Wandke C , Barisic M , Sigl R , Rauch V , Wolf F , Amaro AC , Tan CH , Pereira AJ , Kutay U , Maiato H , Meraldi P , Geley S .

W 1101 Austrian Science Fund FWF, FWF_W 1101 Austrian Science Fund FWF

	Figure 1. Chromokinesin knockdown results in prometaphase arrest. (A) Total cell extracts prepared from HeLa cells 36 h post-transfection (pt) with siRNAs were analyzed by immunoblotting for KIF4A, hKID, and α-tubulin. (B) HeLa cells transfected with 50 nM of siRNAs targeting hKID, KIF4A, or both (KK) were methanol-fixed 40 h pt, DNA stained, and the mitotic index and (C) the mitotic phases determined (mean of three independent experiments + SEM). (D) Maximum intensity projections (MIP) of deconvolved z-stacks showing representative mitotic spindles of different siRNA treatments. Arrows indicate the misoriented chromosome arms and uncongressed chromosomes in hKID/KIF4A RNAi. Arrowheads mark stabilized, aberrant nonkinetochore MTs in KIF4A knockdown cells. Note the increased interkinetochore distance (sister separation) in KIF4A and KK knockdown cells (arrowhead with asterisk). Bar, 5 µm. (E) Quantification of chromosome congression failures from mitotic cells of three independent experiments. (F) HeLa cells were transfected with different siRNAs and treated with 15 nM nocodazole or solvent 24 h later. After 12 h treatment, cells were imaged and analyzed for their mitotic index. Mean ± SEM of three independent experiments. Student’s t test significance values: *, P < 0.05; **, P < 0.01; ***, P < 0.001.
	Figure 2. Generation of a human KIF4A knockout cell line. (A) Cloning strategy for the generation of a recombinant adeno-associated virus (rAAV) gene targeting construct. (B) HCT116 WT and KIF4A KO clone F1 were analyzed by PCR (top), immunoblotting (middle), and immunofluorescence (bottom), demonstrating correct targeting and the resulting loss of KIF4A protein. (C) HCT116 wild-type and KIF4A KO cells were immunostained for α-tubulin (green), kinetochores (red), pericentrin (pink), and DNA (blue) and confocal z-stacks of anaphase and telophase cells acquired. Deconvolved MIPs are shown. Arrows indicate disorganized midzone MTs. Bar, 5 µm. (D) HCT116 wild-type and five different KIF4A KO clones were seeded at 0.5 × 105 cells per well and cell numbers determined over 7 d. Graphs represent the mean of four independent experiments (±SEM). (E) HCT116 wild-type and KIF4A KO cells were monitored by phase-contrast video live-cell microscopy during a period of 16 h. Mitosis was slightly prolonged in HCT116 KIF4A KO cells as shown in the box-and-whisker plot. (F) Cells from E were classified dependent on their fate during and after mitosis, respectively.
	Figure 3. HCT116 KIF4A knockout cells show mitotic delay after depletion of hKID. (A) Selected spindles of HCT116 wild-type and KIF4A KO cells after treatment with mock or hKID-siRNA. Shown are MIPs of deconvolved confocal z-stacks. Arrows indicate aberrant nonkinetochore MTs in KIF4A KO cells. Bar, 5 µm. (B) HCT116 wild-type or KIF4A KO cells were transfected with 50 nM mock or hKID-siRNA, methanol-fixed 40 h pt, and stained for DNA. Mitotic index and mitotic phases of four independent experiments + SEM are shown in B and C, respectively. *, Student’s t test value P < 0.05; **, P < 0.01.
	Figure 4. Loss of chromokinesins causes misorientation and entanglement of chromosome arms. Selected frames from 4D fluorescence microscopy time-lapse sequence of HeLa H2B-GFP cells 30 h after hKID/KIF4A siRNA transfection. 30 z-sections were taken every 15 s (for a total time of 30 min), processed by deconvolution, and carefully analyzed in 3D over time using Imaris to distinguish different chromosomes. The first frame shows a cell with unaligned chromosomes stuck at the poles. The white box indicates the area of the inset with three chromosomes, labeled in blue, red, and green. The following frames show that these chromosomes remain stuck although their arms get extended as if pulled from the opposite spindle pole. Only after a long delay, the red chromosome becomes free and congresses to the metaphase plate. Time is min:s. Bar, 5 µm.
	Figure 5. Loss of hKID and KIF4A induces irregular chromosome oscillations. HeLa CenpA-EGFP cells were transfected with 50 nM siRNA targeting hKID, KIF4A, or both and imaged 40 h pt. Three independent live-cell imaging experiments were performed for each siRNA treatment. Kinetochore behavior and dynamics were determined using MaKi software. (A) Interkinetochore distance was analyzed from cells of three independent experiments. Columns represent mean + SD. (B) Time-lapse movie frames of kinetochore pairs of control (mock transfected), hKID, KIF4A, and KK siRNA-transfected cells. Boxes show enlarged views of kinetochore pairs. Bar, 2 µm. (C) Oscillatory movements of selected sister pairs after different siRNA treatments were plotted against time (in frames; one frame is 7.5 s). Shown are the x-coordinates of each sister relative to the metaphase plate. Arrows indicate irregular breathings. ***, Student’s t test value P < 0.001.
	Figure 7. Loss of KIF4A decreases MT dynamics. (A) HeLa cells were transfected with KIF4A RNAi or mock, stained for EB1 and kinetochores (CREST antiserum) and imaged by confocal microscopy. Representative maximum intensity projections are shown. Bar, 10 µm. (B) HeLa EB1-GFP cells were transfected with either KIF4A siRNA or mock and imaged 36 h pt. MT plus-ends, marked by EB1, were followed over several frames and MT length and stability were calculated. Bar, 5 µm. Shown is mean of three independent experiments ± SEM.
	Figure 8. Loss of KIF4A decreases MT flux. (A) DIC and fluorescence images show representative control and KIF4A-depleted cells in late prometaphase/metaphase. Images were captured before (preconversion) and at various time points (indicated in minutes and seconds) after photo-conversion of mEos-α-tubulin. MT flux was measured by photo-conversion of regions from both half-spindles in U2OS-mEos-tubulin cells and photo-converted mEos-tubulin was tracked over a time interval of 3 min (arrows). Bottom panels of each merged frame show separated green and red channels. Bar, 5 µm. (B) Representative fluorescence intensity curves of control vs. KIF4A-depleted U2OS-mEos-tubulin cell created immediately after photo-conversion and 120 s later. Fluorescence intensity peaks are generated by manual estimation of the peak region, followed by fitting of the intensity data points to a parabola. The maximum of the parabola was used as the effective peak position. (C) Both KIF4A and KIF4A/hKID double-knockdown late prometaphase/metaphase U2OS-mEos-tubulin cells display significant reduction of kinetochore-MT poleward flux in comparison with controls (P < 0.001). Error bars represent SEM derived from three independent experiments each containing 11–26 cells.
	Figure 6. Loss of KIF4A increases sister separation and prolongs the breathing period. (A) Kinetochore distribution across a fitted metaphase plate was determined by measuring the sister center position and kinetochore positions. Both values are displayed as the twofold standard deviation of the mean ± SD. (B and C) Dynamic parameters were determined by calculating the sister displacement cross-correlation (B) and sister kinetochore autocorrelation values (C). Methanol-fixed wild-type cells (gray, dashed line) were used as negative control. (D) Oscillation and breathing speed for different siRNA treatments. Columns represent mean + SD. (E) Table summarizing the kinetochore oscillation parameters. *, Student’s t test value P < 0.05; **, P < 0.01; ***, P < 0.001.
	Figure 9. Model for chromokinesin function in chromosome congression. The left-hand side of the scheme depicts the proposed functions of hKID and KIF4A. On aligned chromosomes, hKID stabilizes the orientation of chromosome arms and by generating PEF that pushes the spindle poles apart. KIF4A limits the elongation of MTs near chromatin and could thereby control the number of MT plus-ends interacting with chromosomes or with MTs from the opposite pole (sliding interpolar MTs). Via MT cross-linking proteins MT-dependent forces are distributed throughout the spindle, which is required for regular chromosome oscillations. Reduced kinetochore MT flux after loss of KIF4A could either be due to changes in the dynamics of MTs interacting with chromosome arms or could be caused by a tighter binding of MTs to kinetochores due to kinetochore stretching (yellow symbols). In combination with loss of hKID, this results in chromosome attachment, congression, and segregation failures.

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