a direct role of mad1 in the spindle assmely checkpoint beyond mad2 kinetochore recruitment

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

http://dx.doi.org/10.1002/embr.201338114

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


(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

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KT-M

ad2

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-Mad

2

Input IP (Venus)

130

95

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ad2

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KT-Mad

2 R13

3A

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to

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ap

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se (

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A

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-24 0 24 48

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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

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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



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.




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

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ad2

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KT-O-M

ad2

0

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1000B

C

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+ + + +siMad1:

NE

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ap

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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***




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.

◂

▸




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




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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info:doi/10.1002/embr.201338114

a direct role of mad1 in the spindle assmely checkpoint beyond mad2 kinetochore recruitment

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