Journal: bioRxiv

Article Title: A BubR1-independent pathway for CENP-E targeting to the outer corona of kinetochores

doi: 10.1101/2023.05.10.540161

Figure Lengend Snippet: (A) Representative immunofluorescence images of HeLa cells transfected with CENP-E constructs after depletion of BubR1, Dynein Heavy chain (DHC) or BubR1/DHC and treated with nocodazole for 2 hours. The cells were stained for endogenous CENP-E, CENP-C and DNA. (B) Scatter dot plot showing quantification of CENP-E intensity normalized to CENP-C, in the absence of endogenous CENP-E. Median and 95% confidence interval are presented. Cells were treated for 2 hours with nocodazole. Each point represents the intensity of CENP-E over CENP-C at one kinetochore. Black line represents the mean and whiskers represent the standard deviation. Asterisks indicate ordinary Kruskal-Wallis test significance value. ****P<0.0001, *P=0.02. n=150 kinetochores for each condition. (C) (D) Scatter dot plot showing quantification of CENP-E intensity normalized to CENP-C for cells in prometaphase, metaphase or metaphase depleted with Dynein, after treatment with control or DHC siRNA for 48 hours. n=201, 202 and 200 respectively. Median and 95% confidence interval are presented. Asterisks indicate ordinary one-way ANOVA test significance value. **** P<0.0001. (E) Representative immunofluorescence images of HeLa cells after depletion with control or Dynein Heavy Chain siRNA and treated with 200nM GSK923295 for 30q minutes. (F) Scatter dot plot showing quantification of CENP-E intensity around spindle poles, median and 95% confidence interval. n= of 83 and 113 cells measured respectively. For each cell, the intensity around both spindle poles marked by Centrin was measured and averaged. Scale bars: 10 μm. Asterisks indicate T-test significance value. ****P<0.0001.

Article Snippet: To generate detachable kinetochore crescents, cells were treated with 3.3 μM nocodazole (Calbiochem) for 3 hours followed by addition of 10 μM RO-3306 (Bio-Techne Ltd) for 25 minutes before fixing ( ; ).

Techniques: Immunofluorescence, Transfection, Construct, Staining, Standard Deviation

Journal: bioRxiv

Article Title: A BubR1-independent pathway for CENP-E targeting to the outer corona of kinetochores

doi: 10.1101/2023.05.10.540161

Figure Lengend Snippet: (A) Representative images of live HeLa cells transfected with GFP-CENP-E constructs, treated with 300 nM nocodazole and with or without the CDK1 inhibitor RO-3306, scale bar: 10 μm. (B) Representative immunofluorescence images of HeLa cells transfected with GFP-CENP-E 2452-2598 and treated with nocodazole and RO-3306, stained with Spindly and CENP-C. Scale bar, 5 μm. (C) Schematic diagram of CENP-E, highlighting the motor (orange) and kinetochore- and corona-targeting domains (purple). (D) X-ray crystallography derived structure of CENP-E 2454-2494 in cartoon representation. The conserved amino acids are represented in ball and stick mode (PDB:8OWI). (E) Model of the CENP-E 2055-2608 generated by combining Alphafold2 models of its monomeric kinetochore targeting domain and dimeric outer corona targeting domain. The conserved residues essential for corona-targeting is in stick and ball orange. The domain crystallized (D) is painted yellow. The residues essential for interaction with BubR1 are in red. (F) Quantification of the kinetochore localization of GFP-CENP-E domain mutants related to images in F and G. (G, H). Representative live-cell images of HeLa cells transfected with GFP-CENP-E point (G) or domain (H) mutants and treated with 300 nM nocodazole for 2 hours. Scalebar, 10 μm.

Article Snippet: To generate detachable kinetochore crescents, cells were treated with 3.3 μM nocodazole (Calbiochem) for 3 hours followed by addition of 10 μM RO-3306 (Bio-Techne Ltd) for 25 minutes before fixing ( ; ).

Techniques: Transfection, Construct, Immunofluorescence, Staining, Derivative Assay, Generated

Journal: bioRxiv

Article Title: A BubR1-independent pathway for CENP-E targeting to the outer corona of kinetochores

doi: 10.1101/2023.05.10.540161

Figure Lengend Snippet: (A) Representative immunofluorescence images of HeLa cells transfected with GFP-CENP-E 2055-2608 wild type and mutants, treated with 300 nM nocodazole. Scalebar, 10 μm. (B) Scatter dot plot showing quantification of GFP-CENP-E 2055-2608 intensity relative to CENP-C at kinetochores. The experiment was done twice. Scale bars: 10 μm. n=150 for each condition, bars represent median and 95% confidence interval. **** P<0.0001. Asterisks indicate ordinary Kruskal-Wallis test significance value. (C) Representative immunofluorescence images of HeLa cells transfected with full-length CENP-E-mNeon wild type and mutants, CENP-C and DNA. Scalebar, 10 μm. (D) Scatter plot showing ratio of fluorescence intensity of mNeon relative to CENP-C for CENP-E wild type and mutants at spindle poles (kinetochore numbers for WT n=194, E4A n=148, TS2AA n=197 and E4A TS2AA n=150). Bars represent median and 95% confidence interval. Asterisks indicate ordinary Kruskal-Wallis test significance value, with **** P<0.0001.

Article Snippet: To generate detachable kinetochore crescents, cells were treated with 3.3 μM nocodazole (Calbiochem) for 3 hours followed by addition of 10 μM RO-3306 (Bio-Techne Ltd) for 25 minutes before fixing ( ; ).

Techniques: Immunofluorescence, Transfection, Fluorescence

Journal:

Article Title: Extracellular Signal-Regulated Kinase Activates Topoisomerase II? through a Mechanism Independent of Phosphorylation

doi:

Figure Lengend Snippet: Coimmunoprecipitation of endogenous topoisomerase IIα and ERK2 from nuclear extracts. ERK2 was immunoprecipitated from nuclear extracts prepared from nocodazole-treated NIH 3T3 cells; this was followed by immunoblotting to visualize both ERK2 and coimmunoprecipitating topoisomerase (Topo) IIα. Lanes 3 and 4 show increasing amounts of immunoprecipitated ERK2 and coimmunoprecipitated topoisomerase IIα. A small amount of topoisomerase IIα nonspecifically bound to the protein A-(Prot. A)-Sepharose resin (lane 2). Ten percent of the extract volume used for the immunoprecipitations served as a loading control (lane 1).

Article Snippet: For coimmunoprecipitations of endogenous ERK2 and topoisomerase IIα, nuclear extracts from nocodazole-treated NIH 3T3 cells were incubated with 0, 0.2, or 2 μg of anti-ERK2 antibody (C-14; Santa Cruz Biotechnology) for 2 h on ice; this was followed by addition of 20 μl of protein A-Sepharose (Pharmacia) that had been pretreated with BSA at 0.5 mg/ml.

Techniques: Immunoprecipitation, Western Blot

Journal: Cell reports

Article Title: The Sar1 GTPase is dispensable for COPII-dependent cargo export from the ER

doi: 10.1016/j.celrep.2023.112635

Figure Lengend Snippet:

Article Snippet: Nocodazole , Cell Signaling Technology , 2190S.

Techniques: Recombinant, CRISPR, Transfection, Plasmid Preparation, Electron Microscopy, Reverse Transcription, Software

Journal: Life Science Alliance

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

doi: 10.26508/lsa.202402927

Figure Lengend Snippet: (A) Schematic representation of human CENP-C. The Mis12C-binding domain (M12BD, amino acids 1–71) is highlighted in CENP-C WT. The N-terminal region (amino acids 1–75) was deleted in CENP-C ∆M12BD . FLAG-tagged CENP-C WT or ∆M12BD was introduced into the CENP-C locus in RPE-1 cells expressing mScarlet-CENP-A and GFP-H2A (CENP-C WT or CENP-C ∆M12BD RPE-1 cells, respectively; see ). (B) DSN1 localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. DSN1 was stained with an antibody against DSN1 (green). mScarlet-CENP-A was used as 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 t test, CENP-C WT cells: n = 10; CENP-C ∆M12BD RPE-1 cells: n = 10). (C) KNL1 localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. KNL1 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 t test, CENP-C WT cells: n = 10; CENP-C ∆M12BD cells: n = 10. (D) Hec1 localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. Hec1 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 t test, CENP-C WT cells: n = 10; CENP-C ∆M12BD RPE-1 cells: n = 10. (E) Representative time-lapse images of mitotic progression in CENP-C WT or CENP-C ∆M12BD cells. DNA was visualized with GFP-H2A. Images were projected using maximum intensity projection and deconvolved. Time is relative to the nuclear envelope breakdown (NEBD). Scale bar, 10 μm. (F) Mitotic duration from the NEBD to the anaphase onset in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. The time-lapse images were analyzed to measure the time from the NEBD to the anaphase onset. Two independent clones of CENP-C WT or CENP-C ∆M12BD RPE-1 cells were tested (mean and SD, two-tailed t test, CENP-C WT RPE-1 cell clone1: n = 120; CENP-C WT RPE-1 cell clone2: n = 105; CENP-C ∆M12BD RPE-1 cell clone1: n = 105; CENP-C ∆M12BD RPE-1 cell clone2: n = 132). (G) Chromosome segregation errors in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. Lagging chromosomes and chromosome bridges during anaphase in the cells analyzed in (F) were scored. Representative images are shown. Scale bar, 5 μm. (H) Micronucleus formation in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. The cells were fixed, and the interphase cells with micronuclei were scored (CENP-C WT RPE-1 cell clone1: n = 1,527; CENP-C WT RPE-1 cell clone2: n = 976; CENP-C ∆M12BD RPE-1 cell clone1: n = 1,633; CENP-C ∆M12BD RPE-1 cell clone2: n = 1,076). Scale bar, 10 μm. (I) K-fiber in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. CENP-C WT or CENP-C ∆M12BD RPE-1 cells expressing mScarlet CENP-A were fixed after CaCl 2 treatment and stained with an anti-alpha-tubulin antibody. Scale bar, 5 μm. The means of tubulin signal intensities of the spindle in a cell were quantified as K-fiber signals (mean and SD, two-tailed t test, CENP-C WT RPE-1 cells: n = 10; CENP-C ∆M12BD RPE-1 cells: n = 10). (J) Cell viability of CENP-C WT or CENP-C ∆M12BD RPE-1 cells treated with various concentrations of nocodazole. Viable cells were measured 3 d 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 t test).

Article Snippet: To detect Aurora B at kinetochore-proximal pool , nocodazole treatment followed with 5 μM 5-iodotubercidin (5-ITu; TOCRIS), a Haspin inhibitor, for 30 min. To spread and swell chromosomes, the method was modified from the protocol described previously ( Preprint ).

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

Journal: Life Science Alliance

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

doi: 10.26508/lsa.202402927

Figure Lengend Snippet: (A) Schematic representation of human CENP-T. Human CENP-T WT has two Ndc80C-binding regions (NBD-1 or NBD-2: amino acids 6–31 or 76–105) and a Mis12-binding domain. Each NBD was deleted in CENP-T ∆NBD−1 or CENP-T ∆NBD−2 RPE-1 cells, respectively. mScarlet-fused CENP-T WT or each mutant was introduced into the CENP-T locus in RPE-1 cells expressing OsTIR1 and GFP-mAID-CENP-T (CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells, respectively; see ). (B) Hec1 localization in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. Hec1 was stained with an antibody against Hec1 (green). mScarlet-CENP-T was used as 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 RPE-1 cells: n = 10; CENP-T ∆NBD−1 RPE-1 cells: n = 10; CENP-T ∆NBD−2 RPE-1 cells: n = 10). (C) DSN1 localization in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. DSN1 was stained with an antibody against DSN1 (green). 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 ∆NBD−1 RPE-1 cells: n = 10; CENP-T ∆NBD−2 cells: n = 10. (D) KNL1 localization in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. KNL1 was stained with an antibody against KNL1 (green). 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 RPE-1 cells: n = 10; CENP-T ∆NBD−1 RPE-1 cells: n = 10; CENP-T ∆NBD−2 RPE-1 cells: n = 10. (E) K-fiber in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells were fixed after CaCl 2 treatment and stained with an anti-alpha-tubulin antibody (green). CENP-T fused with mScarlet was used as a kinetochore marker (CENP-T, red). Scale bar, 5 μm. The means of 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 RPE-1 cells: n = 10; CENP-T ∆NBD−1 RPE-1 cells: n = 10; CENP-T ∆NBD−2 RPE-1 cells: n = 10). (F) Cell viability of CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells treated with various concentrations of nocodazole. Viable cells were measured 3 d 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 versus ∆NBD-1; blue: WT versus ∆NBD-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 ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. DNA was visualized with SPY505-DNA. Images were projected using maximum intensity projection and deconvolved. Time is relative to the nuclear envelope breakdown (NEBD). Scale bar, 10 μm. (H) Mitotic duration from the NEBD to the anaphase onset in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. The time-lapse images were analyzed to measure the time from the NEBD to the anaphase onset (mean and SD, one-way ANOVA with Dunnett’s multiple comparison test, CENP-T WT RPE-1 cells: n = 117; CENP-T ∆NBD−1 RPE-1 cells: n = 120; CENP-T ∆NBD−2 RPE-1 cells: n = 110). (I) Chromosome segregation errors in CENP-T WT , CENP-T ∆NBD−1 , or CENP-T ∆NBD−2 RPE-1 cells. 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: To detect Aurora B at kinetochore-proximal pool , nocodazole treatment followed with 5 μM 5-iodotubercidin (5-ITu; TOCRIS), a Haspin inhibitor, for 30 min. To spread and swell chromosomes, the method was modified from the protocol described previously ( Preprint ).

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

Journal: Life Science Alliance

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

doi: 10.26508/lsa.202402927

Figure Lengend Snippet: (A) BubR1 localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. BubR1 was stained with an antibody against BubR1 (green). mScarlet-CENP-A was used as a kinetochore marker (CENP-A, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. BubR1 signal intensities at kinetochores were quantified (mean and SD, two-tailed t test, CENP-C WT RPE-1 cells: n = 323 kinetochores from 5 cells; CENP-C ∆M12BD RPE-1 cells: n = 363 kinetochores from 5 cells). (B) Ska3 localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. Ska3 was stained with an antibody against Ska3 (green). mScarlet-CENP-A was used as a kinetochore marker (CENP-A, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. Ska3 signal intensities at kinetochores were quantified (mean and SD, two-tailed t test, CENP-C WT RPE-1 cells: n = 419 kinetochores from 5 cells; CENP-C ∆M12BD RPE-1 cells: n = 422 kinetochores from 5 cells). (C) PLK1 localization in CENP-C WT or CENP-C ∆M12BD RPE-1 cells. CENP-C WT or CENP-C ∆M12BD RPE-1 cells were synchronized using a double thymidine block. After the washout of the second thymidine treatment for 6 h, the cells were treated with 1 μg/ml nocodazole for 4 h, as indicated in the scheme. PLK1 was stained with an antibody against PLK1 (green). mScarlet-CENP-A was used as a kinetochore marker (CENP-A, red). DNA was stained with DAPI (blue). Scale bar, 5 μm. The insets show an enlarged pair kinetochore (scale bar, 1 μm). PLK1 signal intensities at kinetochores were quantified (mean and SD, two-tailed t test, CENP-C WT RPE-1 cells: n = 359 kinetochores from 5 cells; CENP-C ∆M12BD RPE-1 cells: n = 361 kinetochores from 5 cells).

Article Snippet: To detect Aurora B at kinetochore-proximal pool , nocodazole treatment followed with 5 μM 5-iodotubercidin (5-ITu; TOCRIS), a Haspin inhibitor, for 30 min. To spread and swell chromosomes, the method was modified from the protocol described previously ( Preprint ).

Techniques: Staining, Marker, Two Tailed Test, Blocking Assay