PMC

In metaphase, chromosome oscillations are caused by chromokinesin-based polar ejection forces and kinetochore-derived forces. These activities are controlled by phosphorylation downstream of CDK1. At anaphase onset, CDK1 is inactivated and phosphatases reverse the phosphorylation of its substrates to downregulate polar ejection forces and kinetochore-derived forces that act through microtubule polymerization. This allows chromosomes to display net motion towards the spindle poles. In the contrast, in the presence of the phosphatase inhibitor Okadaic acid, dephosphorylation is delayed such that chromokinesins and kinetochore-derived forces remain active. This maintains a metaphase-like oscillatory chromosome behavior in anaphase even after sister chromatid separation. Thus, mitosis is characterized by two distinct phases of chromosome motion – metaphase oscillations to align the chromosomes and poleward anaphase motion to segregate the chromosomes – and the switch in movement behavior is controlled by a regulatory transition.

(A) Still images from a time-lapse movie of HeLa cells expressing 3xGFP-CENP-A, 3xGFP-centrin. Box indicates the section used to generate the kymograph. (B) Color-coded kymograph of the time-lapse movie from A. (C) Representative image of time-lapse series displayed in A overlaid with selected tracks of particles. (D) Graph showing the distances over time for the distance between spindle poles (top; to measure spindle elongation) and the kinetochore to pole distance (bottom; to visualize chromosome motion) using tracks of a HeLa cell. The average kinetochore to pole distance is indicated as a black line, with individual kinetochores indicated in color. The time of anaphase onset is indicated by the dashed line. (E) Average spindle pole-to-pole distance (upper graph) and kinetochore to pole distance (lower graph) for HeLa cells undergoing anaphase (n=10). Colored dotted lines indicate standard deviation between cells. (F–H) Still images from a time-lapse movie of an hTERT RPE-1 cell expressing 3xGFP-CENP-A, 3xGFP-centrin. (G) Color-coded kymograph of time-lapse displayed in F. (H) Average spindle pole to pole distance (upper graph) and kinetochore to pole distance (lower graph) of hTERT RPE-1 undergoing anaphase (n=10). Blue dotted lines indicate standard deviation between cells. (I) Direct comparison of average kinetochore to pole distances over time for HeLa (from E) and hTERT-RPE cells data (from H). (J) Percentage of poleward motion over time. Scale bars, 2 μm. See also , , and .

(A) Still image from a representative time-lapse movie of a HeLa cell (3xGFP-CENP-A, 3xGFP-centrin; n=20) following depletion of the cohesin subunit RAD21 (48 h) displaying tracks until current time point of selected kinetochores used to generate the kinetochore to spindle pole distance graph (right). (B) Image of a HeLa cell (3xGFP-CENP-A, 3xGFP-centrin) before laser ablation (orange hair cross) to inactivate one of 2 sister kinetochores (n=29 experiments). Arrowhead indicates the released kinetochore (red) or unaffected kinetochores (blue) which were tracked to generate spindle to pole distance graph (right). (C) Maximal intensity projections of still images from representative time-lapse sequences of HeLa cells expressing mNeonGreen-PICH, 3xGFP-centrin entering anaphase in presence of DMSO (n=10) or 1 μM of the topoisomerase inhibitor ICRF-133 (n=14). (D) Color-coded kymographs of HeLa cells (3xGFP-CENP-A, 3xGFP-centrin) from anaphase onwards treated with DMSO (n=5) or ICRF-193 (n=7). (E) Still image from a time-lapse movie of a HeLa cell (3xGFP-CENP-A, 3xGFP-centrin) treated with S-trityl-L-cysteine (STLC) to generate a monopolar spindle (n=11) showing tracks of the selected kinetochores used to generate kinetochore to spindle pole distance graph (right). (F) Image from time-lapse movie of a monopolar HeLa cell (3xGFP-CENP-A, 3xGFP-centrin) treated with STLC and the Mps1 inhibitor AZ3146 (n=8). Selected tracks were used to generate the kinetochore to spindle pole distance graph (right). Green arrowheads highlight spindle poles. t=0 is beginning of movie. Scale bars, 2 μm. See also , and .

(A) (left) Images of untreated HeLa cells (3xGFP-CENP-A, 3xGFP-centrin) or cells treated with Okadaic acid (n≥10 cells). Selected kinetochore tracks until current time point are displayed. (right) Color-coded kymographs from the corresponding movies starting at anaphase onset. (B) Selected representative curves of individual kinetochore to pole distances from the cells shown in A. Color shades are used to distinguish different tracks. t=0 was set to anaphase onset. (C) Comparison of distribution of motion stages and velocity for the 240 seconds post anaphase onset in untreated or Okadaic acid treated HeLa cells (3xGFP-CENP-A, 3xGFP-centrin; n≥10 each). (D) Comparison of the proportion of anti-poleward motion during metaphase, untreated anaphase (n=10), or Okadaic acid-treated (n=18) anaphase HeLa cells (3xGFP-CENP-A, 3xGFP-centrin). (E) Kymographs as in A for cells expressing either wild type Cyclin B, or a non-degradable Cyclin B mutant. Arrowheads highlight spindle poles (green). Unpaired t tests were applied for comparison (**** p<0.0001; ** p=0.0031; Not significant (n.s.) C: p=0.235, D: p=0.117). Standard deviations were measured across cells using the average behavior for kinetochores in each cell. Scale bars, 2 μm. See also , and .

(A) Representative color-coded kymographs of HeLa cells (3xGFP-CENP-A, 3xGFP-centrin) undergoing anaphase either for control cells (left) or KID and KIF4A double knockout cells (KID+KIF4A KO; right) treated with DMSO (upper panels; n=10 or 6) or Okadaic acid (lower panels; n≥10). (B) Graph showing selected representative kinetochore to pole distances from A. (C) Kymographs as A displaying cells treated with non-targeting control siRNAs (left) or KIF18A siRNA after 24h (right) incubated in DMSO (upper panels; n=6 or 18) or Okadaic acid (lower panels; n=6 or 5, respectively) and MPS1 inhibitor AZ3146. (D) Graph showing selected representative kinetochore to pole distances from Okadaic acid treated cells as displayed in C. (E) Kymographs as A displaying cells in which either wild type mCherry-Ska1 (left) or a Ska1ΔMTBD mutant (right) replaces endogenous Ska1(48h RNAi). Cells were treated with AZ3146 and DMSO (upper panels; n=3 or 4) or AZ3146 and Okadaic acid (lower panels; n=8 or 5, respectively). (F) Graph showing selected representative kinetochore to pole distances from E. (G) Diagrams display fraction of anti-poleward state of kinetochores 240 seconds post anaphase onset for conditions A–F. Unpaired t tests were performed to HeLa cells (n=18): KID+KIF4A KO (n=12) *p=0.0225; KIF18A RNAi (n=5) *p=0.0285; Ska1ΔMTBD (n=5) **p=0.0096. (H) Diagram display velocity of kinetochore motion 240 seconds post anaphase onset for conditions A–F. Unpaired t-tests were performed for poleward motion to HeLa cells (n=18): KID+KIF4A KO (n=12) n.s. p=0.854; KIF18A RNAi (n=5) ****p<0.0001; Ska1ΔMTBD (n=5) ****p<0.0001 and for anti-poleward motion: KID+KIF4A KO (n=12) **p=0.0033; KIF18A RNAi (n=5) n.s. p=0.3492; Ska1ΔMTBD (n=5) ****p<0.0001. Arrowheads highlight spindle poles (green) and examples of anti-poleward motion (white). Scale bars, 2 μm. See also , and .