Fig 2: MKlp2 localizes to the equatorial cortex prior to furrow ingression and promotes furrow ingression.(A, B) Immunofluorescence analysis. Asynchronously growing HeLa cells were fixed with ice-cold methanol (A, panel a; B) or 10% TCA (A, panel b) and stained with the indicated antibodies. Arrows indicate the equatorial cortex. (B) X-Z shows a cross-section at the equator (white arrow) with an intensity profile of the corresponding section (bottom). (C) Images of Dox-induced Flag-MKlp2 in MKlp2-depleted HeLa cells using siRNA transfection. Dox-inducible HeLa cells were transfected for 4 h with siRNA to depleted endogenous MKlp2. Subsequently, cells were treated with Dox for 20 h before fixing with ice-cold methanol. Flag-MKlp2(1-890) accumulated at the equatorial cortex as indicated with arrows (panel a), whereas Flag-MKlp2(1-842) only showed punctate staining patterns close to the equatorial cortex (panel b, arrow heads in inset). However, both proteins were comparably localized to the spindle midzone. For panels A and C, images were acquired using confocal microscopy. For panel B, images were acquired using 3D-SIM. Insets represent the boxed areas. White bars represent 5 µm. (D) HeLa cells were transfected with non-silencing control siRNA or co-transfected with MKlp1 and MKlp2 siRNAs after the first thymidine block and were treated with Dox during the second thymidine block (see Materials and Methods). The cells were synchronously released from the G1/S boundary and subjected to time-lapse live-cell imaging. The cytokinesis progression of cells that were selected at random, and the time they spent in ingression before regression is plotted (top graph). The average duration of furrow in ingression is based on three independent experiments (total n>100 per condition, +/− standard deviation) (bottom graph). To determine the statistical significance of the duration of furrow in ingression, Student’s t-test was performed. P values are indicated.

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: MKlp2 recruits and provides continuous Aurora B kinase activity to the growing furrow for furrow propagation and completion.(A–D) Time-lapse live-cell images. HeLa cells were transfected with expression vectors encoding mCherry-MKlp2 and GFP-UtrCH (cortical marker), and 24 h after transfection, the cells were subjected to monopolar cytokinesis. In panel A, arrows indicate where a typical polarized furrow was completed. Asterisks indicate the site where mCherry-MKlp2 accumulated at the cell cortex and the furrow. Arrow heads in panels A, C and D denote the sites of the growing furrow before adding ZM447439 or hesperadin. For panel B, Purv A and ZM447439 were added concurrently. For panels C and D, ZM447439 or hesperadin was added sequentially after purvalanol A treatment for 10 min. The fate of the same cell was continuously monitored. White bars represent 5 µm. (E) Monopolar cells (from A–D) were scored as cells that completed monopolar cytokinesis (polarized MPC), formed ectopic furrows without a polarized cortex (non-polarized with ectopic furrowing), no significant furrowing activity without a polarized cortex (non-polarized without furrowing), or formed polarized furrows but subsequently regressed (regressed after polarization). The average percentages based on three independent experiments (total n>100 per condition, +/− standard deviation) are shown. (F) Proposed model. (a) In monopolar cytokinesis, inhibition of Cdk1 triggers a symmetry-breaking reaction that initiates polarization of the monopolar spindles and the cell cortex. Although monopolar spindles are symmetrically positioned, they become asymmetric upon Cdk1 inhibition. Thus, the original event triggering the asymmetry of monopolar spindles remains unclear. (b) During this reaction, MKlp2 mediates Aurora B translocation from the monopolar spindles (or the spindle midzone) towards the cell cortex (or the equatorial cortex) in an interdependent manner. At the ends of monopolar spindles that contact the cell cortex, MKlp2 may act as the bridge between the asymmetrically polarizing monopolar spindles and the actomyosin filaments at the cell cortex. This event may stabilize the polarizing monopolar spindles towards the cell cortex. In turn, it facilitates a rapid accumulation of MKlp2-Aurora B to the cell cortex in a polarized manner. This cortical accumulation of MKlp2-Aurora B promotes furrow formation (or might constrict contractile ring in bipolar cytokinesis) via the kinase activity of Aurora B towards currently unidentified target(s). (c) Once the furrow is formed, MKlp2 targets Aurora B to actomyosin filaments in the gap region between the stably polarized monopolar spindles and the furrowing cortical cap. This event may continuously provide Aurora B kinase activity to the growing furrow for propagation and completion. Interestingly, the kinase activity of Aurora B at the growing furrow may also be essential for maintaining the monopolar spindles polarized towards the furrow (Movie S1, S2), indicating that positive feedback loops may exist that maintain polarization between the monopolar spindles and the growing furrow.

Fig 5: KIF20A interacts with Myosin II and stabilizes Myosin II at the Golgi. a Yeast two-hybrid interactions between the tail domain of Myosin II (1147–1653 fragment) and different domains of KIF20A. The Saccharomyces cerevisiae reporter strain L40 was co-transformed with a plasmid encoding fusion proteins to detect interactions between amino acids 1147 and 1653 of human myosin IIA heavy chain and the motor domain (1–530), the tail domain (529–887), and the RAB6 binding domain (RBD) (529–665) of KIF20A. Growth on medium lacking histidine (-W -L -H) indicates an interaction between the encoded proteins. b Endogenous Myosin II is pulled-down by GFP-KIF20A. GFP-KIF20A or GFP-transfected HeLa cell extracts were immunoprecipitated using the GFP-trap system. Myosin II bound to GFP-KIF20A was revealed by western blot analysis using anti-Myosin II antibody. GFP-KIF20A and KIF20A were revealed by anti-KIF20A antibody. Note that endogenous KIF20A is found in the IP fraction because it likely can form dimers with GFP-KIF20A. Input represents a 5% load of the total cell extracts used in all conditions. c Staining for endogenous Giantin (green) and endogenous Myosin II using the AD7 antibody (red) in Rat Clone 9 cells 3 days after transfection with specific KIF20A siRNAs. Bar, 10 µm. Of note, KIF20A is required for cytokinesis and KIF20A depletion leads to binucleated cells56,57. Quantification of Golgi-associated Myosin II fluorescence intensity in cells treated as described above (mean ± SEM, n = 23–66 cells). *P = 0.02 (Student’s t test). MW molecular weight in kDa