Journal: bioRxiv

Article Title: CENP-C-Mis12 complex establishes a regulatory loop through Aurora B for chromosome segregation

doi: 10.1101/2024.05.29.596395

Figure Lengend Snippet: (A) Schematic representation of human CENP-C. The Mis12C-binding domain (M12BD, amino acids 1-71) is highlighted in CENP-C wild-type (WT). The N-terminus region (amino acid 1-75) was deleted in CENP-C ΔM12BD . FLAG-tagged CENP-C WT or ΔM12BD was introduced into the CENP-C locus in RPE-1 cells expressing mScarlet-CENP-A and GFP-H2A (CENP-C WT or CENP-C ΔM12BD cells, respectively. See ). (B) DSN1 localization in CENP-C WT or CENP-C ΔM12BD cells. DSN1, a subunit of the Mis12 complex, was stained with an antibody against DSN1 (green). mScarlet-CENP-A is a kinetochore marker (CENP-A, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. DSN1 signal intensities at mitotic kinetochores were quantified (Mean and SD, two-tailed Student’s t-test, CENP-C WT cells: n = 10, CENP-C ΔM12BD cells: n = 10). (C) KNL1 localization in CENP-C WT or CENP-C ΔM12BD cells. KNL1, a subunit of the KNL1 complex, was stained with an antibody against KNL1. KNL1 localization at mitotic kinetochores was examined and quantified as in (B). Scale bar, 5 μm. Mean and SD, two-tailed Student’s t-test, CENP-C WT cells: n = 10, CENP-C ΔM12BD cells: n = 10. (D) Hec1 localization in CENP-C WT or CENP-C ΔM12BD cells. Hec1, a subunit of the Ndc80C, was stained with an antibody against Hec1. Hec1 localization at mitotic kinetochores was examined and quantified as in (B). Scale bar, 5 μm. Mean and SD, two-tailed Student’s t-test, CENP-C WT cells: n = 10, CENP-C ΔM12BD cells: n = 10. (E) Representative time-lapse images of mitotic progression in CENP-C WT or CENP-C ΔM12BD cells. DNA was visualized with GFP-H2A. Images were projected using maximum intensity projection and deconvoluted. Time is relative to nuclear envelope breakdown (NEBD). Scale bar, 10 μm. (F) Mitotic duration from NEBD to anaphase onset in CENP-C WT or CENP-C ΔM12BD cells. The time-lapse images were analyzed to measure the time from NEBD to anaphase onset. Two independent clones of CENP-C WT or CENP-C ΔM12BD cells were tested (Mean and SD, two-tailed Student’s t-test, CENP-C WT cells clone1: n = 120, CENP-C WT cells clone2: n = 105, CENP-C ΔM12BD cells clone1: n = 105, CENP-C ΔM12BD cells clone2: n = 132). (G) Chromosome segregation errors in CENP-C WT or CENP-C ΔM12BD cells. The lagging chromosomes and chromosome bridges during anaphase in the cells analyzed in (F) were scored. Representative images are shown. Scale bar 5 μm. (H) Micronuclei formation CENP-C WT or CENP-C ΔM12BD cells. The cells were fixed and the interphase cells with micronuclei were scored (CENP-C WT cells clone1: n = 1527, CENP-C WT cells clone2: n = 976, CENP-C ΔM12BD cells clone1: n = 1633, CENP-C ΔM12BD cells clone2: n = 1076). Scale bar, 10 μm. (I) K-fiber in CENP-C WT or CENP-C ΔM12BD cells. CENP-C WT or CENP-C ΔM12BD cells expressing mScarlet CENP-A were fixed after CaCl 2 treatment and stained with an anti-alpha-tubulin antibody. Scale bar, 5 μm. The mean tubulin signal intensities of the spindle in a cell were quantified as K-fiber signals (Mean and SD, two-tailed Student’s t-test, CENP-C WT cells: n = 10, CENP-C ΔM12BD cells: n = 10). (J) Cell viability of CENP-C WT or CENP-C ΔM12BD cells treated with various concentrations of nocodazole. Viable cells were measured three days after nocodazole addition. Three independent experiments were performed (Mean and SD, two-way ANOVA with Šídák’s multiple comparison test, **: p = 0.0022). IC 50 indicates the average of concentration to reduce cell viability to 50% from three independent experiments (Mean and SD, two-tailed Student’s t-test).

Article Snippet: The primary antibodies used in this study were rabbit anti-mouse CENP-C (1:5000), Guinea pig anti-human CENP-C (1:10,000) , mouse anti-human CENP-A (1:3000) , rabbit anti-human DSN1 (1:5000) (a gift from Iain Cheeseman, Whitehead Institute, MIT) , rat anti-RFP (1:1000) (Chromotek), rat anti-human CENP-T (1:1000) (a gift from Kinya Yoda, Nagoya University, Nagoya, Japan), rat anti-histone H3 (1:5000) (a gift from Hiroshi Kimura, Tokyo Tech, Tokyo, Japan) , and mouse anti-α-tubulin (1:10,000) (Sigma).

Techniques: Binding Assay, Expressing, Staining, Marker, Two Tailed Test, Clone Assay, Comparison, Concentration Assay

Journal: bioRxiv

Article Title: CENP-C-Mis12 complex establishes a regulatory loop through Aurora B for chromosome segregation

doi: 10.1101/2024.05.29.596395

Figure Lengend Snippet: (A) Schematic representation of human CENP-T. human CENP-T wild-type (WT) has two Ndc80C binding regions (NBD-1 or -2: amino acids 6-31 or 76-105) and a Mis12-binding domain. Each NBD was deleted in CENP-T ΔNBD-1 or CENP-T ΔNBD-2 , respectively. mScarlet-fused CENP-T WT or each mutant was introduced into CENP-T locus in RPE-1 cells expressing OsTIR1 and GFP-mAID-CENP-T (CENP-T WT , CENP-T ΔNBD-1 , or CENP-T ΔNBD-2 cells, respectively. See ). (B) Hec1 localization in CENP-T WT , CENP-T ΔNBD- 1 , or CENP-T ΔNBD-2 cells. Hec1, a subunit of Ndc80C, was stained with an antibody against Hec1 (green). mScarlet-CENP-T is a kinetochore marker (CENP-T, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. Hec1 signal intensities at mitotic kinetochores were quantified (Mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT cells: n = 10, CENP-T ΔNBD-1 cells: n = 10, CENP-T ΔNBD-2 cells: n = 10). (C) DSN1 localization in CENP-T WT , CENP-T ΔNBD-1 , or CENP-T ΔNBD-2 cells. DSN1, a subunit of Mis12C, was stained with an antibody against DSN1. DSN1 localization at mitotic kinetochores was examined and quantified as in (B). Scale bar, 5 μm. Mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT cells: n = 10, CENP-T ΔNBD-1 cells: n = 10, CENP-T ΔNBD-2 cells: n = 10. (D) KNL1 localization in CENP-T WT , CENP-T ΔNBD-1 , or CENP-T ΔNBD-2 cells. KNL1, a subunit of KNL1C, was stained with an antibody against KNL1. KNL1 localization at mitotic kinetochores was examined and quantified as in (B). Scale bar, 5 μm. Mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT cells: n = 10, CENP-T ΔNBD-1 cells: n = 10, CENP-T ΔNBD-2 cells: n = 10. (E) K-fiber in CENP-T WT , CENP-T ΔNBD-1 , or CENP-T ΔNBD-2 cells. CENP-T WT , CENP-T ΔNBD-1 , or CENP-T ΔNBD-2 cells were fixed after CaCl 2 treatment and stained with an anti-alpha-tubulin antibody. CENP-T fused with mScarlet is a kinetochore marker (CENP-T, red). Scale bar, 5 μm. The mean tubulin signal intensities of the spindle in a cell were quantified as K-fiber signals (Mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT cells: n = 10, CENP-T ΔNBD-1 cells: n = 10, CENP-T ΔNBD-2 cells: n = 10). (F) Cell viability of CENP-T WT , CENP-T ΔNBD-1 , or CENP-T ΔNBD-2 cells treated with various concentrations of nocodazole. Viable cells were measured three days after nocodazole addition. Four independent experiments were performed (Mean and SD, two-way ANOVA with Dunnett’s multiple comparison test, ***: p = 0.0007, ****: p < 0.0001. Red: WT vs ΔNBD-1, Blue: WT vs ΔNBD-2). IC 50 indicates the average of nocodazole concentration to reduce cell viability to 50% from four independent experiments (Mean and SD, one-way ANOVA with Dunnett’s multiple comparison test). (G) Representative time-lapse images of mitotic progression in CENP-T WT , CENP-T ΔNBD-1 , or CENP-T ΔNBD-2 cells. DNA was visualized with SPY505-DNA. Images were projected using maximum intensity projection and deconvoluted. Time is relative to nuclear envelope breakdown (NEBD). Scale bar, 10 μm. (H) Mitotic duration from NEBD to anaphase onset in CENP-T WT , CENP-T ΔNBD-1 , or CENP-T ΔNBD-2 cells. The time-lapse images were analyzed to measure the time from NEBD to anaphase onset (Mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT cells: n = 117, CENP-T ΔNBD-1 cells: n = 120, CENP-T ΔNBD-2 cells: n = 110). (I) Chromosome segregation errors in CENP-T WT , CENP-T ΔNBD-1, or CENP-T ΔNBD-2 cells. The lagging chromosomes and chromosome bridges during anaphase in the cells analyzed in (H) were scored. Representative images are shown. Scale bar 5 μm.

Article Snippet: The primary antibodies used in this study were rabbit anti-mouse CENP-C (1:5000), Guinea pig anti-human CENP-C (1:10,000) , mouse anti-human CENP-A (1:3000) , rabbit anti-human DSN1 (1:5000) (a gift from Iain Cheeseman, Whitehead Institute, MIT) , rat anti-RFP (1:1000) (Chromotek), rat anti-human CENP-T (1:1000) (a gift from Kinya Yoda, Nagoya University, Nagoya, Japan), rat anti-histone H3 (1:5000) (a gift from Hiroshi Kimura, Tokyo Tech, Tokyo, Japan) , and mouse anti-α-tubulin (1:10,000) (Sigma).

Techniques: Binding Assay, Mutagenesis, Expressing, Staining, Marker, Comparison, Concentration Assay

Journal: bioRxiv

Article Title: CENP-C-Mis12 complex establishes a regulatory loop through Aurora B for chromosome segregation

doi: 10.1101/2024.05.29.596395

Figure Lengend Snippet: (A) Schematic representation for forced binding of Mis12C to CENP-C in HeLa cells. To validate the idea that the CENP-C-Mis12C interaction positively regulates Aurora B localization, we utilized HeLa cells in which Aurora B activity is low at centromeres, leading to chromosome instability. The CENP-C-Mis12C interaction was increased by expressing a DSN1 mutant lacking the basic motif (ΔBasic motif) in HeLa cells and examined the Aurora B levels and efficiency of kinetochore-microtubule error correction. Dsn1 is a subunit of Mis12C. The basic motif of DSN1 masks the CENP-C-binding surface of Mis12C, preventing the CENP-C-Mis12C interaction. The deletion of the basic motif increases the binding affinity between CENP-C and Mis12C . (B) DSN1 localization in DSN1 WT or DSN1 ΔBasic motif HeLa cells. DSN1, a subunit of the Mis12 complex, was stained with an antibody against DSN1 (red). DNA was stained with DAPI (blue). CENP-A was stained by an antibody against CENP-A as a kinetochore marker (green). Scale bar, 5 μm. DSN1 signal intensities at mitotic kinetochores were quantified (Mean and SD, two-tailed Student’s t-test, DSN1-C WT : n = 10 cells, DSN1 ΔBasic motif : n = 10 cells). (C) Bub1 localization in DSN1 WT or DSN1 ΔBasic motif HeLa cells. Bub1 was stained with an antibody against Bub1 (green). DSN1 was stained as a kinetochore marker (red). DNA was stained with DAPI (blue). Scale bar, 5 μm. Bub1 signal intensities at kinetochores were quantified (Mean and SD, two-tailed Student’s t-test, DSN1-C WT : n = 523 kinetochores from 6 cells, DSN1 ΔBasic motif : n = 501 kinetochores from 6 cells). (D) Aurora B and H2AT120ph localization in DSN1 WT or DSN1 ΔBasic motif HeLa cells. Aurora B and H2AT120ph were stained with their antibodies (green and cyan). mScarlet-DSN1 is a kinetochore marker (DSN1, red). DNA was stained with DAPI (blue). Scale bar, 10 μm. The insets show an enlarged single chromosome (Scale bar, 2 μm). The signal intensities of Aurora B at centromeres and H2AT120ph at kinetochore-proximal centromeres were quantified (Mean and SD, two-tailed Student’s t-test, DSN1-C WT : n = 385 centromeres, 651 kinetochores from 6 cells for Aurora B, H2AT120ph, respectively, DSN1 ΔBasic motif : n = 379 centromeres, 668 kinetochores from 6 cells for Aurora B, H2AT120ph, respectively). (E) Error correction assay in DSN1 WT or DSN1 ΔBasic motif HeLa cells. The cells were treated with monastrol for 4 h and then released and incubated in a medium with MG132 for 30 or 45 min. The cells were fixed, and microtubules (green) and DNA (blue) were stained with an antibody against alpha-tubulin and DAPI, respectively. mScarlet-DSN1 is a kinetochore marker (DSN1, red). The cells with more than two unaligned chromosomes were defined as “cells with unaligned chromosomes.” Arrows indicate unaligned chromosomes. Scale bar 5 μm. Mitotic cells with unaligned chromosomes were quantified. Four independent experiments were performed (Mean and SD, two-tailed Student’s t-test). (F) Model for a positive regulatory loop to facilitate Aurora B localization at the centromere through the CENP-C-Mis12C interaction. The regulatory system is required for efficient error correction of kinetochore-microtubule attachment, leading to accurate chromosome segregation.

Article Snippet: The primary antibodies used in this study were rabbit anti-mouse CENP-C (1:5000), Guinea pig anti-human CENP-C (1:10,000) , mouse anti-human CENP-A (1:3000) , rabbit anti-human DSN1 (1:5000) (a gift from Iain Cheeseman, Whitehead Institute, MIT) , rat anti-RFP (1:1000) (Chromotek), rat anti-human CENP-T (1:1000) (a gift from Kinya Yoda, Nagoya University, Nagoya, Japan), rat anti-histone H3 (1:5000) (a gift from Hiroshi Kimura, Tokyo Tech, Tokyo, Japan) , and mouse anti-α-tubulin (1:10,000) (Sigma).

Techniques: Binding Assay, Activity Assay, Expressing, Mutagenesis, Staining, Marker, Two Tailed Test, Incubation

Journal: bioRxiv

Article Title: CENP-C-Mis12 complex establishes a regulatory loop through Aurora B for chromosome segregation

doi: 10.1101/2024.05.29.596395

Figure Lengend Snippet: (A) Strategy to generate DSN1 WT or DSN1 ΔBasic motif HeLa cells. (See also ). (B) Schematic representation of mScarlet-DSN1 cDNA targeting the endogenous DSN1 locus. To express mScarlet-tagged DSN1 wiled-type (WT) or a basic motif deletion mutant (ΔBasic motif: Δ91-113) under the control of the endogenous DSN1 promoter, mScarlet-DSN1 WT or ΔBasic motif, cDNA was targeted into DSN1 locus by CRISPR/Cas9-mediated homologous recombination. Since the targeting constructs have puromycin resistance genes ( PuroR ) or neomycin resistance genes ( NeoR ), targeted cells were selected using these selection markers. The gRNA sequence and position of primers for genotyping are shown. (C) Genotyping PCR and immunoblotting for mScarlet-DSN1 in mScarlet-DSN1 -introduced HeLa cells. The genotype in isolated single clones was examined using the primers shown in (B). In immunoblots, DSN1 was detected by an antibody against DSN1 or mScarlet (RFP), and α-tubulin was probed as a loading control. (D) Hec1 and KNL1 localization in DSN1 WT or DSN1 ΔBasic motif HeLa cells. Hec1, a subunit of the Ndc80C (green), and KNL1, a subunit of the KNL1C (cyan), were stained with antibodies against Hec1 and KNL1, respectively. mScarlet-DSN1 is a kinetochore marker (DSN1, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. Hec1 or KNL1 signal intensities at mitotic kinetochores were quantified. A representative result from two independent experiments is shown (Mean and SD, two-tailed Student’s t-test, DSN1 WT : n = 7, DSN1 ΔBasic motif : n = 7).

Article Snippet: The primary antibodies used in this study were rabbit anti-mouse CENP-C (1:5000), Guinea pig anti-human CENP-C (1:10,000) , mouse anti-human CENP-A (1:3000) , rabbit anti-human DSN1 (1:5000) (a gift from Iain Cheeseman, Whitehead Institute, MIT) , rat anti-RFP (1:1000) (Chromotek), rat anti-human CENP-T (1:1000) (a gift from Kinya Yoda, Nagoya University, Nagoya, Japan), rat anti-histone H3 (1:5000) (a gift from Hiroshi Kimura, Tokyo Tech, Tokyo, Japan) , and mouse anti-α-tubulin (1:10,000) (Sigma).

Techniques: Mutagenesis, Control, CRISPR, Homologous Recombination, Construct, Selection, Sequencing, Western Blot, Isolation, Clone Assay, Staining, Marker, Two Tailed Test