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
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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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

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

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