Table 1.
Composition of samples for phosphatase treatment.
Fig 1.
Mitotic delay in CAMSAP2 knockout Caco-2 cells.
(A) Endogenous localization of CAMSAP2 and CAMSAP3 in Caco-2 cells. Cells were fixed with methanol and stained for CAMSAP2 or CAMSAP3 (red), α-tubulin (green), and DAPI (blue). Boxed regions have been enlarged, brightness-adjusted, and shown in insets to visualize CAMSAP2 and CAMSAP3 punctae at the end of microtubules. Scale bars; 10 μm. (B) Western blots showing mitotic phosphorylation of CAMSAP2 and CAMSAP3. Caco-2 WT cells were synchronized with STLC. Cell homogenates were treated with or without protein phosphatase (λPP) and with or without its inhibitor EDTA. Asynchronized cell lysates were loaded in the far left. Blots with α-tubulin are shown as loading controls. MW, kDa. (C) Growth curves for wild type (WT), CAMSAP2 knockout (KO), and CAMSAP3 KO cells. Cell concentrations at indicated hours after cell seeding were counted. Mean ± SD of three biological replicates is shown at each timepoint. *P < 0.05, Student’s t-test. Three independent experiments were performed, and the representative is shown. (D) Mitotic index of WT, CAMSAP2 KO, and CAMSAP3 KO cells. Cells were fixed with 2% PFA (paraformaldehyde) and stained for α-tubulin (green), phospho histone H3 (red), and DAPI (blue). Arrowheads indicate phospho histone H3 positive chromosomes. Percentages of cells with phospho histone H3 signals in the population were calculated as mitotic index. n = 431–638 cells per experiment, five independent experiments. Boxplots indicate 25th percentile, median, and 75th percentile values. *P < 0.05, n.s.: P > 0.05, Student’s t-test. Scale bar; 20 μm.
Fig 2.
Defective spindle morphology in CAMSAP2 KO cells.
(A) Representative images of WT and CAMSAP2 KO spindles. Notably, 2% PFA-fixed cells were stained for α-tubulin (green), Mad2 (red), and DAPI (blue). Scale bar; 10 μm. (B) The spindle length of cells prepared in (A) measured based on α-tubulin signals. Mad2-negative spindles were exclusively chosen for quantification as of metaphase. n = 79 (WT) and 86 (CAMSAP2 KO) cells from four independent experiments were analyzed. Boxplots indicate 25th percentile, median, and 75th percentile values. ****P < 0.0001, Welch’s t-test. (C) Areas of chromosomes in metaphase cells prepared in (A), measured from DAPI signals. Mad2-negative spindles were exclusively chosen for quantification. n = 79 (WT) and 86 (CAMSAP2 KO) cells from four independent experiments. Boxplots indicate 25th percentile, median, and 75th percentile values. ****P < 0.0001, Welch’s t-test. (D) Orientation of polar microtubules extending inward from the spindle. Representative images acquired with the super-resolution confocal microscope LSM980 Airyscan are shown. Cells were fixed with 2% PFA and stained for α-tubulin (green) and DAPI (blue). The centrosome regions are boxed and enlarged below. Radar charts show distribution of angles of polar microtubules emanating towards chromosomes. Astral microtubules are not included. The origin of microtubules (the spindle pole) was centered in the chart and labels on the radar axis indicate frequencies. The sum of all the frequencies equals 1.0. Each microtubule emanating from the pole (traced in red) is taken to measure its orientation. n = 158 microtubules from 9 cells (WT) and 139 microtubules from 9 cells (CAMSAP2 KO) from four independent experiments. Scale bar; 10 μm. (E) Orientation of astral microtubules as in (D). Boxed regions are enlarged and brightness is adjusted to visualize astral microtubules. Radar charts show distribution of angles of astral microtubules. n = 255 microtubules from 11 cells (WT) and 336 from 12 cells (CAMSAP2 KO) from four independent experiments. Scale bars; 10 μm. (F, G) The length (F) and number (G) of astral microtubules captured in (E) are quantified. For (F), n = 263 microtubules from 9 cells (WT) and 324 from 15 cells (CAMSAP2 KO) from three independent experiments. Boxplots indicate 25th percentile, median, and 75th percentile values. ****P < 0.0001, Welch’s t-test. For (G), n = 9 cells (WT) and 15 cells (CAMSAP2 KO) from three independent experiments. **P < 0.01, Student’s t-test. (H) Definition of the degree of displaced centrosomes. Cells were fixed with 2% PFA and stained for α-tubulin (green), γ-tubulin (red), and DAPI (blue). The γ-tubulin punctate signals were defined as centrosomes, and the distance between two γ-tubulin dots was defined as the γ-tubulin distance (red bidirectional arrows). The distance between two α-tubulin signals at the tips of the spindle was defined as the α-tubulin distance (green bidirectional arrows). Degree of displaced centrosome was calculated as the ratio of γ-tubulin distance to α-tubulin distance. n = 57 (WT) and 59 (CAMSAP2 KO) cells from three independent experiments. Boxplots indicate 25th, median, and 75th percentile values. ****P < 0.0001, Welch’s t-test. Scale bar; 10 μm.
Fig 3.
KIF2A depletion partially suppresses mitotic defects of CAMSAP2 KO cells.
(A) Mitotic index of WT and CAMSAP2 KO cells with (si KIF2A) or without (si Control) KIF2A knockdown. Cells were fixed with 2% PFA and stained for β-tubulin (green), phospho histone H3 (red), and DAPI (blue). Arrowheads, chromosomes with phospho histone H3 signals. Mitotic index was measured as in Fig 1D. n = 490–786 cells per experiment, three independent experiments. Boxplots indicate 25th percentile, median, and 75th percentile values. ***P < 0.001, **P < 0.01, n.s. > 0.05, Two-way ANOVA followed by Tukey’s multiple comparison tests. Scale bar; 20 μm. (B) Spindle length in WT and CAMSAP2 KO cells with and without KIF2A knockdown. Methanol-fixed cells were stained for α-tubulin (green), BubR1 (red), and DAPI (blue). BubR1-negative spindles were exclusively chosen as metaphase cells for size measurement. n = 35 (si-control WT), 42 (si KIF2A WT), 39 (si-control CAMSAP2 KO), and 48 (si KIF2A CAMSAP2 KO) cells from three independent experiments. Boxplots indicate 25th, median, and 75th percentile values. ****P < 0.0001, n.s.: P > 0.05, Two-way ANOVA followed by Tukey’s multiple comparison tests. (C) Astral microtubules in WT and CAMSAP2 KO cells with or without KIF2A knockdown. Cells were fixed with 2% PFA and stained for α-tubulin (green) and DAPI (blue). Representative images acquired using an LSM980 Airyscan are shown. Boxed regions are enlarged below, and brightness is adjusted for visualization of astral microtubules. Scale bars; 10 μm. To quantify the length, n = 160 microtubules from 9 cells (si-control WT), 153 from 9 cells (si KIF2A WT), 181 from 8 cells (si-control CAMSAP2 KO), and 144 from 7 cells (si KIF2A CAMSAP2 KO) from three independent experiments. Boxplots indicate 25th, median, and 75th percentile values. **** P < 0.0001; n.s.: P > 0.05, Two-way ANOVA followed by Tukey’s multiple comparison tests. (D) The position of centrosomes (γ-tubulin signals) in CAMSAP2 KIF2A co-depleted cells. The degree of displaced centrosomes was calculated similarly, as shown in Fig 2H. n = 68 (si-control WT), 65 (si KIF2A WT), 72 (si-control CAMSAP2 KO), and 65 (si KIF2A CAMSAP2 KO) cells from three independent experiments. Boxplots indicate 25th, median, and 75th percentile values. ****P < 0.0001, n.s.: P > 0.05, Two-way ANOVA followed by Tukey’s multiple comparison tests.
Fig 4.
Decreased spindle microtubules in CAMSAP2 KO cells.
(A) Signal intensity of α-tubulin immunostained in WT and CAMSAP2 KO cells quantified along the spindle axis (red lines). Notably, 2% PFA-fixed cells stained for α-tubulin (green) and DAPI (blue). Images analyzed in Fig 2A were used for quantification. Heatmaps indicate gradient fluorescence intensities of α-tubulin signals. Lower graphs show in gray, each spindle; black (WT) and red (CAMSAP2 KO), average of all spindles. Background intensities were subtracted, which were then normalized by the maximum values of the spindles. Mad2-negative spindles were chosen as metaphase ones for quantification. n = 79 (WT) and 86 (CAMSAP2 KO) cells from four independent experiments. Scale bar; 10 μm. (B) Representative images of spindle untreated (control) and cold treated WT and CAMSAP2 KO cells. Notably, 2% PFA-fixed cells were stained for α-tubulin (green), CREST (red), and DAPI (blue). Scale bar; 10 μm. (C) Signal intensities of α-tubulin in spindles of (B). The mean intensity in the spindle excluding poles were measured. The boxed region is shown magnified to the right. The background intensity off the spindle was subtracted. n = 57 (control WT), 71 (cold treated WT), 83 (control CAMSAP2 KO), and 76 (cold treated CAMSAP2 KO) cells from three independent experiments. Boxplots indicate 25th percentile, median, and 75th percentile values. *P < 0.05, control WT vs cold treated WT: Welch’s t-test, n.s.: P > 0.05, control CAMSAP2 KO vs cold treated CAMSAP2 KO: Student’s t-test. (D) γ-tubulin intensities on the spindle. Notably, 2% PFA-fixed cells were stained for α-tubulin (green), γ-tubulin (red), and DAPI (blue). γ-tubulin signal intensities on the spindle excluding the poles were quantified. The background intensity off the spindle was subtracted. n = 88 (WT) and 76 (CAMSAP2 KO) cells from three independent experiments. Boxplots indicate 25th percentile, median, and 75th percentile values. ****P < 0.0001, Welch’s t-test, Scale bar; 10 μm. (E) Visualization of bridging fibers in the spindle. Representative images of single z-planes acquired with LSM980 Airyscan are shown. Notably, 2% PFA-fixed cells were stained for α-tubulin (green), CREST (red), and DAPI (blue). Insets are enlarged with adjusted brightness, showing sister kinetochore fibers and bridging fibers behind the sister-kinetochore fibers (arrowheads). Frequencies of sister kinetochore pairs accompanying bridging fibers behind. n = 56 pairs of kinetochores from 18 cells (WT) and 53 from 14 cells (CAMSAP2 KO) from four independent experiments. Boxplots indicate 25th, median, and 75th percentile values. *P < 0.05, Student’s t-test. Scale bar; 10 μm. (F) α-tubulin intensity of bridging fibers (left) and kinetochore fibers (right). The intensity was measured according to a previous report with some modifications [14]. For the intensities of bridging fibers (left), 38 fibers from 17 cells (WT) and 15 from 10 cells (CAMSAP2 KO) were obtained from three independent experiments. Pairs without visible bridging fibers were excluded from quantification. For kinetochore fibers (right), 86 kinetochore fibers from 17 cells (WT) and 59 from 13 cells (CAMSAP2 KO) from three independent experiments. Boxplots indicate 25th, median, and 75th percentile values. *P < 0.05, Student’s t-test.
Fig 5.
Defects in chromosome segregation with slowed anaphase spindle elongation in CAMSAP2 KO.
(A) Alignment and segregation of chromosomes during mitotic progression of WT and CAMSAP2 KO cells visualized with Hoechst staining. ‘First alignment’ was defined as the timepoint when chromosomes first aligned on the equatorial plane. ‘Anaphase onset’ was defined as the timepoint when the chromosomes began to separate (t = 0). ‘Completion of chromosome separation’ was defined as the timepoint when the chromosome masses completely separated poleward. time; mm:ss, Scale bar; 10 μm. (B) Duration from first alignment to anaphase onset. n = 26 cells from eight independent experiments (WT) and 25 from nine independent experiments (CAMSAP2). (C) Duration from anaphase onset to completion of chromosome separation. Cells showing cytokinetic failures or no segregation (see D) are not included. n = 23 cells from eight independent experiments (WT) and 21 from nine independent experiments (CAMSAP2). Boxplots indicate 25th percentile, median, and 75th percentile values. *P < 0.05, Student’s t-test. (D) Patterns of chromosome segregation in WT and CAMSAP2 KO cells were classified into the indicated categories. Red arrowheads indicate lagging chromosomes and yellow arrowheads indicate a chromosomal bridge. Percentages of each category are shown in the right. n = 26 cells (WT) from eight independent experiments and 26 (CAMSAP2 KO) from nine independent experiments. Scale bar; 10 μm. (E) Time-lapse images of EB1-mCherry2 (red) expressed in WT and CAMSAP2 KO cells. DNA (blue) was stained with Hoechst. Arrowheads indicate spindle poles. Schematics designate how to calculate the relative spindle length at each timepoint. Scale bar; 10 μm. (F) Relative spindle length after anaphase onset calculated as in (E). Gray, each spindle. Black (WT) and red (CAMSAP2 KO), average of all the samples. n = 18 cells (WT) from six independent experiments and 22 cells (CAMSAP2 KO) from five independent experiments. (G) Average speed of spindle elongation for 5 minutes in anaphase shown in (F). n = 16 cells (WT) from six independent experiments and 20 cells (CAMSAP2 KO) from four independent experiments. Boxplots indicate 25th percentile, median, and 75th percentile values. **P < 0.01, Student’s t-test. (H) Temporal kinetics of the spindle elongation speed. Each gray dot corresponds to the value for each spindle. Black (WT) and red (CAMSAP2 KO) lines represent mean values of all the samples. The mean lines are selected to draw the graph at the bottom for simplicity. (G) and (H) share the dataset for quantification.