1. Volume 28, Issue 17, Pages 2685-2696.e4 (September 2018)
Loss of Kif18A Results in Spindle Assembly Checkpoint Activation at Microtubule- Attached Kinetochores 
Louise M.E. Janssen, Tessa V. Averink, Vincent A. Blomen, Thijn R. Brummelkamp, René H. Medema, Jonne A. Raaijmakers 
Current Biology 
Volume 28, Issue 17, Pages e4 (September 2018)
DOI: /j.cub
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2. Current Biology 2018 28, 2685-2696.e4DOI: (10.1016/j.cub.2018.06.026)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1
The Identification of Essential Genes Reveals KIF18A and ASTRIN as Synthetic Viable Interactors with SAC Deficiency
(A) Representative plots of individual insertional mutagenesis screens in HAP1 WT, ΔMad1, and ΔMad2 cells. Each dot represents a single gene, and the number of integrations per gene is plotted on the x axis. On the y axis, the relative amount of sense integrations over the total amount of integrations is plotted. The synthetic viable hits KIF18A and ASTRIN are highlighted in red.
(B) The average ratio of sense gene trap integrations over the total amount of integrations is plotted for both KIF18A and ASTRIN. The data are extracted from 4 individual WT, 2 individual ΔMad1, and 2 individual ΔMad2 insertional mutagenesis screens. Error bars represent SD of two independent experiments.
(C) Colony formation assay of HAP1 WT and ΔMad2 cells treated with non-targeting siRNA (siNT) or siKif18A for 5 days, stained with crystal violet and quantified with Fiji.
See also Figures S1 and S4.
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4. Figure 2
Depletion of Kif18A Leads to a Prolonged Mitotic Arrest and Subsequent Cell Death
(A) Mitotic timing and cell fate of individual cells followed by live-cell imaging of 3 independent experiments (n = ∼90 cells per condition). Each dot represents an individual cell. Cell fate was determined by either anaphase progression (NEB-Ana), start of NEB followed by interphase without anaphase (slippage), or DNA hypercondensation and fragmentation (cell death). Bars represent average time spent in mitosis.
(B) Representative live-cell microscopy images of WT HAP1 cells treated with siNT or siKif18A for 48 hr. After siKif18A treatment, chromosome oscillations become visible (indicated with black arrow). One example is shown where cells progress into anaphase after a prolonged mitosis without segregation errors (t = 105), and one example is shown where prolonged mitosis results in cell death, indicated by DNA fragmentation at t = 295. The scale bar represents 10 μm.
(C) Quantification of chromosome segregation errors in WT HAP1 cells treated with siNT or siKif18A for 48 hr, ΔMad2, and two different ΔMad2ΔKif18A clones: cA1 and cB1. Segregation errors were scored during live-cell imaging and consist of anaphase bridges and lagging chromosomes. Error bars represent SD of 3 independent experiments (n = ∼90 per condition). An unpaired Student’s t test showed no significant difference between WT HAP1 cells and WT HAP1 cells treated with siKif18A or a difference between ΔMad2 and the ΔMad2ΔKif18A clones A1/B1.
(D) Western blot analysis confirmed the successful generation of Kif18A and Mad2 double-knockout (ΔMad2ΔKif18A) generation in HAP1 cells. Cell lysates were immunoblotted for Kif18A, Mad2, and α-tubulin.
(E) Doubling time as determined with a Lionheart microscope (1 independent experiment with samples plated in triplicate). An unpaired Student’s t test showed a significant difference between WT and ΔMad2 cells (∗p < 0.05). No significant difference in doubling time between ΔMad2 and the ΔMad2ΔKif18A clones A1/B1 was observed.
See also Figures S1–S4 and Videos S1, S2, S3, S4, and S5.
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5. Figure 3
DOX-Inducible MAD2 Expression Induces Lethality in ΔKIF18AΔMAD2 Cells
(A) Western blot analysis confirmed the doxycycline (DOX)-inducible GFP and Mad2 expression in WT, ΔMad2, ΔMad2ΔKif18A_A1, and ΔMad2ΔKif18A_B1 cells. Cell lysates were immunoblotted for GFP and Mad2. s.e. indicates short exposure; l.e. indicates long exposure.
(B) Mitotic timing of nocodazole-treated WT HAP1 and ΔMad2ΔKif18A cells, treated with or without DOX to induce Mad2 expression (n = ∼15 cells per condition in 1 independent experiment).
(C) Mitotic timing and cell fate of individual cells treated with or without DOX (8 hr prior to live-cell imaging) to induce Mad2 expression (n = ∼30 cells per condition in one independent experiment). Each dot represents an individual cell.
(D) Quantification of segregation errors of cells shown in (C).
(E) Percentage of GFP (and thus Mad2) expressing cells over time, determined by FACS over a period of 7 days.
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6. Figure 4
Mad1-Positive Kinetochores of Kif18A-Depleted Cells Display Decreased Tension
(A) Representative images of the quantification of the number of Mad-positive kinetochores in ΔMad2ΔKif18A_A1 cells. The scale bar represents 5 μm.
(B) Quantification of immunofluorescent staining of Mad1 at kinetochores in WT HAP1 cells treated with siNT (48 hr), Eg5 inhibitor STLC (10 μM; 4 hr), nocodazole (50 nm; 4 hr), siKif18A (48 hr), and ΔMad2ΔKif18A_A1/B1 cells. All conditions were pre-treated for 1 hr with MG132 before fixation to arrest cells in mitosis. Error bars represent SD of 4 independent experiments (n = ∼15 cells and 100 kinetochores per condition).
(C) Measurements of inter-kinetochore (KT) distance of WT HAP1 cells treated with siNT (48 hr), Eg5 inhibitor STLC (10 μM; 4 hr), nocodazole (50 nM; 4 hr), siKif18A (48 hr), and ΔMad2ΔKif18A_A1. Measurements between Crest pairs were separated in Mad1-positive and Mad1-negative kinetochores. Error bars represent SD of minimally 15 kinetochore pairs per condition. p values were determined by unpaired Student’s t test comparing all experimental conditions against WT + siNT. ∗∗∗∗p <
(D) Measurements of intra-KT distance of WT HAP1 cells treated with siNT (48 hr), Eg5 inhibitor STLC (10 μM; 4 hr), nocodazole (50 nM; 4 hr), ΔMad2ΔKif18A_A1, and ΔMad2ΔKif18A_B1. Distance measurements between Hec1 and CENPC were separated in Bubr1-positive and Bubr1-negative kinetochores. Error bars represent SD of minimally 15 kinetochore pairs per condition. p values were determined by unpaired Student’s t test comparing against WT + siNT. ∗∗∗∗p <
(E) Representative images of immunofluorescent staining of astrin, Mad1, and Crest in WT HAP1 cells treated with siNT or siKif18A for 48 hr. The scale bar represents 5 μm.
(F) Quantification of astrin levels on Mad1-positive and Mad1-negative kinetochores in WT HAP1 cells. Error bars represent SD of 15 kinetochore pairs in 1 experiment.
(G) Quantification of astrin levels on Mad1-positive and Mad1-negative kinetochores in ΔMad2ΔKif18A cells. Error bars represent SD of 15 kinetochore pairs in 1 experiment.
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7. Figure 5
Kif18A Loss Results in Recruitment of Mad1 in the Presence of Microtubule Connections
(A) Representative immunofluorescent images of WT HAP1, WT HAP1 nocodazole-treated (50 nM; 4 hr), and ΔMad2ΔKif18A_A1 cells, stained for tubulin, Mad1, and Crest. The scale bars represent 5 μm in main figure and 0.8 μm in inlay.
(B) Quantification of the amount of k-Mt attachments in Mad1-positive kinetochores of WT HAP1 cells treated with Eg5-inhibitor STLC (10 μM; 4 hr), nocodazole (50 nM; 4 hr), siKif18A (48 hr), and ΔMad2ΔKif18A_A1 cells. All conditions were pre-treated with MG132 for 1 hr, after which cells were calcium treated and fixed before staining the cells for microtubules (α-tubulin), SAC activity (Mad1), and centromeres (Crest). Error bars represent SD of 3 independent experiments (n = 20 cells and 120 kinetochores per condition). p values were determined by paired t test, comparing Kif18A-depleted cells against WT HAP1 cells treated with Eg5 inhibitor STLC. ∗∗∗p <
(C) Graphs showing measurements of k-fiber thickness and intensity (n = 5 cells and ∼25 kinetochores per condition) in WT HAP1 cells treated with Eg5 inhibitor STLC (10 μM; 4 hr), nocodazole (50 nm; 4 hr), siKif18A (48 hr), and ΔMad2ΔKif18A_A1 cells, all treated for 1 hr with MG132 before fixation.
See also Figures S1, S3, and S4.
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8. Figure 6
Loss of Kif18A Results in SAC Activation at Microtubule-Attached Kinetochores
Schematic overview of hypothetical model of Kif18A loss. In WT cells, Kif18A is recruited to the plus ends of microtubules when chromosomes are properly aligned at the metaphase plate, leading to a reduction of chromosome oscillations, stabilization of k-Mt attachments, and the generation of tension, which silences the SAC and promotes anaphase onset. Upon loss of Kif18A, the oscillatory movements of chromosomes are no longer reduced and tension defects are found at the checkpoint signaling kinetochores. Thus, the tension defect is able to activate or maintain the SAC, leading to a mitotic arrest and subsequent cell death without destabilizing the k-Mt attachment.
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