) | 75.4 (±80.5) | 102.2 (±118.9) | 159.1 (±197.2) | 184.9 (±232.1) | |
-
*
Numbers are given as mean ±STD.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, background (HeLa, Kyoto) | Gerlich Lab | IMBA, Vienna, Austria | - | - |
| Software, algorithm | SerialEM Boulder Laboratory for 3-Dimensional Electron Microscopy of cells Colorado, USA | https://bio3d.colorado.edu/ Mastronarde, 2003 | - | - |
| Software, algorithm | IMOD Boulder Laboratory for 3-Dimensional Electron Microscopy of cells Colorado, USA | http://bio3d.colorado.edu/ Kremer et al., 1996 | - | - |
| Software, algorithm | Amira Thermo Fisher Scientific, USA | https://www.zib.de/software/amira Stalling et al., 2005 | - | - |
| Software, algorithm | ASGA Robert Kiewisz / Müller - Reichert Lab Dresden, Germany | https://github.com/RRobert92/ Kiewisz and Müller-Reichert, 2021 | - | https://kiewisz.shinyapps.io/ASGA |
| Software, algorithm | ASGA - 3D Viewer Robert Kiewisz / Müller - Reichert Lab Dresden, Germany | https://github.com/RRobert92/ Kiewisz and Müller-Reichert, 2022 | - | https://cfci.shinyapps.io/ASGA_3DViewer/ |
Table 12
Quantification of KMT ultrastructure before and after application of Z-expansion to the 3D models.
| Data set | Length of KMTs [µm]* | Length of non- KMTs [µm]* | No. of KMTs per kinetochore* | No. of KMTs in the MT-centrosome interaction area* | Mean KMT minus-end distance to poles [µm] | No. of KMTs associated with poles [%] | No. of non-KMTs associated with poles [%] | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Before | After | Before | After | Before | After | Before | After | Before | After | Before | After | Before | After | |
| Spindle #1 | 3.23 (±1.49) | 3.59 (±1.57) | 2.03 (±1.6) | 2.13 (±1.67) | 8.04 (±1.86) | 8.04 (±1.86) | 4.1 (±1.8) | 5.0 (±1.8) | 1.16 | 1.72 | 62.2 | 61.2 | 44.5 | 44.3 |
| Spindle #2 | 3.69 (±1.87) | 3.82 (±1.97) | 1.85 (±1.55) | 1.95 (±1.60) | 9.75 (±2.18) | 9.75 (±2.18) | 2.4 (±2.0) | 3.1 (±2.3) | 2.47 | 2.87 | 53.6 | 31.5 | 28.8 | 28.6 |
| Spindle #3 | 4.03 (±1.79) | 4.27 (±1.93) | 1.91 (±1.80) | 2.07 (±1.93) | 7.49 (±1.91) | 7.49 (±1.91) | 3.4 (±1.8) | 4.1 (±2.0) | 1.35 | 2.12 | 62.0 | 54.2 | 42.3 | 41.9 |
-
*
Numbers are given as mean ±STD.
Table 13
Quantification of k-fiber organization before and after application of Z-expansion to the 3D models.
| Data set | Density of KMTs at the kinetochore [KMT/µm2]* | KMT-KMT distance at the kinetochore [nm]* | Global tortuosity of KMTs* | % of curved KMTs* | Area of k-fibers [µm2]* | % of KMTs in a k-fibers* | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Before | After | Before | After | Before | After | Before | After | Before | After | Before | After | |
| Spindle #1 | 151 (±74) | 122 (±62) | 61 (±11) | 67 (±20) | 1.09 (±0.10) | 1.11 (±0.11) | 36.1 | 39.8 | 0.063 (±0.09) | 0.08 (±0.1) | 34 (±27) | 64 (±27) |
| Spindle #2 | 137 (±68) | 99 (±45) | 65 (±12) | 78 (±23) | 1.06 (±0.06) | 1.07 (±0.07) | 21.4 | 28.4 | 0.068 (±0.10) | 0.09 (±0.11) | 70 (±25) | 70 (±25) |
| Spindle #3 | 175 (±123) | 117 (±72) | 66 (±12) | 76 (±23) | 1.11 (±0.11) | 1.13 (±0.13) | 39.5 | 47.1 | 0.080 (±0.15) | 0.12 (±0.24) | 59 (±39) | 59 (±29) |
-
*
Numbers are given as mean ±STD.
