A Direct Role of Mad1 in the Spindle Assmely Checkpoint Beyond Mad2 Kinetochore Recruitment
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Transcript of A Direct Role of Mad1 in the Spindle Assmely Checkpoint Beyond Mad2 Kinetochore Recruitment
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Scientific Report
A direct role of Mad1 in the spindle assemblycheckpoint beyond Mad2 kinetochore recruitmentThomas Kruse†, Marie Sofie Yoo Larsen†, Garry G Sedgwick, J�on Otti Sigurdsson, Werner Streicher,
Jesper V Olsen & Jakob Nilsson*
Abstract
The spindle assembly checkpoint (SAC) ensures accurate chromo-some segregation by delaying entry into anaphase until all sisterchromatids have become bi-oriented. A key component of the SACis the Mad2 protein, which can adopt either an inactive open(O-Mad2) or active closed (C-Mad2) conformation. The conversionof O-Mad2 into C-Mad2 at unattached kinetochores is thought tobe a key step in activating the SAC. The template model proposesthat this is achieved by the recruitment of soluble O-Mad2 toC-Mad2 bound at kinetochores through its interaction with Mad1.Whether Mad1 has additional roles in the SAC beyond recruitmentof C-Mad2 to kinetochores has not yet been addressed. Here, weshow that Mad1 is required for mitotic arrest even when C-Mad2is artificially recruited to kinetochores, indicating that it hasindeed an additional function in promoting the checkpoint. TheC-terminal globular domain of Mad1 and conserved residues inthis region are required for this unexpected function of Mad1.
Keywords Mad1; Mad2; mitosis; SAC
Subject Categories Cell Cycle; Chromatin, Epigenetics, Genomics &
Functional Genomics
DOI 10.1002/embr.201338101 | Received 15 October 2013 | Revised 9 January
2014 | Accepted 9 January 2014 | Published online 28 January 2014
EMBO Reports (2014) 15, 282–290
See also: S Heinrich et al (March 2014)
Introduction
The SAC ensures accurate chromosome segregation by delaying
anaphase entry by inhibiting Cdc20, the mitotic co-activator of the
anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin
ligase essential for targeting cyclin B1 and securin for degradation
[1]. Cdc20 is inhibited by the direct binding of Mad2 and the
BubR1-Bub3 checkpoint proteins forming the mitotic checkpoint
complex (MCC) [2–6]. Current models propose that the Mad2-Cdc20
complex represents the initial inhibitory complex formed that is
then converted into the MCC by binding of BubR1-Bub3. Following
this Mad2 is removed by a p31-dependent mechanism to generate
the Cdc20-BubR1-Bub3 complex potentially representing the final
inhibitor [7–10].
Given the importance of the Mad2-Cdc20 complex, it is critical to
understand how unattached kinetochores catalytically generate this
complex. An important feature of Mad2 is that it exists in at least two
conformations, namely an active closed conformation (C-Mad2) that
is the conformation observed when Mad2 is bound to its ligands
Mad1 and Cdc20, and an inactive open conformation (O-Mad2),
which is the predominant conformation of soluble Mad2 [11–15].
The Mad1-Mad2 complex is an extremely stable complex displaying
little exchange of bound C-Mad2, and Mad1 makes contacts with the
kinetochore to position C-Mad2 at this structure [16–18]. Based on
the observation that C-Mad2 can catalyze the conversion of O-Mad2
into C-Mad2-Cdc20 in vitro and that C-Mad2 and O-Mad2 can dimer-
ize the “template model” proposes that unattached kinetochores act
to generate C-Mad2 by recruitment of O-Mad2 to the C-Mad2-Mad1
complex localized at unattached kinetochores [19–21]. This model
explains the need for both soluble Mad2 and the Mad1-Mad2 com-
plex, the observed FRAP kinetics of Mad2, and the requirement for
the Mad2 dimerization interface for a functional SAC [20,22–25].
In the template model, the active molecule at the kinetochore is
C-Mad2, while Mad1 merely acts to bring this molecule to the
kinetochore. In agreement with this, no differences in the ability to
promote O-Mad2 conversion have been observed when C-Mad2 and
C-Mad2-Mad1 were compared in in vitro assays [19].
Surprisingly, we show here that Mad1 is absolutely essential for
generating an active SAC even when C-Mad2 is constitutively recruited
to kinetochores. We find that the C-terminal globular domain of Mad1
and conserved residues in this region are critical for this role of Mad1 in
theSAC.Ourwork revealsanunexpecteddirect roleofMad1 in theSAC.
Results and Discussion
Constitutive recruitment of Mad2 to kinetochores results in amitotic arrest
To investigate whether the only function of Mad1 in the SAC is to
recruit Mad2 to kinetochores, we needed to bypass the require-
The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark*Corresponding author. Tel: +45 35325053; Fax: +45 35325001; E-mail: [email protected]†These authors contributed equally.
EMBO reports Vol 15 | No 3 | 2014 ª 2014 The Authors282
Published online: January 29, 2014
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ment of Mad1 for Mad2 kinetochore targeting. To this end, we tar-
geted Mad2 to kinetochores by fusing it to the C-terminus of the
outer kinetochore protein Ndc80 (referred to as KT-Mad2). The
KT-Mad2 fusion protein localized strongly to kinetochores, and
even at low levels, a strong mitotic arrest was observed with all
chromosomes aligned on the metaphase plate (Fig 1A and B). The
expression level of KT-Mad2 in the stable cell line was very low
compared to endogenous Mad2 (Supplementary Fig S1A). This
metaphase arrest persisted for hours until the metaphase plate col-
lapsed likely due to cohesion fatigue [26,27]. When we expressed
soluble Mad2, C-Mad2 (Mad2 L13A), or O-Mad2 (Mad2 V193N),
only negligible effects on mitotic progression were observed and
similarly targeting Mad2 to chromosomes via fusion to H2B did
not arrest cells in mitosis (Fig 1B). Analysis of recombinant Mad2
L13A and Mad2 V193N on a Resource Q column confirmed that
they were largely in the closed or open confirmation, respectively,
similar to what has been reported (Supplementary Fig S1B) [21].
The failure of soluble Mad2 proteins to induce a metaphase arrest
was not due to low expression levels as they were expressed at
much higher levels than KT-Mad2 (Supplementary Fig S1C). These
results show that Mad2 needs to be specifically targeted to kinet-
ochores to induce a mitotic arrest similar to what has been
described for Mad1 and Mps1 [28,29].
To investigate the conformational requirements of the kineto-
chore-targeted Mad2, we used the same approach to target
C-Mad2, O-Mad2, and Mad2 R133A that has a mutation in the
dimerization interface, hereby preventing Mad2 dimerization
[16,21]. While targeting of C-Mad2 to kinetochores produced a
strong metaphase arrest, cells expressing similar levels of targeted
O-Mad2 or Mad2 R133A did not arrest (Fig 1C). Purification of the
different Ndc80 fusions from stable cell lines arrested with noco-
dazole revealed that the tethered Mad2 molecules behaved as
expected in that only KT-Mad2 and KT-C-Mad2 bound to Mad1
(Fig 1D). These observations are in agreement with the template
model and provide further in vivo evidence for this model.
Mad1 is required for the KT-Mad2-induced mitotic arrest
Since the Ndc80 complex is essential for microtubule binding and
end-on attachment, we wanted to exclude that the observed arrest
upon kinetochore targeting of active Mad2 was due to a secondary
defect in kinetochore–microtubule interactions. To exclude this,
we analyzed the metaphase-arrested cells by immunofluorescence
after cold treatment, as this will depolymerize non-kinetochore
microtubules. Cells transfected with the different Mad2 tethering
constructs all exhibited robust K-fibers and end-on attachment to
kinetochores, and in addition, measurement of the distance
between sister kinetochore pairs revealed that tension was applied
(Fig 2A and B). Measuring the time from nuclear envelope break-
down to alignment of all chromosomes at the metaphase plate
revealed no major difference between cells expressing Ndc80-
Venus and KT-Mad2 (Fig 2C). These results and the observation
that KT-O-Mad2 does not affect chromosome segregation argue
that the arrest observed in KT-Mad2 and KT-C-Mad2 is due to the
persistent presence of these proteins rather than subtle defects in
kinetochore–microtubule interactions.
Given that KT-Mad2 and KT-C-Mad2 bind Mad1, we could now
test whether Mad1 was still required to obtain a strong metaphase
arrest. When we depleted Mad1 in cells by approximately 90%
using RNAi, the cell lines expressing KT-Mad2 and KT-C-Mad2 no
longer arrested for a prolonged time and the levels of Mad1 bound
by these molecules were strongly reduced (Fig 3A–C). A similar
result was obtained using KT-Mad2 L13Q, a mutation that also
maintains Mad2 in the closed conformation (Supplementary Fig S1D)
[21]. Thus, Mad1 binding to the kinetochore-targeted Mad2 mole-
cules is essential for inducing a prolonged mitotic arrest. In addition
to a requirement for Mad1, all other checkpoint components we
tested: Mps1, Bub1, BubR1, and soluble Mad2, were required for a
strong mitotic arrest (Fig 3D). This shows that KT-Mad2 induces a
SAC arrest requiring all components of the pathway. In the
metaphase-arrested cells, we could still detect Bub1 and BubR1 at
kinetochores although at lower levels than in prometaphase cells
(Supplementary Fig S2A and B). Their localization is likely also
critical for the observed metaphase arrest.
That Mad1 was still required for a SAC arrest despite the con-
tinuous kinetochore targeting of C-Mad2 suggested additional criti-
cal roles of Mad1 in the SAC beyond Mad2 kinetochore
recruitment. To make this conclusion, we needed to rule out that
the requirement of Mad1 did not reflect that KT-Mad2 and KT-C-
Mad2 depended on Mad1 binding for maintaining the closed con-
formation. To address this, we used a mouse monoclonal anti-
body generated in the laboratory specific for the closed
conformation of Mad2 (see Supplementary Fig 2C–E for character-
ization of this antibody). We stained metaphase plates from cells
expressing the different KT-Mad2 molecules in the presence or
absence of Mad1 depletion. The antibody did not stain cells
expressing KT-O-Mad2 as expected, while clear kinetochore stain-
ing coinciding with the GFP signal was observed in cells express-
ing KT-Mad2 and KT-C-Mad2 (Fig 3E and F). The levels of closed
Mad2 at kinetochores were higher in KT-C-Mad2-expressing cells
than in KT-Mad2-expressing cells, showing that the L13A muta-
tion maintains Mad2 in a closed conformation in vivo similar to
what was observed in our biochemical analysis of Mad2 L13A
(Supplementary Fig S1B). Upon Mad1 depletion, the staining with
the closed specific Mad2 antibody decreased in KT-Mad2 cells,
but not in KT-C-Mad2 (Fig 3E and F). Furthermore, the level of
p31, a C-Mad2-specific ligand, at metaphase kinetochores in KT-
C-Mad2-expressing cells was not affected by Mad1 depletion
(Supplementary Fig S3A). Comparison of the level of C-Mad2 at
kinetochores in KT-C-Mad2 and KT-Mad1 revealed no major
differences (Supplementary Fig S3B). Combined, these results
reveal that in KT-C-Mad2 the closed conformation of Mad2 is
maintained when Mad1 is depleted and can provide levels of
C-Mad2 at kinetochores to the same degree as KT-Mad1. The
dependency on Mad1 for an arrest in KT-C-Mad2-expressing cells
therefore argues for additional roles of Mad1 in the SAC beyond
C-Mad2 kinetochore recruitment.
The C-terminal domain of Mad1 is critical for a functional SAC
To gain further mechanistic insight into how Mad1 acts in the SAC,
we first investigated which domains of Mad1 are required for its
function in the SAC when its role in kinetochore recruitment of
C-Mad2 is bypassed. Mad1 is a 718 amino acid long protein consisting
of a long coiled-coil region preceding the Mad2-binding site followed
by two alpha helixes that pair and end in a small globular domain
Thomas Kruse et al A direct role of Mad1 in the SAC EMBO reports
ª 2014 The Authors EMBO reports Vol 15 | No 3 | 2014 283
Published online: January 29, 2014
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(Fig 4A) [12,30]. We generated a panel of N-terminal and C-terminal
truncations of Mad1 all containing the Mad2-binding site. As a con-
trol, we also generated the full-length Mad1 protein with a mutated
Mad2-binding site (Fig 4A). The Mad1 constructs were made resis-
tant to the Mad1 RNAi oligo and tagged with mTurquoise (TFP) at
the N-terminus. All the constructs were able to bind Mad2 as
expected (Fig 4B).
We then depleted Mad1 from the cell line expressing KT-Mad2
and complemented the cells with the different Mad1 constructs
(Fig 4C). As overexpression of Mad1 abrogates the SAC due to
sequestering of soluble Mad2, only cells expressing low levels of
Mad1 were able to restore the metaphase arrest and all con-
structs were analyzed in this range of expression. All Mad1 con-
structs were recruited to the kinetochores by KT-Mad2 except
C
D
BubR1
Mad1
Cdc20
p31
Mad2
GFP
KT-C
-Mad
2
KT-M
ad2
Ndc
80
Ndc
80
KT-O
-Mad
2
KT-C
-Mad
2
KT-M
ad2
KT-O
-Mad
2
Input IP (Venus)
130
95
55
34
25
130
KT-C-M
ad2
KT-O-M
ad2
KT-Mad
2 R13
3A
0
100
200
300
400
500
600
700
NE
BD
to
an
ap
ha
se (
min
ute
s)
A
24 0 24 48 240 480
-24 0 24 48
DIC
Venus
Histone
DIC
Venus
Histone
KT-Mad2 (Ndc80-Venus-Mad2)
KT-O-Mad2
Legends
Kinetochore C-Mad2 MicrotubuleO-Mad2Ndc80-Complex Mad1
B
NE
BD
to
an
ap
ha
se (
min
ute
s)
KT-Mad
2
Untethered
Ndc80
Mad
2
C-Mad
2
O-Mad
2
H2B-M
ad2
0
100
200
300
400
500
600
700
800Exit from mitosis during recording
Arrested in mitosis at the end of recording
Figure 1. Targeting of Mad2 to kinetochores results in a SAC arrest.
A Still images from a time-lapse movie of a stable HeLa cell line expressing the KT-Mad2 (Ndc80-Venus-Mad2) fusion protein or the KT-O-Mad2 protein, and ahistone marker. Time is in minutes and t = 0 at NEBD.
B, C NEBD-Anaphase times in stable HeLa cell lines expressing the indicated Mad2 constructs as measured by time-lapse microscopy. Each dot represents a single celland red dots are cells that were still arrested when the recording ended. The red line indicates the median.
D The indicated Ndc80 fusion proteins were purified using GFP binder resin from nocodazole-arrested cells and analyzed for their ability to bind the indicatedproteins by Western blot.
EMBO reports A direct role of Mad1 in the SAC Thomas Kruse et al
EMBO reports Vol 15 | No 3 | 2014 ª 2014 The Authors284
Published online: January 29, 2014
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Mad1 D1-500 (Fig 4D). As expected, full-length Mad1 restored
the KT-Mad2-induced arrest and this depended on its Mad2-bind-
ing site. More importantly, the C-terminal globular domain was
absolutely required for restoring the arrest as cells complemented
with Mad1 1-639 divided with almost normal mitotic timing
(Fig 4C). A similar requirement for the C-terminal globular
domain of Mad1 was observed in KT-C-Mad2-expressing cells
(Supplementary Fig S3C).
Ndc80
A
C
B
KT-Mad2
KT-C-Mad2
KT-O-Mad2
Nocodazole
0.0
0.5
1.0
1.5
Ndc80
KT-Mad
2
KT-C-M
ad2
KT-O-M
ad2
Ndc80
KT-Mad
2
KT-C-M
ad2
KT-O-M
ad2
Nocod
azole
Dis
tanc
e be
twee
n ki
neto
chor
e pa
irs (
µm)
NE
BD
to M
etap
hase
(M
inut
es)
Tubulin CREST GFP TubulinCREST
DAPI
10µm
****
****
0
5
10
15
20
25
30
35
40
45
50
ns
Figure 2. Normal kinetochore–microtubule interactions in tethered Mad2 cell lines.
A Cells were transfected with the indicated Ndc80 fusion proteins or treated with nocodazole and treated on ice prior to fixation and stained with the indicatedantibodies to monitor microtubule–kinetochore interactions.
B The distance between kinetochore pairs on metaphase plates was determined by measuring the distance between the two CREST pairs from the images in (A). Atleast 60 pairs were measured from at least 9 different cells for each condition, and a red line indicates the mean and standard deviation is indicated. Each dotrepresents one kinetochore pair. A t-test was used to compare the different conditions *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001, ns: non-significant P > 0.05.
C The time from NEBD to the alignment of all chromosomes on the metaphase plate was measured from the time-lapse movies for the indicated cell lines. The medianis indicated by the red line and was 20 minutes for all conditions.
Thomas Kruse et al A direct role of Mad1 in the SAC EMBO reports
ª 2014 The Authors EMBO reports Vol 15 | No 3 | 2014 285
Published online: January 29, 2014
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Mad1
Ndc80
Tubulin
siMad1:
KT-M
ad2
Ndc
80
KT-C
-Mad
2KT
-O-M
ad2
A
-18 0 24 42
KT-Mad2 + siMad1
DIC
Venus
siLuc
Rever
sine
siBub
R1
siMad
2 #1
siMad
2 #2
siBub
1
siMad
2 #1+
#2
0
100
200
300
400
500
600
700
800
900
Ndc80
Ndc80
KT-Mad2
KT-Mad
2
KT-C-M
ad2
KT-C-M
ad2
KT-O-M
ad2
KT-O-M
ad2
0
100
200
300
400
500
600
700
800
900
1000B
C
D
+ + + +siMad1:
NE
BD
to
an
ap
ha
se (
min
ute
s)N
EB
D t
o a
na
ph
ase
(m
inu
tes)
- + - + - + - +
KT-M
ad2
Ndc
80
KT-C
-Mad
2KT
-O-M
ad2
- + - + - + - +
siMad1
Input IP (Venus)
KT-Mad2
KT-O-Mad2siLUC
DAPI GFP C-Mad2 DAPI GFP C-Mad2
KT-Mad2siLUC
KT-Mad2siMAD1
KT-C-Mad2siLUC
KT-C-Mad2siMAD1
10
0
10
20
30
40
50
KT-O-M
ad2
+ +
KT-C-M
ad2
KT-Mad
2KT-M
ad2
KT-C-M
ad2
siMad1:
C-M
ad
2 i
nte
nsi
ty n
orm
aliz
ed
to
GF
P
E F
10 µm 10 µm
Mad1 KD
24h 30h 18h 6h
Mad1 KD
Thymidine+
DoxycyclineThymidineRelease Analyze
****ns***
EMBO reports A direct role of Mad1 in the SAC Thomas Kruse et al
EMBO reports Vol 15 | No 3 | 2014 ª 2014 The Authors286
Published online: January 29, 2014
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In a complementary approach, we fused the different Mad1
truncations to the C-terminus of Ndc80-Venus to monitor their
ability to induce a mitotic arrest (referred to as KT-Mad1). Similar
to what has been reported for a Mis12-Mad1 fusion protein [28],
the constitutive targeting of Mad1 to Ndc80 resulted in a strong
metaphase arrest (Fig 4E–F). Again in this assay, we observed a
clear dependency on the C-terminal globular domain of Mad1 to
obtain a metaphase arrest. The targeted Mad1 constructs, except
for Mad1 DMad2, recruited C-Mad2 to kinetochores as evidenced
by quantitative immunofluorescence with the closed specific
Mad2 antibody (Supplementary Fig S4A and B), reaffirming that
failure in inducing an arrest is not due to the lack of C-Mad2 at
the kinetochore. Also, we observed that mutation of conserved
surface-exposed residues in the C-terminal globular domain (resi-
dues 710, 712, 714) strongly reduced the ability of tethered Mad1
to induce a metaphase arrest (Fig 4F). These residues were also
required for soluble Mad1 function (Supplementary Fig S4C). In
addition, two mutations, Mad1 L571D and Mad1 L575D,
described to affect the pairing of the two terminal alpha helices
[12], which positions the C-terminal domain, had reduced activity
in this assay (Fig 4F).
The results presented in this study reveal an essential role of
Mad1 in the SAC unrelated to its role in recruiting C-Mad2 to kinet-
ochores. We show a critical role for the globular C-terminal domain
of Mad1 and conserved residues within this domain that have been
shown not to affect kinetochore recruitment or dimerization of
Mad1 [30]. Similar conclusions have been obtained by the Hauf
laboratory in fission yeast [31]. Potentially, this function of the
Mad1 C-terminal domain is conserved.
Previous work from the Hardwick laboratory has revealed a check-
point-dependent complex between Mad1 and Bub1 in budding yeast
that depends on the conserved RLK motif of Mad1 (residues 617-619
in human Mad1) [32]. Using in vitro translated proteins, an interac-
tion has also been reported for the human proteins [33]. In budding
yeast, the Mad1-Bub1 interaction is required for a functional SAC in
part due to the fact that the interaction is required for Mad1 kineto-
chore localization. Since we observe a dependency for Bub1 even
when the Mad1-Mad2 complex is tethered, it could be that the Mad1-
Bub1 interaction is required for a functional SAC in addition to play-
ing a role in Mad1-Mad2 recruitment. However, analysis of endoge-
nous and exogenous Mad1 by exhaustive mass spectrometry as well
as extensive yeast two-hybrid screens has failed to detect Bub1 as a
binding partner for Mad1. Potentially, this interaction in human cells
is very weak compared to budding yeast or the role of the Mad1 C-ter-
minal domain observed here is unrelated to Bub1 binding. Defining
this function of Mad1 will be an important future goal.
Materials and Methods
Cloning
Using a forward primer for Venus and a reverse primer for
either Mad2 or Mad1, the appropriate Venus-Mad fragments were
amplified and inserted into pcDNA5/FRT/TO Ndc80-Venus [34] by
using ApaI and NotI restriction enzymes that removed Venus and
allowed in-frame insertion of the various Venus-Mad PCR products.
All constructs were fully sequenced.
Figure 3. Mad1 is required for a SAC arrest despite Mad2 persisting on kinetochores.
A Still images from time-lapse movies of a stable HeLa cell line expressing KT-Mad2 where Mad1 has been depleted by RNAi. The protocol for depletion of Mad1 isdepicted above.
B NEBD-Anaphase times were measured from time-lapse movies for the indicated Ndc80 fusions either depleted of Mad1 or control-depleted. Each dot represents asingle cell and red dots represent cells that were still arrested when the recording ended. The median is indicated by a red line.
C Purification of the indicated Ndc80 fusions from stable HeLa cell lines treated with nocodazole using GFP binder. Cells were either depleted of Mad1 or treated witha control oligo as indicated and probed for Ndc80 and Mad1.
D A stable HeLa cell line expressing KT-Mad2 was depleted of soluble Mad2 using two different oligos targeting the 3′ UTR of Mad2 or depleted of BubR1 or Bub1using RNAi. Mps1 was inhibited using reversine. For each condition, the NEBD-Anaphase time was measured from time-lapse movies and each dot represents asingle cell analyzed.
E The indicated Ndc80 fusions were transfected into HeLa cells and either control-depleted or depleted of Mad1 using RNAi. The cells were fixed and stained forexpression of the fusion protein (GFP) and closed Mad2 (C-Mad2).
F The level of C-Mad2 and GFP signal was quantified from deconvoluted images using 3 z-stacks 200 nm apart encompassing the bulk of kinetochore signal, andthen the C-Mad2 signal was normalized to GFP. Each dot represents a single kinetochore, and at least 47 kinetochores from at least seven different cells wereanalyzed. The mean is indicated by a red line and standard deviation as well. A t-test was used to compare the different conditions ***P ≤ 0.001, ****P ≤ 0.0001,ns: non-significant P > 0.05.
Figure 4. The globular C-terminal domain of Mad1 is essential for a functional SAC.
A Schematic of the different Mad1 constructs with the Mad2-binding site and globular domain (CTD) indicated.B Stable cell lines expressing the indicated FLAG-Venus-Mad1 constructs were purified from nocodazole-arrested cells, and their ability to bind Mad2 was determined
by Western blotting.C The stable KT-Mad2 cell line was depleted for Mad1 and complemented with the indicated Mad1 constructs. NEBD-Anaphase times were determined for the
different Mad1 constructs expressing similar levels. The red line indicates the median, and each dot represents a single cell analyzed.D Still images from time-lapse analysis of stable Venus-Mad1 cell lines transfected with TFP-tagged KT-Mad2.E Still images from time-lapse movies of a cell expressing Ndc80-Venus-Mad1 (KT-Mad1) with time given in minutes and time at NEBD set to zero.F NEBD-Anaphase times determined from time-lapse movies of cells expressing the indicated Ndc80 fusion proteins. Each dot represents a single cell analyzed and
red dots represent cells still arrested in mitosis when the recording ended. The red lines indicate the median.
◂
▸
Thomas Kruse et al A direct role of Mad1 in the SAC EMBO reports
ª 2014 The Authors EMBO reports Vol 15 | No 3 | 2014 287
Published online: January 29, 2014
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A
200
300
400
500
1-639
1-598
Mad2
B
DIC KT-Mad2 Mad1
FL
Mad1 constructs:
Δ100
Δ200
Δ300
Δ400
Δ500
1-639
1-598
D
CTD
CTD
CTD
CTD
CTD
CTD
1 718
718
718
718
718
718
639
598
Mad2 binding site mutated
FL
Mad1 constructs:
100100
200
300
400
500
1
1
CTD1
C
F
E
KT-Mad1
DIC
Venus
24 0 80 240 400
Mad1 constructs:
NE
BD
to a
naph
ase
(min
utes
)
0
100
200
300
400
500
600
700
800
900
KT-Mad2 + siMad1
Mad1 constructs:
FLAG(Mad1)
Mad2
Δ100
FL Δ300
Δ200
Δ500
Δ400
1-59
8
1-63
9
Δ100
FL Δ300
Δ200
Δ500
Δ400
1-59
8
1-63
9130
95
26
72
55
Input IP (Venus)
siMad1Mad1
complement?
Mad1 KD
24h 30h 18h 6h
Mad1 KDComplementingMad1 construct
Thymidine+
DoxycyclineThymidineRelease Analyze
KT-Mad1:
NE
BD
to a
naph
ase
(min
utes
)
siMad1
0
100
200
300
400
500
600
700
800
900
KVLHMSLNP
AAAHMSLNP
540 548
FLΔ1
00Δ2
00Δ3
00Δ4
00Δ5
001-
639
1-59
8
ΔMad
2
FLΔ1
00Δ2
00Δ3
00Δ4
00Δ5
00
ΔMad
2
L571
D
L575
D
F712A
/R71
4A
E710A
/F71
2A1-
639
1-59
8
EMBO reports A direct role of Mad1 in the SAC Thomas Kruse et al
EMBO reports Vol 15 | No 3 | 2014 ª 2014 The Authors288
Published online: January 29, 2014
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Antibodies
All antibodies used are specified in Supplementary Data 1.
RNAi depletion and rescue
For efficient Mad1 depletion, cells were subjected to a double
knock-down protocol using 10 nM of Mad1 siRNA oligo (Ambion
Silencer Select, s15905) with transfection on days one and two.
As a control, 50 nM of Luciferase siRNA oligo (SIGMA, VC300B2)
was used. Cells were analyzed on day three or four as indicated.
In RNAi rescue experiments, cells were co-transfected with the
Mad1 siRNA oligo and the complementing plasmid constructs on
day two.
For depletion of Mad2, BubR1, and Bub1, the following oligos
were used from Sigma: 5′ GAUGGUGAAUUGUGGAAUA (BubR1), 5′
GAGUGAUCACGAUUUCUAA (Bub1), 5′ CCUGAAAUCAAGUCAU-
CUA (MAD2 #1), 5′ ACUGAACUGUGUUAAUUG (MAD2 #2).
Purification of complexes/immunoprecipitation analysis
Cells were lysed in lysis buffer (50 mM Tris–HCl pH 7.5, 150 mM
NaCl, 1 mM EDTA, 1 mM DDT, and 0.1% NP40). Complexes were
immunoprecipitated in lysis buffer with antibodies coupled to Pro-
tein G-Sepharose 4B (Invitrogen) or GFP-Trap (ChromoTek) beads
as indicated and incubated at 4°C for 2 h or 30 min, respectively.
Precipitated protein complexes were washed three times in lysis buf-
fer and eluted in 2× SDS sample buffer.
Immunofluorescence
After thymidine treatment, cells were released and given MG132
for 2 hours prior to fixation to keep cells in mitosis. The cells were
pre-fixed for 20 s in 4% formaldehyde, permeabilized in 0.5% Tri-
ton X-100, and then fixed for 20 min in 4% formaldehyde. For cold
treatment, cells were put on ice 10 min prior to fixation. The fixed
cells were quenched with 25 mM glycine for 20 min, incubated
with primary antibodies for 2 h at room temperature or overnight
at 4°C, followed by 1 h of incubation with appropriate secondary
antibodies and DAPI (1:1,000). For detection of the transfected
constructs, GFP antibody or GFP-booster (1:200, ChromoTek) was
used.
Supplementary information for this article is available online:
http://embor.embopress.org
AcknowledgementsWe thank Stephen Taylor for providing the HeLa/FRT/TRex cell line and Silke
Hauf for sharing unpublished results. Mia F. Nielsen and Tine K. Nielsen kindly
prepared recombinant Mad2 protein. This work was supported by grants to JN
from the Novo Nordisk Foundation and the Lundbeck Foundation.
Author contributionTK performed biochemical analysis and live cell analysis of tethered proteins.
MSYL performed immunofluorescence analysis and helped with live cell analy-
sis. GGS and WS performed the characterization of the C-Mad2 antibody. JOS
and JVS assisted with MS analysis of Mad1 complexes. JN assisted with clon-
ings and designing of experiments and wrote the paper.
Conflict of interestThe authors declare that they have no conflict of interest.
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Published online: January 29, 2014