Fig 1.
KBP is a novel interaction partner of Kif15.
(A-B) Non-transfected or stable Flag-Kif15 expressing HEK293T cells were synchronized in mitosis (A) or interphase (B) and the corresponding cell lysates were used for anti-Flag immunoprecipitation. Cell lysates and immunoprecipitated proteins were resolved by SDS-PAGE and detected by immunoblotting with anti-Kif15 or anti-KBP antibodies. (C) Cell lysates of HEK293T cells synchronized in mitosis were subjected to anti-KBP immunoprecipitation. Cell lysates and immunoprecipitated proteins were resolved by SDS-PAGE and detected by immunoblotting with anti-Kif15 and anti-KBP antibodies. (D) Non-transfected or transiently expressing Flag-Kif15 HEK293T were synchronized in mitosis. Cell lysates were collected and incubated in the presence or absence of λ-phosphatase. Treated and non-treated cell lysates were subjected to Flag immunoprecipitation. Cell lysates and immunoprecipitated proteins were resolved by SDS-PAGE and detected by immunoblotting with anti-Kif15 and anti-KBP antibodies. (E) HEK293T cells were transiently transfected with GFP-KBP and different truncation constructs of Flag-Kif15 and synchronized in mitosis. Cell lysates were subjected to Flag immunoprecipitation. Cell lysates and immunoprecipitated proteins were resolved by SDS-PAGE and detected by immunoblotting with anti-Flag and anti-KBP antibodies. (F) Western blot analysis of selected fractions from gel filtration and sucrose gradient centrifugation experiments using purified recombinant Kif15-M-His, KBP-His individually or in combination as indicated on the left. Proteins were detected by immunoblotting using anti-His (Kif15-M) and anti-KBP antibodies as indicated on the right. The sucrose gradient fractions corresponding to 6 to 20% sucrose are shown.
Fig 2.
KBP promotes localization of Kif15 to the spindle midzone.
(A) Left panel: Immunoblot of SDS-PAGE resolved HeLa cell lysate with affinity-purified anti-KBP antibody. One single band of about 72kDa is recognized. Right panel: Immunoblot of control or KBP silenced HeLa cell lysates. Membranes were probed with anti-KBP and anti-tubulin, as a loading control, antibodies. (B) Cell cycle regulation of KBP expression. HeLa cells were synchronized by double thymidine block and released for the indicated time periods. Cell lysates were SDS-PAGE resolved and immunoblotted with anti-KBP antibody. Anti-Cyclin B1 staining was used to monitor progression through the cell cycle and anti-tubulin staining was used as a loading control (* indicates the Cyclin B1 signal). (C) Immunofluorescence showing representative images of prometa-, meta-, ana- and telophase cells. Cells were pre-extracted, fixed and stained with anti-KBP and anti-tubulin antibodies (scale bar, 5 μm). (D) Immunofluorescence showing representative images of metaphase cells in control silenced or KBP silenced cells. Cells were pre-extracted, fixed and stained with anti-KBP and anti-tubulin antibodies (scale bar, 5 μm). (E) Upper panel: Immunofluorescence of control, KBP, Ki67 or KBP/Ki67-silenced cells showing the reduced Kif15 signal at the metaphase plate (scale bar 5, μm). Lower panel: Quantification of Kif15 signal in a region central in the metaphase spindles (as indicated by the grey box in the scheme). Kif15 signal was measured as intensity profiles along the spindle axis. Plotted is the average Kif15 signal with standard deviation (four independent experiments with at least 39 cells per condition, per experiment for KBP silencing, and one experiment with at least 61 cells per condition for Ki67 or KBP/Ki67 silencing).
Fig 3.
KBP and Kif15 function in efficient chromosome alignment.
(A) Scheme outlining the experimental set-up of the live-cell imaging. (B) Quantification of prometaphase duration as observed in the live-cell imaging in control (C), KBP-, Kif15- or KBP/Kif15-silenced cells. Prometaphase was defined as the time needed for cells to progress from NEBD till anaphase onset. Time points are represented in box-and-whisker plots. Boxes show the upper and lower quartiles (25–75%) with a line at the median, whiskers extend from the 5th to the 95th percentile. Dots represent the outliers (data were compared using Mann-Whitney U tests, ** means p ≤ 0.01, **** means p ≤ 0.0001). (C) Representative stills from the live-cell imaging of control, KBP-, Kif15- or KBP/Kif15-silenced cells. Arrowheads highlight chromosomes that show less efficient alignment to the metaphase plate. Time is indicated in the upper right corner as hh:mm. Scale bars represent 5μm.
Fig 4.
Chromosomally localized Kif15 is required for chromosome alignment.
(A) Representative stills from the live-cell imaging of Ki67-silenced cells. Arrowheads indicate chromosomes that show less efficient alignment to the metaphase plate. Time is indicated in the upper left corner as hh:mm. Scale bar represents 5μm. (B) Quantification of prometaphase duration as observed in the live-cell imaging in control or Ki67-silenced cells. Prometaphase was defined as the time needed for cells to progress from NEBD till anaphase onset. Time points are represented in box-and-whisker plots. Boxes show the upper and lower quartiles (25–75%) with a line at the median, whiskers extend from the 5th to the 95th percentile. Dots represent the outliers (data were compared using Mann-Whitney U tests, **** means p ≤ 0.0001). (C) Upper panel: Representative immunofluorescence images showing bipolar spindles with fully aligned or misaligned chromosomes. Lower panel: Quantification of chromosome alignment in control, KBP, Kif15 or Ki67-silenced cells (scale bar 5, μm). Cells with bipolar spindles were categorized based upon the degree of chromosome alignment, i.e. fully aligned chromosomes (metaphase cells), misaligned chromosomes (most chromosomes have congressed to the metaphase plate, few chromosomes are not aligned yet). Graph shows the average and standard deviation of three independent experiments with at least 61 cells counted per condition, per experiment (data were analyzed using t tests, no significant differences were observed).
Fig 5.
KBP and Kif15 affect K-fiber stability.
(A) Left panel: Immunofluorescence images of Nuf2-silenced cells showing three different categories of chromosome alignment, i.e. siNuf2 spindles with all chromosomes aligned (fully aligned), with most of the chromosomes aligned (mostly aligned) or with scattered chromosomes (severely misaligned) (scale bar, 5 μm). Right panel: Quantification of the proportion of cells with chromosomes aligned following the specified classification upon in Nuf2 silencing or Nuf2 and Kif15, KBP or Ki67 co-silencing. Graph represents the averages and standard deviations of four independent experiments, counting at least 100 cells per condition, per experiment (data were analyzed using t tests, no significant differences were observed). (B) Scheme outlining the set-up of the K-fiber measurement experiments. (C) Left panel: Immunofluorescence showing representative cells of control, KBP or Kif15-silenced cells treated with STLC and cold before fixation (scale bar, 5 μm). Right panel: Quantification of K-fiber lengths as measured in the immunofluorescence images, represented in box-and-whisker plots. Boxes show the upper and lower quartiles (25–75%) with a line at the median, whiskers extend from the 5th to the 95th percentile. Dots represent the outliers. Data from three independent experiments with at least 20 cells per experiment analyzed (>314 K-fibers measured per condition, per experiment; data were compared using Mann-Whitney U tests, **** means p ≤ 0.0001). (D) Left panel: Immunofluorescence images showing representative cells with intact versus weak K-fibers after 10 min cold treatment before pre-extraction (5 min on ice) and fixation (scale bar, 5 μm). Right panel: Quantification of the normalized tubulin fluorescence intensity in cold-treated spindles upon control, Kif15, KBP or Ki67 silencing. Data are represented in box-and-wisker plots. Boxes show the upper and lower quartiles (25–75%) with a line at the median, whiskers extend from the 5th to the 95th percentile. Dots represent the outliers. Data from three independent experiments with at least 52 cells quantified per condition, per experiment (data were compared using Mann-Whitney U tests, **** means p ≤ 0.0001).