1. Cell Biology
Download icon

Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

  1. Giulia Vallardi
  2. Lindsey A Allan
  3. Lisa Crozier
  4. Adrian T Saurin  Is a corresponding author
  1. University of Dundee, United Kingdom
Research Article
Cite this article as: eLife 2019;8:e42619 doi: 10.7554/eLife.42619
7 figures, 1 table and 2 additional files

Figures

Figure 1 with 5 supplements
A subset of PP2A-B56 complexes control spindle assembly checkpoint silencing and chromosome alignment.

(A and B) Representative images (A) and line plots (B) of nocodazole-arrested Flp-in HeLa cells expressing YFP-B56 (B56α, B56β, B56γ1, B56γ3, B56δ and B56ε). For line plots, five kinetochore pairs were analysed per cell, for a total of 10 cells per experiment. Graphs represent the mean intensities (±SD) from 3 independent experiments. Intensity is normalized to the maximum signal in each channel in each experiment. (C–G) Flp-in HeLa cells treated with siRNA against B56pool, all B56 isoforms except B56α, or all B56 isoforms except B56γ, were analysed for sister chromatid cohesion, SAC strength, Knl1-MELT dephosphorylation and chromosomal alignment. (C) Quantification of percentage of chromosome spreads that contain at least one split centromere. Graph represents mean data (+SD) from 3 independent experiments with 50 metaphase spreads quantified per condition per experiment. (D) Time-lapse analysis of cells entering mitosis in the presence of nocodazole and 2.5 µM AZ-3146. The graph represents the cumulative data from 50 cells, which is representative of 3 independent experiments. Representative images (E) and quantification (F) of relative kinetochore intensities of Knl1-pMELT in cells arrested in prometaphase with nocodazole and treated with MG132 for 30 min, followed by 2.5 µM AZ-3146 for the indicated amount of time. 10 cells were quantified per experiment and the graph displays the mean (+SD) of 3 independent experiments. The individual data points for each experiment can be found in the source data. (G) Quantification of chromosome misalignment in cells arrested in metaphase with MG-132. At least 100 cells were scored per condition per experiment and graph represents the mean (-SD) of 3 independent experiments. Misalignments were score as mild (1 to 2 misaligned chromosomes), intermediate (3 to 5 misaligned chromosomes), and severe (>5 misaligned chromosomes). Asterisks indicate significance (Figure 1c: Welch’s t -test, unpaired, Figure 1f: Mann-Whitney test); ns p>0.05, *p≤0.05, **p≤0.01, ****p≤0.0001. Scale bars, 5µm.

https://doi.org/10.7554/eLife.42619.003
Figure 1—figure supplement 1
Alignment of B56 isoforms to show that Sgo1 and BubR1 interacting regions are conserved.

Alignment of the BubR1 binding pocket (A) and the Sgo1-binding region (B) in B56 isoforms.

https://doi.org/10.7554/eLife.42619.004
Figure 1—figure supplement 2
Western blot showing knockdown of different B56 isoforms.

Immunoblot of whole cell lysates from nocodazole-arrested HeLa Flp-in cells treated with the indicated siRNA and probed for B56α, B56γ, B56δ, B56ε or tubulin (note B56β was undetectable). Asterix (*) indicate non-specific bands that do not change upon B56 siRNA.

https://doi.org/10.7554/eLife.42619.005
Figure 1—figure supplement 3
Endogenous tagging of B56α and B56γ with YFP confirms centromere and kinetochore localisation.

(A) Schematic representation of the strategy used for CRISPR/Cas9 YFP-tagging of B56 isoforms α and γ at the N-terminus. (B) Immunoblot of whole cell lysates from nocodazole-arrested HeLa cells with or without YFP-endogenously tagged B56α (enB56α) or B56γ (enB56γ). (C and D) Representative images (C) and line plots (D) of nocodazole-arrested Flp-in HeLa cells expressing endogenously tagged YFP-B56α (enB56α) or YFP-B56γ (enB56γ). For line plots, 5 kinetochore pairs were analysed per cell, for a total of 10 cells per experiment. Graphs represent the mean intensities (±SD) from 3 independent experiments. Intensity is normalized to the maximum signal in each channel in each experiment. Scale bars, 5µm. 

https://doi.org/10.7554/eLife.42619.006
Figure 1—figure supplement 4
Overexpression of YFP-B56α can rescue kinetochore functions.

(A-D) Flp-in HeLa cells treated with siRNA against B56 pool and induced to re-express YFP-B56α or YFP-B56γ were analysed for SAC silencing and chromosomal alignment. Representative images (A) and quantification of relative kinetochore intensities (B) of Knl1-pMELT in cells arrested in prometaphase with nocodazole and treated with MG132 for 30 min followed by 2.5 μM AZ-3146 for the indicated amount of time. 10 cells were quantified per experiment and graphs represent the mean (+SD) of 4 independent experiments. Scale bars, 5µm. (C) Time-lapse analysis of cells entering mitosis in the presence of nocodazole and 2.5 µM AZ-3146. The graph represents the cumulative data from 50 cells, which is representative of 3 independent experiments. (D) Quantification of chromosome alignment in cells arrested in metaphase with MG-132. At least 100 cells were scored per condition per experiment and graph represents the mean (-SD) of 3 independent experiments. Misalignments were score as mild (1 to 2 misaligned chromosomes), intermediate (3 to 5 misaligned chromosomes), and severe (>5 misaligned chromosomes). Asterisks indicate significance (Mann-Whitney test); ****p≤0.0001.

https://doi.org/10.7554/eLife.42619.007
Figure 1—figure supplement 5
Overexpression of YFP-B56α enhances B56α levels at centromeres and kinetochores.

(A) Immunoblot of whole cell lysates from nocodazole-arrested Flp-in HeLa cells treated with the indicated siRNA expressing endogenous YFP-B56α (enB56α) or exogenous YFP-B56α (ex B56α). Blot was probed for B56α or actin. (B–D) Localization of endogenous and exogenous YFP-B56α. Representative images (B) and quantification of relative centromere/kinetochore intensity (C) of B56α in cells arrested in prometaphase with nocodazole. 10 cells were quantified per experiment and graphs represent the mean (±SD) of 3 independent experiments. Scale bars, 5µm. (D) Line plot analysis: five kinetochore pairs were analysed per cell, for a total of 10 cells per experiment. Graphs represent the mean intensities (±SD) from three independent experiments. Intensity is normalized to the maximum signal present in each channel within the endogenous B56α experiment. Asterisks indicate significance (Mann-Whitney test); ****p≤0.0001.

https://doi.org/10.7554/eLife.42619.008
Figure 2 with 1 supplement
Sgo2 specifically localizes B56α to centromeres.

(A-G) The effect of Sgo1 and/or Sgo2 knockdown on YFP-B56α localisation in Flp-in HeLa cells. Representative images (A, C, F) and quantifications (B, D, G) of relative kinetochore intensity of B56α in cells arrested in prometaphase with nocodazole after knockdown of Sgo2 (A, B), Sgo1 (C, D), or Sgo1 +Sgo2 (F, G). (E) shows line plots of Sgo2 and B56α localisation following Sgo1 knockdown; 5 kinetochore pairs were analysed per cell, for a total of 10 cells per experiment. Graphs represent the mean intensities (±SD) from 3 independent experiments. Intensity is normalized to the maximum signal present in each channel within the endogenous B56α experiment. (H–M) Flp-in HeLa cells expressing YFP-B56α or YFP-B56γ were transfected with the CB-Sgo2 (H–K) or gChr7 +Cas9 DARPIN (L, M) and analysed for B56 recruitment in cells arrested in prometaphase with nocodazole. (H), (L), and J). are representative images; I) and K) are quantifications of relative centromere/kinetochore intensity of the indicated antigen; and M) is quantification of intensity of Sgo2 over B56 at the Chr7 locus. For all centromere/kinetochore intensity graphs, each dot represents a cell and 10 cells were quantified per experiment for at least 3 independent experiments. The spread of dots indicates the biological variation between individual cells and the errors bars display the variation between the experimental repeats (displayed as -/+SD of the experimental means). Asterisks indicate significance (Mann-Whitney test); ns p>0.05, *p≤0.05, ****p≤0.0001. Scale bars, 5µm.

https://doi.org/10.7554/eLife.42619.009
Figure 2—figure supplement 1
Cenp B-Sgo1 recruits both B56α and B56γ to centromeres.

(A-D) Flp-in HeLa cells expressing YFP-B56α or YFP-B56γ were transfected with CB-Sgo1 and analysed for B56 recruitment. Representative images (A and C) and quantifications (B and D) of relative centromere/kinetochore intensity of the indicated antigen in cells arrested in prometaphase with nocodazole. 10 cells were quantified per experiment and graphs represent the mean (±SD) of 4 independent experiments. Asterisks indicate significance (Mann-Whitney test); *****p≤0.0001. Scale bars, 5µm.

https://doi.org/10.7554/eLife.42619.010
Figure 3 with 2 supplements
BubR1 and Sgo1 localize B56γ to kinetochores.

B56γ kinetochore localisation in Flp-in HeLa cells after BubR1 knockdown (A, B, E) or mutation of the LxxIxE binding pocket (H187A: C), (D, F) in cells arrested in prometaphase with nocodazole. For each condition, representative images (A, C), quantification of relative centromere/kinetochore levels (B, D) and line plot analysis (E, F) depicts the levels and distribution of the indicated antigens. (G–J): representative images (G, I) and quantification of relative centromere/kinetochore intensities (H, J) YFP-B56γ WT or H187A following Sgo1 knockdown (G, H) or mutation of the Sgo1 binding region (ΔSgo1). For all centromere/kinetochore intensity graphs, each dot represents a cell and 10 cells were quantified per experiment from at least 3 independent experiments. The spread of dots indicates the biological variation between individual cells and the errors bars display the variation between experimental repeats (displayed as -/+SD of the experimental means). For the line plot analysis, 5 kinetochore pairs were analysed per cell, for a total of 10 cells per experiment. The graph represents the mean intensities (±SD) from at least 3 independent experiments. Intensity is normalized to the maximum signal in each channel in each experiment. Asterisks indicate significance (Mann-Whitney test); ****p≤0.0001. Scale bars, 5µm.

https://doi.org/10.7554/eLife.42619.011
Figure 3—figure supplement 1
Knl1 and Bub1 depletion completely removes B56γ from kinetochores.

(A-H) Flp-in HeLa cells expressing YFP-B56γ were treated with the indicated siRNA. Representative images (A, C, E and G) and quantifications of relative centromere/kinetochore intensity (B, D, F and H) of the indicated antigen in cells arrested in prometaphase with nocodazole. On the graphs, each dot represents a cell and 10 cells were quantified per experiment from at least 3 independent experiments. The spread of dots indicates the biological variation between individual cells and the errors bars display the variation between experimental repeats (displayed as -/+SD of the experimental means). Asterisks indicate significance (Mann-Whitney test); ns p>0.05, *p≤0.05, ****p≤0.0001. Scale bars, 5µm.

https://doi.org/10.7554/eLife.42619.012
Figure 3—figure supplement 2
Mutation of the Sgo1 binding region in B56γ perturbs binding to CB-Sgo1.

Flp-in HeLa cells expressing YFP-B56γ or YFP-B56γΔSgo1 were untransfected or transfected with CB-Sgo1. Representative images (A, C) and quantification (B, D) of relative kinetochore intensity of the indicated antigen in interphase cells or cells arrested in prometaphase with nocodazole. On the graphs, each dot represents a cell and 10 cells were quantified per experiment from at least three independent experiments. The spread of dots indicates the biological variation between individual cells and the errors bars display the variation between experimental repeats (displayed as -/+SD of the experimental means). Asterisks indicate significance (Mann-Whitney test); ns p>0.05, *p≤0.05, ****p≤0.0001. Scale bars, 5µm.

https://doi.org/10.7554/eLife.42619.013
Specific binding of B56γ to kinetochores reflects an enhanced ability to bind LxxIxE motifs.

(A) Immunoblot of the indicated proteins, containing a LxxIxE motif (Hertz et al., 2016), following YFP immunoprecipitation from nocodazole-arrested Flp-in HeLa cells expressing YFP-B56α or YFP-B56γ. (B) Quantification of the mean normalised intensity (+SD) of the indicated antigens in B56α immunoprecipitates, relative to B56γ immunoprecipitates, from at least 3 independent experiments. Representative images (C) and line plot analysis (D) of YFP-B56α in Flp-in HeLa cells arrested in nocodazole and treated with the indicated siRNA. Each line plot graph represents the mean intensities (±SD) from 3 independent experiments. 5 kinetochore pairs were analysed per cell, for a total of 10 cells per experiment. Intensity is normalized to the maximum signal in each channel in each experiment. Asterisks indicate significance (Welch’s t -test, unpaired); *p≤0.05, **p≤0.01, ****p≤0.0001. Scale bars, 5µm.

https://doi.org/10.7554/eLife.42619.014
Figure 5 with 2 supplements
A C-terminal loop in B56 specifies B56 localization to centromeres or kinetochores.

B56 localisation in B56α-γ chimaeras spanning the entire B56 (Ch1-4: A–C), a region at the C-terminus (Ch4a-4d: D–F). (A, D) Schematic representation of the B56α-γ chimaeras created. Representative images (B, E) and line plot analysis (C, F) to show the B56 localisation pattern in each chimaera. (G). Alignment of B56 isoforms within region 4d that controls centromere/kinetochore localisation. (G–H): Effect of 4 point-mutations within region 4d to convert B56α to the correspond B56γ sequence (B56αTKHG). Representative images (H) and line plot analysis (I) of B56α WT or B56αTKHG in cells arrested in prometaphase with nocodazole. Each graph represents the mean intensities (±SD) from 3 independent experiments. 5 kinetochore pairs were analysed per cell, for a total of 10 cells per experiment. Intensity is normalized to the maximum signal in each channel in each experiment. Scale bars, 5µm.

https://doi.org/10.7554/eLife.42619.015
Figure 5—figure supplement 1
Region four is sufficient to induce B56 localization to the centromere or kinetochore.

Schematic representation of the B56α-γ chimeras alongside representative images and line plot analysis of these chimeras in cells arrested in prometaphase with nocodazole. Each graph represents the mean intensities (±SD) from 2 independent experiments. five kinetochore pairs were analysed per cell, for a total of 10 cells per experiment. Intensity is normalized to the maximum signal in each channel in each experiment. Scale bars, 5µm.

https://doi.org/10.7554/eLife.42619.016
Figure 5—figure supplement 2
Holoenzyme assembly and mitotic exit is unperturbed by B56α TKHG mutation.

(A) Immunoblot of the PP2A subunits following YFP immunoprecipitation from nocodazole-arrested Flp-in HeLa cells expressing YFP-B56α WT or YFP-B56α TKHG. (B) Flp-in HeLa cells treated with siRNA against B56 pool and induced to re-express indicted antigens were subjected to time-lapse analysis to determine mitotic timing. The graph represents the cumulative data from 50 cells, which is representative of 3 independent experiments.

https://doi.org/10.7554/eLife.42619.017
Figure 6 with 2 supplements
A C-terminal loop in B56 regulates binding to Sgo2 and LxxIxE motifs to specify centromere/kinetochore localisation.

(A-D) Flp-in HeLa cells expressing either YFP-B56α WT or TKHG were transfected with the CB-Sgo2 and analysed for B56 recruitment (A, B) or gChr7 +dCas9 DARPIN to assess YFP-B56α:Sgo2 co-localisation (C, D). Representative images (A, C) and quantification of relative kinetochore intensity (B) or intensity of Sgo2 over B56α at the Chr7 locus (D). For the intensity graphs in B) and D), each dot represents a cell and 10 cells were quantified per experiment from at least 3 independent experiments. The spread of dots indicates the biological variation between individual cells and the errors bars display the variation between experimental repeats (displayed as -/+SD of the experimental means). (E) Immunoblot of the indicated antigens following immunoprecipitation of YFP from nocodazole-arrested Flp-in HeLa cells expressing YFP- B56γ, YFP-B56α WT or YFP-B56α-TKHG. (F) Quantification of the mean normalised intensity (+SD) of indicated antigens in B56α WT or B56α TKHG immunoprecipitates, relative to B56γ, from at least 4 experiments. (G) Crystal structure of PP2A-B56γ [accession code 2NPP (Xu et al., 2006)] with annotation to indicate the regions that specify localisation to centromeres or kinetochores (see Figure 6—figure supplement 1 for details of the additional centromere/kinetochore specification regions). Note, the structure is meant only as a guide because the critical regions may be present within B56α, which has not been structurally solved. The B56α WT values are also used in some of the points plotted in Figure 2m. Asterisks indicate significance (Mann-Whitney test, except Figure 6f: Welch’s t -test, unpaired); ns p>0.05, *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. Scale bars, 5µm.

https://doi.org/10.7554/eLife.42619.018
Figure 6—figure supplement 1
Additional residues from B56α are required to switch the localisation of B56γ.

(A) Schematic, representative images and line plot analysis of B56γ and B56γ EPVA localisation in nocodazole-arrested Flp-in HeLa cells. (B) Schematic, representative images and line plot analysis of additional B56γ-α chimaeras to define the extra regions in B56α needed to fully switch B56γ from kinetochores to centromeres. Each graph represents the mean intensities (±SD) from at least 2 independent experiments. 5 kinetochore pairs were analysed per cell, for a total of 10 cells per experiment. Intensity is normalized to the maximum signal in each channel in each experiment. Scale bars, 5µm.

https://doi.org/10.7554/eLife.42619.019
Figure 6—figure supplement 2
Sgo2 depletion does not enhance the ability of B56α to bind BubR1 or LxxIxE motifs.

Immunoblot of LxxIxE containing proteins (GEF-H1, BubR1 and RepoMan) following YFP immunoprecipitation from nocodazole-arrested Flp-in HeLa cells expressing YFP-B56α or YFP-B56γ and subjected to control or Sgo2 siRNA, as indicated.

https://doi.org/10.7554/eLife.42619.020

Tables

Key resources table
Reagent type
or resource
DesignationSource or referenceIdentifiersAdditional information
Cell line
(H.sapiens)
HeLa Flp-inTighe et al. (2008)
Recombinant
DNA reagent
pcDNA5-YFP-B56
α, β, γ1, γ3, δ and ε.
This paperB56 from pCEP-4xHA-B56
(Addgene 14532–14537) cloned
into pcDNA5-LAP-BubR1WT
(Nijenhuis et al., 2014),
Not1-Apa1 sites.
Recombinant DNA
reagent
pcDNA5-YFP-B56α−(TKHG)This paperSite-directed mutagenesis of
pcDNA5-YFP-B56α: E405T, P409K,
V412H, A413G
Recombinant DNA
reagent
pcDNA5-YFP-B56α-(γ4)This paperSee
Figure 5—figure supplement 1
Recombinant DNA
reagent
pcDNA5-YFP-B56γ-H187AThis paperSite-directed mutagenesis
of pcDNA5-YFP-B56γ
Recombinant DNA
reagent
pcDNA5-YFP-B56γ-ΔSgo1This paperSite-directed mutagenesis of
pcDNA5-YFP-B56γ: Y391F, L394S,
M398Q.
Recombinant DNA
reagent
pcDNA5-YFP-B56γ-H187A-ΔSgo1This paperSite-directed mutagenesis of
pcDNA5-YFP-B56γ-H187A: Y391F,
L394S, M398Q.
Recombinant DNA
reagent
pcDNA5-YFP-B56γ-(α4)This paperSee
Figure 5—figure supplement 1
Recombinant DNA
reagent
pcDNA5-YFP-B56γ-(α4.1)This paperSee
Figure 6—figure supplement 1
Recombinant DNA
reagent
pcDNA5-YFP-B56γ-(α4.2)This paperSee
Figure 6—figure supplement 1
Recombinant DNA
reagent
pcDNA5-YFP-B56γ-(α4.3)This paperSee
Figure 6—figure supplement 1
Recombinant DNA
reagent
pcDNA5-YFP-B56γ−(EPVA)This paperSite-directed mutagenesis of
pcDNA5-YFP-B56γ: T631E, K635P,
H638V, G639A.
Recombinant DNA
reagent
pcDNA5-YFP-B56-Ch1This paperSee Figure 5.
Recombinant DNA
reagent
pcDNA5-YFP-B56-Ch2This paperSee Figure 5.
Recombinant DNA
reagent
pcDNA5-YFP-B56-Ch3This paperSee Figure 5.
Recombinant DNA
reagent
pcDNA5-YFP-B56-Ch4This paperSee Figure 5.
Recombinant DNA
reagent
pcDNA5-YFP-B56-Ch4aThis paperSee Figure 5.
Recombinant DNA
reagent
pcDNA5-YFP-B56-Ch4bThis paperSee Figure 5.
Recombinant DNA
reagent
pcDNA5-YFP-B56-Ch4cThis paperSee Figure 5.
Recombinant DNA
reagent
pcDNA5-YFP-B56-Ch4dThis paperSee Figure 5.
Recombinant DNA
reagent
pcDNA5-vsv-CENP-
B-Sgo2-mCherry
This paperPCR Sgo2 from pDONR-Sgo2
(gift T.J.Yen) into pcDNA5-vsv-
CENP-B-Sgo1-mCherry
Recombinant DNA
reagent
pcDNA5-vsv-CENP-B-
Sgo1-mCherry
Meppelink et al. (2015)
Recombinant DNA
reagent
pHAGE-TO-dCas9-
DARPIN-flag
This paperProgenitor plasmid: pHAGE-TO-
dCas9-3xmCherry (Addgene
64108). 3xmCherry replaced with
synthesised DARPIN-Flag
(Brauchle et al., 2014).
Sequence-based
reagent
gRNA targeting a
repetetive region on
chromosome 7
Chen et al. (2016)GCTCTTATGGTGAGAGTGT
Sequence-based
reagent
B56 Knockin gRNAsThis paperB56a: gatgtcgtcgtcgtcgccgccgg.
B56g: gtcaacatctagacttcagcggg
Sequence-based
reagent
siRNAsFoley et al. (2011)B56α (PPP2R5A),
5’-UGAAUGAACUGGUUGAGUA-3’;
B56β (PPP2R5B),
5’-GAACAAUGAGUAUAUCCUA-3’;
B56γ (PPP2R5C),
5’-GGAAGAUGAACCAACGUUA-3’;
B56δ (PPP2R5D),
5’-UGACUGAGCCGGUAAUUGU-3’;
B56ε (PPP2R5E),
5’-GCACAGCUGGCAUAUUGUA-3’;
Sequence-based
reagent
siRNAsKitajima et al. (2006)Sgo2,
5’-GCACUACCACUUUGAAUAA-3’;
Sequence-based
reagent
siRNAsDharmacon, J-015475–12Sgo1,
5’-GAUGACAGCUCCAGAAAUU-3’;
Sequence-based
reagent
siRNAsNijenhuis et al. (2014)BubR1,
5’-AGAUCCUGGCUAACUGUUC-3’
Sequence-based
reagent
siRNAsVleugel et al. (2013)Knl1,
5’-GCAUGUAUCUCUUAAGGAA-3’;
Bub1
5’-GAAUGUAAGCGUUCACGAA-3’;
Sequence-based
reagent
siRNAsDharmacon (D-001830)Control (GAPDH),
5’-GUCAACGGAUUUGGUCGUA-3’
AntibodyMouse monoclonal
anti-GFP (clone 4E12/8)
Peter Parker, Francis
Crick Institute
1:1000
AntibodyChicken polyclonal
anti-GFP
AbcamAbcam: ab13970,
RRID:AB_300798
1:5000
AntibodyMouse monoclonal
anti-Sgo1 (clone 3C11)
AbnovaAbnova:
H001516480M01
1:1000
AntibodyRabbit polyclonal
anti-Sgo2
BethylBethyl:
A301-262A,
RRID:AB_890650
1:1000
AntibodyMouse monoclonal
anti-BubR1 (clone 8G1)
EMD MilliporeEMD Millipore:
05–898,
RRID:AB_417374
1:1000
AntibodyMouse monoclonal
anti-VSV (clone P5D4)
SigmaSigma: V5507,
RRID:AB_261877
1:1000
AntibodyRabbit polyclonal
anti-Knl1
AbcamAbcam:
ab70537,
RRID:AB_1209410
1:1000
AntibodyRabbit polyclonal
anti-Bub1
BethylBethy;l:
A300-373A,
RRID:AB_2065943
1:1000
AntibodyMouse monoclonal
anti-FLAG (clone M2)
SigmaSigma: F3165,
RRID:AB_259529
1:10000
AntibodyGuinea Pig polyclonal
anti-Cenp-C
MBLMBL: PD0301:5000
AntibodyRabbit polyclonal
anti-pMELT-Knl1
(phospho-T943 and -
T1155)
Nijenhuis et al. (2014)1:1000
AntibodyRabbit polyclonal
anti-GFP
Geert Kops, Hubrecht
Institute
1:5000
AntibodyMouse monoclonal
anti-B56γ (clone A-11)
Santa Cruz
Biotechnology
Santa Cruz
Biotechnology:
sc-374379,
RRID:AB_10988028
1:1000
AntibodyMouse monoclonal
anti-B56α (clone 23)
BD BiosciencesBD Biosciences:
610615,
RRID:AB_397947
1:1000
AntibodyMouse monoclonal
anti-B56δ (clone H-11)
Santa Cruz
Biotechnology
Santa Cruz
Biotechnology:
sc-271363,
RRID:AB_10611062
1:1000
AntibodyRabbit polyclonal
anti-B56ε
AvivaAviva:
ARP56694-P50
1:1000
AntibodyMouse monoclonal
anti-PPP2CA
(clone 1D6)
EMD MilliporeEMD Millipore:
05–421,
RRID:AB_309726
1:5000
AntibodyRabbit polyclonal
anti-PPP2R1A
(clone 81G5)
Cell Signaling
Technology
Cell Signaling
Technology: 2041,
RRID:AB_2168121
1:1000
AntibodyRabbit polyclonal
anti-BubR1
BethylBethyl:
A300-386A,
RRID:AB_386097
1:1000
AntibodyRabbit polyclonal
anti-Axin
Cell Signaling
Technology
Cell Signaling
Technology: C76H11,
RRID:AB_2274550
1:1000
AntibodyRabbit polyclonal
anti-GEF-H1
AbcamAbcam: ab1557851:1000
AntibodyRabbit polyclonal
anti-Kif4A
BethylBethyl: A301-074A,
RRID:AB_2280904
1:1000
AntibodyRabbit polyclonal
anti-Repoman
SigmaSigma: HPA030049,
RRID:AB_10600862
1:1000
AntibodyRabbit polyclonal
anti-Actin
SigmaSigma: A2066,
RRID:AB_476693
1:5000
AntibodyMouse monoclonal
anti-α-Tubulin
(clone B-5-1-2)
SigmaSigma: T5168,
RRID:AB_477579
1:5000
AntibodyAlexa-fluor488
anti-mouse
ThermoFisher
Scientific
Invitrogen: A11029,
RRID:AB_138404
1:1000
AntibodyAlexa-fluor488
anti-rabbit
ThermoFisher
Scientific
Invitrogen: A11034,
RRID:AB_2576217
1:1000
AntibodyAlexa-fluor488
anti-chicken
ThermoFisher
Scientific
Invitrogen: A11039,
RRID:AB_142924
1:1000
AntibodyAlexa-fluor488
anti-guinea pig
ThermoFisher
Scientific
Invitrogen: A11073,
RRID:AB_142018
1:1000
AntibodyAlexa-fluor568
anti-mouse
ThermoFisher
Scientific
Invitrogen: A11031,
RRID:AB_144696
1:1000
AntibodyAlexa-fluor568
anti-rabbit
ThermoFisher
Scientific
Invitrogen: A11036,
RRID:AB_10563566
1:1000
AntibodyAlexa-fluor647
anti-guinea pig
ThermoFisher
Scientific
Invitrogen: A21450,
RRID:AB_141882
1:1000
AntibodyHRP-anti-mouseBio-RadBio-Rad: 170–6516,
RRID:AB_11125547
1:2000
AntibodyHRP-anti-rabbitBio-RadBio-Rad: 170–6515,
RRID:AB_11125142
1:5000
Chemical
compound, drug
AZ-3146SelleckchemSelleckchem: S2731
Chemical
compound, drug
Calyculin ALC labsLC labs: C-3987
Chemical
compound, drug
4,6-diamidino-2-
phenylindole (DAPI)
SigmaRoche: 10236276001
Chemical
compound, drug
Dulbecco's Modified
Eagle Medium (DMEM)
ThermoFisher
Scientific
Gibco: 41966029
Chemical
compound, drug
Doxycycline hyclateSigmaSigma: D9891
Chemical
compound, drug
Fetal Bovine SerumThermoFisher
Scientific
Life Technologies:
10270106
Chemical
compound, drug
GFP-Trap magnetic
beads
ChromotekChromotek: GTMA-20
Chemical
compound, drug
Hygromycin BSanta Cruz
Biotechnology
Santa Cruz
Biotechnology:
sc-29067
Chemical
compound, drug
Lipofectamine
RNAiMax
ThermoFisher
Scientific
Invitrogen: 13778150
Chemical
compound, drug
NocodazoleEMD MilliporeEMD Millipore: 487928
Chemical
compound, drug
MG132SelleckcemSelleckchem: S2619
Chemical
compound, drug
Opti-MEM reduced
serum medium
ThermoFisher
Scientific
Gibco: 31985–047
Chemical
compound, drug
penicillin/streptomycinThermoFisher
Scientific
Gibco: 15070–063
Chemical
compound, drug
RO-3306TocrisTocris: 4181
Chemical
compound, drug
ThymidineSigmaSigma: T1895
Software,
algorithm
Kinetochore quantification
macro
Saurin et al. (2011)Software,
AlgorithmMulticolor Line
plot quantification
macro
Kees Straatman (University
of Leicester) with modification
by Balaji Ramalingam
(University of Dundee)
Software,
algorithm
Quantification of
immunoblots
Image Studio Lite
(LI-COR Biosciences)
Software,
algorithm
Microscopy image
processing
Softworx software,
GE Healthcare
Software,
algorithm
Microscopy image
processing
ImageJ, National
Institutes of Health

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for all figures that contain graphical information, which is every figure except Figure 1—figure supplements 1 and 2, and Figure 6 - figure supplement 2

Additional files

Source data 1

The raw data and statistical values from all the individual experiments that are expressed in graphical format.

This files contains the raw data and statistical values from all the graphs displayed in Figures 16; Figure 1—figure supplements 2, 4 and 5; Figure 2—figure supplement 1; Figure 3—figure supplement 2; Figure 5—figure supplements 1 and 2; Figure 6—figure supplement 1.

https://doi.org/10.7554/eLife.42619.021
Transparent reporting form
https://doi.org/10.7554/eLife.42619.022

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)