Journal: Cell reports

Article Title: KIF18A promotes chromosome congression in cooperation with CENP-E downstream of CENP-C

doi: 10.1016/j.celrep.2025.116515

Figure Lengend Snippet: (A) Schematic representation of human CENP-C protein. The Mis12-binding domain (M12BD) of human CENP-C is highlighted in wild-type (WT) CENP-C. Cells expressing CENP-C lacking M12BD (CENP-C ΔM12BD cells) were generated (see also ). (B) Schematic representation of the pooled CRISPR screening in K562 WT or CENP-C ΔM12BD cells. Using CRISPR viability scores, genes which are essential for cell growth in K562 CENP-C ΔM12BD cells but not in K562 WT, cells were selected as candidates whose knockout showed synthetic lethality with CENP-C ΔM12BD . (C) Scatterplots showing the CRISPR viability score in K562 WT versus CENP-C ΔM12BD cell pools. Red dots are essential genes in K562 CENP-C ΔM12BD cells but not in K562 WT cells. (D) Gene set enrichment analysis (GSEA) for 375 candidate genes. The candidate genes were ranked by the magnitude of differences in CRISPR viability score between K562 WT and CENP-C ΔM12BD cells in descending order and applied to GSEA. The top 10 enriched gene sets were highlighted. (E) GSEA result of GOCC_SPINDLE_POLE gene set. The heatmap indicates the CRISPR viability score of the leading-edge genes of the preranking based on the magnitude of differences in CRISPR viability score between K562 WT and CENP-C ΔM12BD cells, in descending order. (F) GSEA result of GOMF_TUBULIN_BINDING gene set. The heatmap shows as in (E).

Article Snippet: To localize CENP-C, CENP-T, CENP-A, DSN1, KNL1, HEC1, and MAD2 in K562 cells, K562 cells were cytospan onto slide glasses (MATSUNAMI, S2111) or coverslips (MATSUNAMI, C024361) and then fixed with 3% paraformaldehyde (PFA; Nacalai Tesque) for 10 min at RT, and rinsed with PBS.

Techniques: Binding Assay, Expressing, Generated, CRISPR, Knock-Out

Journal: Cell reports

Article Title: KIF18A promotes chromosome congression in cooperation with CENP-E downstream of CENP-C

doi: 10.1016/j.celrep.2025.116515

Figure Lengend Snippet: (A) Cell viability of K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days after doxycycline (Dox) addition (mean and SD, two-tailed Student’s t test, CENP-C WT cells: n = 6; CENP-C ΔM12BD cells: n = 6; ** p < 0.01). (B) Representative images of DAPI-stained K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days. Scale bar, 50 μm. (C) Population of normal interphase cells, mitotic cells, and cells with micronuclei in K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days. Error bars indicate SEM. n = 3 independent experiments; 200 cells from each cell line were quantified in each experiment. (D) The growth curve of K562 WT or CENP-C ΔM12BD cells with or without KIF18A knockout. K562 WT or CENP-C ΔM12BD cells were treated with or without Dox ( KIF18A OFF or ON). The cell numbers were normalized to those at time 0 of each line. (E) Cell-cycle distribution of conditional knockout of KIF18A in K562 WT cells at each day after Dox addition, based on FACS analysis. (F) Cell-cycle distribution of conditional knockout of KIF18A in K562 CENP-C ΔM12BD cells at each day after Dox addition, based on FACS analysis. (G and H) Quantification of cells with misaligned chromosomes in K562 WT or CENP-C ΔM12BD cells with or without KIF18A knockout. The experimental scheme is shown. Cells were stained with antibodies against MAD2 (red) to detect misaligned chromosomes and CENP-T (green) as a kinetochore marker. DNA was stained with DAPI (blue). Arrowheads show typical MAD2-positive unaligned chromosomes. Scale bar, 10 μm. The cells with MAD2-positive chromosomes were quantified (H) (mean and SEM, two-tailed Student’s t test; n = 5 independent experiments; n.s., non-significant; ** p < 0.01). (I) Numbers of MAD2 positive kinetochores in each cell in each condition (WT KIF18A ON; WT KIF18A OFF; CENP-C ΔM12BD KIF18A ON; CENP-C ΔM12BD KIF18A OFF) (Mean and SEM, n = 5 independent experiments).

Article Snippet: To localize CENP-C, CENP-T, CENP-A, DSN1, KNL1, HEC1, and MAD2 in K562 cells, K562 cells were cytospan onto slide glasses (MATSUNAMI, S2111) or coverslips (MATSUNAMI, C024361) and then fixed with 3% paraformaldehyde (PFA; Nacalai Tesque) for 10 min at RT, and rinsed with PBS.

Techniques: Knock-Out, Two Tailed Test, Staining, Marker

Journal: Cell reports

Article Title: KIF18A promotes chromosome congression in cooperation with CENP-E downstream of CENP-C

doi: 10.1016/j.celrep.2025.116515

Figure Lengend Snippet: (A) Cell viability of RPE-1, K562, U2OS, A549, TIG-3, HeLa, HT1080, OVCAR-3, HT29, and HCC1806 cells with or without KIF18A knockdown (mean and SD, two-tailed Student’s t test; each sample size: n = 6; ** p < 0.01). (B–D) CENP-E (B), CENP-C (C), and CENP-T (D) localization in RPE-1, K562, U2OS, A549, TIG-3, HeLa, HT1080, OVCAR-3, HT29, and HCC1806 cells. The cell lines were treated with 50 μM monastrol for 2 h to enrich prometaphase cells. Each cell line was cytospun together with monastrol-treated RPE-1 cells expressing mScarlet-CENP-A as an internal control for immunostaining. CENP-E (green) (B), CENP-C (green) (C), and CENP-T (green) (D) were stained with antibodies against each protein. DNA was stained with DAPI (blue). CENP-T or CENP-A was stained as a kinetochore marker (red). Scale bar, 10 μm. CENP-E, CENP-C, or CENP-T signal intensities at mitotic kinetochores were quantified and normalized with those of the internal control RPE-1 mScarlet-CENP-A cells in each sample (mean and SD, two-tailed Student’s t test, each cell line: n = 10 cells; n.s., non-significant; * p < 0.1; ** p < 0.01).

Article Snippet: To localize CENP-C, CENP-T, CENP-A, DSN1, KNL1, HEC1, and MAD2 in K562 cells, K562 cells were cytospan onto slide glasses (MATSUNAMI, S2111) or coverslips (MATSUNAMI, C024361) and then fixed with 3% paraformaldehyde (PFA; Nacalai Tesque) for 10 min at RT, and rinsed with PBS.

Techniques: Knockdown, Two Tailed Test, Expressing, Control, Immunostaining, Staining, Marker

Journal: Cell reports

Article Title: KIF18A promotes chromosome congression in cooperation with CENP-E downstream of CENP-C

doi: 10.1016/j.celrep.2025.116515

Figure Lengend Snippet: In wild-type K562 and RPE-1 cells with KIF18A depletion (middle: + KIF18A KD), although KIF18A depletion enhances microtubule dynamics and increases peripheral chromosomes, CENP-E downstream of CENP-C compensates for KIF18A depletion, supporting chromosome congression and alignment, albeit with a delay in the congression. By contrast, in CENP-E-deficient cells (bottom: K562 CENP-C ΔM12BD and RPE-1 CENP-C ΔM12BD cells), KIF18A depletion disrupts chromosome congression due to weakened CENP-E activity downstream of CENP-C at kinetochores, leading to mitotic arrest and subsequent cell death. Based on these observations, we propose that the CENP-C pathway plays a role in chromosome congression, with KIF18A and CENP-E acting cooperatively to promote the congression of peripheral chromosomes during early prometaphase in wild-type K562 and RPE-1 cells (upper).

Article Snippet: To localize CENP-C, CENP-T, CENP-A, DSN1, KNL1, HEC1, and MAD2 in K562 cells, K562 cells were cytospan onto slide glasses (MATSUNAMI, S2111) or coverslips (MATSUNAMI, C024361) and then fixed with 3% paraformaldehyde (PFA; Nacalai Tesque) for 10 min at RT, and rinsed with PBS.

Techniques: Activity Assay