Fig 2: MKlp2 localizes to the growing furrow in a myosin-II-dependent manner, and it is required for cell polarization during monopolar cytokinesis.(A) Immunofluorescence analysis of HeLa cells before (panel a) or during monopolar cytokinesis (panels b-d). Asynchronously growing HeLa cells were treated with monastrol for 6 h and then treated with Purv A or DMSO for 15 min before fixation in ice-cold methanol. (B, C) HeLa cells were transfected with the indicated siRNAs for 20 h before the addition of monastrol, and monopolar cytokinesis was induced as described in panel A. For panel B, HeLa cells stably expressing GFP-α-tubulin were used. For panels A–C, images were acquired using 3D-SIM. (D) The average percentages calculated based on three independent experiments using non-polarized, polarized (determined by cortically localized Aurora B from panel C) or furrowed cells (determined by myosin-II) are shown with error bars (n>100 cells per condition). (E) Time-lapse live-cell imaging. HeLa cells were transfected with the indicated siRNAs together with the vectors encoding GFP-UtrCH to monitor cortical changes and were subjected to monopolar cytokinesis. Arrows indicate the site where a typical polarized furrow was formed and progressed. (F) Monopolar cells (from panel E) were scored as cells that completed monopolar cytokinesis (polarized MPC) or failed without polarizing the cortex (non-polarized with no MPC). The average percentages based on three independent experiments (total n>100 per condition, +/− standard deviation) are shown. Images were acquired using 2D-SIM. White bars represent 5 µm.

Fig 3: MKlp2 is a novel binding partner of myosin-II in vivo and in vitro.(A) Asynchronously growing HeLa cells were harvested and subjected to immunoprecipitation analysis using antibodies for pre-immune control IgG (lanes 1, 4), myosin-II (lanes 2, 3), and MKlp2 with (lane 5) or without (lane 3) HeLa cell lysates. Note that the 100 kDa band is specific for MKlp2 (lane 2) and not caused by α-Myosin-II antibodies used for immunoprecipitation as it is not detected in lane 3. N.S. indicates non-specific. (B–D) Asynchronously growing HeLa cells were transfected with the indicated expression plasmids, and 24 h after transfection, HeLa cell lysates expressing the indicated MKlp2 or myosin-II proteins were subjected to immunoprecipitation with the indicated antibodies. (E) Autoradiography of in vitro-translated Myc-Myosin-II precipitates with the indicated GST-MKlp2 proteins using GST-pulldown analysis (bottom, visualized with Coomassie Blue staining). Overall, 10% of the input for total in vitro-translated product is shown. (F) F-actin binding assay. Asynchronously growing HeLa cells were transfected with the indicated expression plasmids, and 24 h after transfection, HeLa cell lysates expressing the indicated HA-MKlp2 proteins were supplemented without (lanes 1-6) or with in vitro-polymerized recombinant F-actin (lanes 7-12) and subjected to ultracentrifugation. Supernatant (S) and pellet (P) fractions were subjected to immunoblot analysis.

Fig 4: 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 5: 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.