T H E J O U R N A L O F C E L L B I O L O G Y

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1 T H E J O U R N A L O F C E L L B I O L O G Y Supplemental material Sikirzhytski et al., Figure S1. Behavior and organization of K-fibers in PtK 2 cells during metaphase. (A) Kymograms of individual K-fibers with photobleached marks (PM). K denotes position of the kinetochore. Notice that the bleached mark moves poleward continuously and monotonously as expected for poleward flux of MT subunits. The rate of flux varies significantly among individual K-fibers (0.5, 0.8, and 1.1 µm/min in these examples). (B) The rate of flux does not change during transition from metaphase to anaphase. AO marks anaphase onset. (C) Reorientation of the K-fiber stub toward the opposite spindle pole. Selected frames from a time-lapse recording are similar to Fig. 1 A. In this example, the distal end of a stub created by laser severing of a K-fiber (arrows) makes contact with a MT connected to the opposite spindle pole (29 s). Poleward gliding of the stub alongside this MT transports the stub with the attached kinetochore poleward, ultimately converting a proper amphitelic attachment into a syntelic one. The chromosome subsequently moves poleward and becomes monooriented. Time is in seconds from the time of the laser burst. (D and D ) Minus end of laser-severed K-fibers are decorated with NuMA. (D) Overview (maximal-intensity projection) of a cell with severed K-fiber. (D ) Through-focus series (200-nm steps) for the area boxed in D. Notice accumulation of NuMA at the severed end of the fiber. A single MT appears to interact with the stub. Notice that the distance between sister kinetochores (K1 and K2) is 1.9 µm. S1

2 Figure S2. Behavior of laser-severed K-fibers in rounded and flattened PtK 2 cells during anaphase. (A) K-fiber stubs are transported poleward during anaphase. Similar to Fig. 1 B; however, in this example, the severed K-fiber reaches the pole in a single rapid movement. (A ) Kymogram analysis demonstrates that the K-fiber stub remains motionless for 10 s and then moves poleward at 6 µm/min. Time is in seconds from the time of the laser burst. X marks the laser beam. See Video 5 for the full record. (B) Behavior of chromosomes with severed K-fibers is similar in flattened and rounded cells. In this example, a K-fiber is severed 1 µm from the kinetochore (X) in a PtK 2 cell flattened to 4.5 µm (see Materials and methods). The K-fiber fragment attached to the spindle pole depolymerizes rapidly (blue arrows), whereas the distal end of the stub attached to the chromosome connects to neighboring MTs (yellow arrows). Notice that the initial length of the K-fiber stub is 1 µm but it gradually elongates during the recording. The stub also appears to split into two fibers that maintain multiple connections with neighboring MTs (66 and 107 s). The cell initiates anaphase at 230 s and the chromatid with severed K-fiber is rapidly transported poleward. Notice that it reaches the pole significantly faster than the other chromosomes (arrows in 380 and 444 s). Time in seconds from the laser burst. See Video 6 for the full record. S2

3 Figure S3. Mitosis in RPE1 cells flattened to 3 µm. (A and A ) The spatial distribution of kinetochores and centrosomes during metaphase in rounded (A) versus flattened (A ) cells. Notice that the width and depth of the rounded spindle are similar. Extensive Z-series are necessary for the reliable tracking of kinetochores. The spindle in a cell flattened to 3 µm is longer and wider; however, the chromosomes are properly congressed. The spindle is parallel to the coverslip and the movements of individual chromosomes occur primarily in a single focal plane. (B) No abnormalities are apparent at the earlier stages of mitosis in flattened cells (see cells 1, 2, and 3). However, mitosis in flattened cells is prolonged and spindle poles often fragment at later stages, resulting in multipolar anaphase and cytokinesis (cells 1 and 2). Time in h:min. S3

4 Figure S4. K-fibers lacking attachment to the spindle poles visualized in cold-treated cells. (A) A metaphase RPE1 cell exposed to 0 C for 2 min before fixation. Only cold-stable MTs remain. Multiple short K-fibers forming side attachments to K-fibers of other chromosomes are prominent (arrows). Local maximal-intensity projections (two to four Z-sections) are shown for the boxed areas. (B) Similar to A but the cell is in anaphase. Red, MTs (also shown in grayscale); green, kinetochores (CenpA-GFP) and centrioles (Centrin-GFP); blue, chromosomes (DNA). (C) Similar to A but a metaphase U2OS cell. Multiple short K-fibers forming side attachments to K-fibers of other chromosomes are prominent (arrows). Red, MTs ( -tubulin; also shown in grayscale); green, kinetochores (CenpB-GFP); blue, chromosomes (DNA). S4

5 Figure S5. NuMA concentrates at the ends of K-fibers that lack attachment to the spindle poles. (A) The distribution of the dynein-interacting protein NuMA in cold-treated RPE1 cells (0 C for 2 min) during metaphase. NuMA is highly concentrated at the minus ends of K-fibers attached to spindle poles and is present at a significantly lower concentration in small patches throughout the spindle (A ). Mild accumulation of NuMA is seen at the minus ends of short K-fibers that do not reach the poles. (B) The association of NuMA with a short K-fiber in PtK 2 cell during anaphase. Notice that the short fiber is masked in the total maximal-intensity projection (arrow). (B ) However, it can be detected in the projection of a 1-µm-thick volume. (B ) Premature termination of the K-fiber and the association of the minus end with NuMA becomes apparent in the series of individual focal planes separated by 200-nm steps. S5

6 Video 1. Typical behavior of laser-severed K-fibers during metaphase. Laser-severed K-fibers in PtK 2 cells expressing GFP -tubulin were analyzed by time-lapse spinning-disc confocal (Yokogawa Electric Corporation) microscopy using a microscope (TE-2000E PFS; Nikon). Selected frames from this recording are shown in Fig. 1 A. 0.5-s intervals (49.5 s), 108-nm pixels, 100 NA 1.4 objective. Time in seconds. 0 corresponds to laser irradiation. Video 2. Reorientation of laser-severed K-fiber. Laser-severed K-fibers in PtK 2 cells expressing GFP -tubulin were analyzed by time-lapse confocal microscopy using a microscope (TE-2000E PFS; Nikon). Selected frames from this recording are shown in Fig. S1 C. 1-s intervals (599 s), 108-nm pixels, 100 NA 1.4 objective. Time in seconds. 0 corresponds to laser irradiation. Video 3. Laser operation and live-cell recording followed by fixation and subsequent correlative LM/EM analysis. Lasersevered K-fibers in PtK 2 cells expressing GFP -tubulin were analyzed by time-lapse confocal microscopy using a microscope (TE-2000E PFS; Nikon). X marks the laser beam. Selected frames from this recording are shown in Fig. 1 B. 0.5-s intervals (38.5 s), 108-nm pixels, 100 NA 1.4 objective. Time in seconds. 0 corresponds to laser irradiation. Video 4. Typical behavior of laser-severed K-fibers during anaphase. Laser-severed K-fibers in PtK 2 cells expressing GFP -tubulin were analyzed by time-lapse confocal microscopy using a microscope (TE-2000E PFS; Nikon). Selected frames from this recording are shown in Fig. 1 B. 0.5-s intervals (49.5 s), 108-nm pixels, 100 NA 1.4 objective. Time in seconds. 0 corresponds to laser irradiation. Video 5. Extended rapid poleward movement of laser-severed K-fiber during anaphase. Laser-severed K-fibers in PtK 2 cells expressing GFP -tubulin were analyzed by time-lapse confocal microscopy using a microscope (TE-2000E PFS; Nikon). Selected frames from this recording are shown in Fig. S1 C. 1-s intervals (32 s), 108-nm pixels, 100 NA 1.4 objective. Time in seconds. 0 corresponds to laser irradiation. S6

7 Video 6. Typical behavior of laser-severed K-fibers in a flattened cell. Laser-severed K-fibers in PtK 2 cells expressing GFP -tubulin were analyzed by time-lapse confocal microscopy using a microscope (TE-2000E PFS; Nikon). Selected frames from this recording are shown in Fig. S2 D. 1-s intervals (450 s), 108-nm pixels, 100 NA 1.4 objective. Time in seconds. Laser irradiation immediately after time 0. Flattened PtK 2 cells were obtained by placing a coverslip with microfabricated feet of known height (3 µm) on top of the coverslip with the growing cells. Video 7. Comparison of Pacman versus fiber transport mechanisms of chromosome segregation. Laser-severed K-fibers in PtK 2 cells expressing GFP -tubulin (Takara Bio Inc.) were analyzed by time-lapse confocal microscopy using a microscope (TE-2000E PFS; Nikon). Selected frames from this recording are shown in Fig. 2 (C and D). 1-s intervals (54 s), 108-nm pixels, 100 NA 1.4 objective. Time in seconds. Laser irradiation shortly before time 0. Video 8. Walk-through array-tomography reconstruction of metaphase spindle in an RPE1 cell. Phase-contrast (left) and fluorescence (right) images obtained from serial 200-nm-thin Lowicryl sections. (right) Arrows denote the minus ends of K-fibers that lack direct attachment to the spindle poles. Selected images from this reconstruction are shown in Fig. 5 (A and B). Images recorded on a Revolution spinning-disc confocal system (Andor Technology) at 55-nm pixel size. 100 NA 1.4 objective lens (Nikon). Video 9. Image transformation used for the generation of kymograms of cells with labeled centrosomes and kinetochores. Time-lapse confocal microscopy using a microscope (TE-2000E PFS; Nikon). The panels are maximal intensity projections of 3D volumes recorded at 5-s intervals before (raw) and after (aligned) each volume was rotated and shifted to make the mother centriole in the left centrosome pseudo-stationary and to fix the axis of spindle in the horizontal position. An RPE1 cell expressing CenpA-GFP and centrin-gfp. Time in min:s. 5-s intervals (320 s), 108-nm pixels, 100 NA 1.4 objective, and 500-nm Z-steps. Video 10. Image transformation used for the generation of kymograms in recordings with labeled MTs. Laser-severed K-fibers in PtK 2 cells expressing GFP -tubulin were analyzed by time-lapse confocal microscopy using a microscope (TE-2000E PFS; Nikon). Each frame was rotated and shifted to make the plus end of the K-fiber stub (kinetochore) pseudo-stationary and fix the orientation of the stub in the horizontal position. Time in min:s. 1-s intervals (100 s), 108-nm pixels, 100 NA 1.4 objective. Table S1 shows the results of laser microsurgery experiments. S7