Fig 2: Decreased midzone stability does not affect chromosome segregation velocity during anaphase(A and B) Immunofluorescent (A) and expansion microscopy (B) images of fixed control, PRC1-siRNA-depleted (only immunofluorescence) and KIF4A-siRNA-depleted 40-µM STLC-treated RPE-1 cells stained with AlexaFluor594 conjugated with a-tubulin antibody. Images in (A) and (B) are the maximum projections of color-coded z stacks as shown by the scheme in (B). Arrowheads point to the spindle midzone region.(C) STED images (single z plane) of anaphase spindles labeled with 100 nM SiR-tubulin in live non-induced CRISPR PRC1 KO and induced PRC1 KO RPE-1 cells.(D) Smoothed live-cell images (single z plane) of RPE-1 cells after midzone photoactivation of photoactivatable (PA)-GFP-a-tubulin (green) in the indicated conditions. 100-nM SiR-DNA (magenta) was used for chromosome staining. Time 0 represents the photoactivation onset.(E) Quantification of midzone stability, defined as the ratio of the integrated photoactivated signal in the boxed region (see schemes and boxes in D) at times 30 and 0 s, and chromosome segregation velocity measured in the same time period (see schemes), for the indicated treatments. Statistics: t test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Numbers: 14, 7, 8, 9, 6, 5, 15, and 5 cells, from left to right. Three independent experiments for every condition except MKLP1 + MKLP2, MKLP1 + MKLP2 + EG5 inh. and KIF4+EG5 inh., which were done in two independent experiments regarding siRNAs or non-targeting treatments, while number of independent experiments regarding STLC treatment is equal to the number of cells. Black data dots in every treatment correspond to the measurements from the exemplar cells shown on the time-lapse images and kymographs (D).(F) Chromosome segregation velocity versus midzone stability (data from E) for indicated treatments and linear regression (line); rs, Spearman correlation coefficient, p < 0.001. Scale bars, 1 µm. See also Figure S5.

Fig 3: Chromosome segregation and spindle elongation are dependent upon KIF4A and EG5-generated sliding(A) Montage time-lapse live images of the mitotic spindle midzone region after photoactivation of photoactivatable (PA)-GFP-a-tubulin in the indicated treatments. Top and bottom schemes depict sliding of the photoactivated midzone region. Vertical scale bar, 1 s. Horizontal scale bar, 1 µm.(B) Quantification of sliding velocity, measured as the length change of the photoactivated spot (L, see scheme) in the indicated conditions. Statistics: t test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Numbers: 13, 8, 6, 10, 6, 6, 15, and 6 cells, from left to right. Three independent experiments for every condition except MKLP1 + MKLP2 siRNAs and MKLP1 + MKLP2 + EG5 inh., which were done in two independent experiments regarding siRNAs or non-targeting treatments, while number of independent experiments regarding STLC treatment is equal to the number of cells. Black data dots in every treatment correspond to the measurements from the exemplar cells shown on the time-lapse images and kymographs (A and B).(C and D) Chromosome segregation velocity (C) and centrosome separation velocity (D) versus sliding velocity in the indicated treatments, and linear regression (line); rs, Spearman correlation coefficient, p < 0.001.(E) Proposed model for the motor-driven sliding in the antiparallel midzone region (top scheme) of an anaphase mitotic spindle including two independent sliding modules. Gray arrows point to the direction of overall microtubule motion as a result of forces produced by the motors walking on antiparallel microtubules (black arrows). See also Figure S7.

Fig 4: Depletion of both kinesin-6 MKLP1 and kinesin-6 MKLP2 with inactivation of EG5 induces a partial block in chromosome segregation by disrupting spindle elongation(A) Live-cell images of control, MKLP1- and MKLP2-siRNA co-depleted and MKLP1- and MKLP2 siRNA co-depleted 40-µM STLC-treated RPE-1 cells stably expressing CENP-A-GFP and centrin1-GFP. kin, kinetochore; cen, centrosome. White arrowheads indicate centrioles. Horizontal gray bars in the kymographs indicate the onset of anaphase.(B) Quantification of velocity of spindle elongation (n = 34, 17, 12, and 11 cells, from left to right). Three independent experiments for every condition regarding siRNAs or non-targeting treatments, while number of independent experiments regarding STLC treatment is equal to the number of cells. Black data dots in every treatment correspond to the measurements from the exemplar cells shown on the time-lapse images and kymographs (A). Statistics: t test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).(C) Quantification of net spindle elongation from anaphase onset to 5-min afterward for treatments as indicated (n = 7, 10, 9, and 10 cells, from left to right). Time is shown as minutes. Images are the maximum projection of the acquired z stack. Time 0 represents anaphase onset. Horizontal scale bars, 1 µm. Vertical scale bars, 1 min.(D) Immunofluorescence images of fixed RPE-1 cells depleted of target proteins by treatment with indicated siRNAs stably expressing CENP-A-GFP and centrin1-GFP (magenta) stained with AlexaFluor594 conjugated with antibody specific to the indicated target protein (green KIF4A and red MKLP2). Chromosomes were stained with 1 µg/mL DAPI solution (cyan). Scale bars, 2 µm (D).(E) Quantification of mean signal intensity measured in the spindle midzone of KIF4A (top) and MKLP2 (bottom) antibodies conjugated with AlexaFluor594 versus spindle length in the indicated conditions (8 MKLP2 depleted and 5 control cells). Two independent experiments for every condition regarding siRNAs or non-targeting treatments. See also Figure S4.

Fig 5: Chromosome segregation is compromised after KO of PRC1 and inhibition of EG5(A) Live images of RPE-1 cells in PRC1 non-induced KO, induced PRC1 CRISPR KO and induced PRC1 KO treated with 40-µM STLC imaged 4 days after doxycycline induction. 100-nM SiR-DNA (cyan) was used for chromosome staining.(B) Quantification of chromosome segregation velocity (see scheme) in CRISPR experiments (n = 14, 11, and 11, from left to right). Two independent experiments for every condition except PRC1 KO + Eg5 inh. which was done in three independent experiments, while number of independent experiments regarding STLC treatment is equal to the number of cells. Black data dots in every treatment correspond to the measurements from the exemplar cells shown on the time-lapse images and kymographs (A). Statistics: t test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).(C) Live-cell images of induced RPE-1 PRC1 CRISPR KO and induced PRC1 KO treated with 40-µM STLC. Dashed lines designate the cell borders. 100-nM SiR-DNA was used for chromosome staining. Time is shown as minutes. Images are maximum projections of the acquired z stack. Time 0 represents anaphase onset. Scale bars, 1 µm. See also Figure S2.