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Mol Cell. Author manuscript; available in PMC 2014 March 28.
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Mol Cell. 2013 March 28; 49(6): 1097–1107. doi:10.1016/j.molcel.2013.01.023.

The MRN-CtIP pathway is required for metaphase chromosome


alignment
Lorene Rozier1, Yige Guo1, Shaun Peterson2, Mai Sato2, Richard Baer2, Jean Gautier2,*,
and Yinghui Mao1,*
1Department of Pathology and Cell Biology, Columbia University College of Physicians and

Surgeons, 630 W 168thStreet, New York, NY 10032


2Institute
for Cancer Genetics, Irving Cancer Research Center, 1130 St. Nicholas Avenue, New
York, NY 10032

Summary
Faithful duplication of the genome in S phase followed by its accurate segregation in mitosis is
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essential to maintain genomic integrity. Recent studies have suggested that proteins involved in
DNA transactions are also required for whole chromosome stability. Here we demonstrate that the
MRN (Mre11, Rad50, and Nbs1) complex and CtIP are required for accurate chromosome
segregation. Depletion of Mre11 or CtIP, antibody-mediated inhibition of Mre11, or small
molecule inhibition of MRN using mirin results in metaphase chromosome alignment defects in
Xenopus egg extracts. Similarly, loss of MRN function adversely affects spindle assembly around
DNA-coated beads in egg extracts. Inhibition of MRN function in mammalian cells triggers a
metaphase delay and disrupts the RCC1-dependent RanGTP gradient. Addition of the Mre11
inhibitor mirin to egg extracts and mammalian cells reduces RCC1 association with mitotic
chromosomes. Thus, the MRN-CtIP pathway contributes to Ran-dependent mitotic spindle
assembly by modulating RCC1 chromosome association.

Introduction
Proper mitotic spindle assembly is essential for accurate chromosome segregation. There are
two major pathways of spindle assembly: “Search and capture” and “Self-assembly” (Gadde
and Heald, 2004; Walczak and Heald, 2008). During “Search and capture”, microtubules are
nucleated from centrosomes, captured by kinetochores, and thereby stabilized to form the
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mitotic spindle (Kirschner and Mitchison, 1986). In contrast, the “Self-assembly” model
posits that microtubules are nucleated around chromosomes and sorted into an antiparallel
array to generate the bipolar spindle (Heald et al., 1996). The relative contribution of these
two pathways varies in different systems (Figure 1A). Xenopus egg extracts have the
advantage of allowing independent examination of both pathways during mitosis. On one
hand, the addition of sperm chromosomes to these extracts can induce functional centrosome
formation, kinetochore-mediated spindle assembly and chromosome alignment (Murray,

© 2013 Elsevier Inc. All rights reserved.


*
Address for correspondence: Dr. Yinghui Mao, Department of Pathology and Cell Biology, Columbia University College of
Physicians and Surgeons, 630 W 168th Street, P&S 14-460, New York, NY 10032, Tel 212-305-7914, Fax 212-342-5459,
ym2183@columbia.edu. Dr. Jean Gautier, Institute for Cancer Genetics, Irving Cancer, Research Center, Columbia University, 1130
St. Nicholas Avenue, Room 602, New York, NY 10032, Tel 212-851-4564, Fax 212-851-5284, jg130@columbia.edu.
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Rozier et al. Page 2

1991). On the other hand, adding plasmid DNA-coated beads to Xenopus egg extracts can
drive bipolar microtubule structure formation in the absence of centrosomes and
kinetochores (Heald et al., 1996). This chromatin-dependent spindle formation relies
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primarily upon the establishment of a RanGTP gradient (Carazo-Salas et al., 1999; Ohba et
al., 1999; Wilde and Zheng, 1999), which is generated by the chromatin-bound guanine
nucleotide exchange factor (GEF) RCC1 (Li et al., 2003).

The MRN complex is an evolutionary conserved protein complex composed of Mre11,


Rad50, and Nbs1 (Xrs2 in budding yeast) (Symington, 2002). Mutations in the genes
encoding the yeast MRX proteins result in genomic instability, increased sensitivity to
ionizing radiation, telomere shortening, and meiosis defects, indicating that the MRX
complex is involved in multiple aspects of DNA end metabolism, including double-strand
break (DSB) sensing, end-processing, and repair (Assenmacher and Hopfner, 2004;
D’Amours and Jackson, 2002; Stracker et al., 2004; Symington and Gautier, 2011).
Disruption of any component of the MRN complex yields early embryonic lethality in mice
(Kang et al., 2002; Luo et al., 1999; Xiao and Weaver, 1997) and unviable vertebrate cell
lines (Yamaguchi-Iwai et al., 1999). Hypomorphic mutations in the mre11, rad50 or nbs1
genes result in rare autosomal recessive genetic diseases, Ataxia-Telangiectasia-like disorder
and Nijmegen breakage syndrome (Stewart et al., 1999; Varon et al., 1998; Waltes et al.,
2009; Wang et al., 2004). These disorders are characterized by chromosomal instability,
hypersensitivity to ionizing radiation, and radio-resistant DNA synthesis, suggesting that the
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MRN complex can function to suppress the onset of DNA damage-induced tumorigenesis.
In addition, chromosomal instability is also associated with mutations in components of the
MRN complex in sporadic tumors (Wang et al., 2004). The CtIP protein cooperates with
MRN to perform a subset of its functions, especially the resection of DSBs to yield a 3′
ssDNA overhang required for homology-directed repair (HDR) (Chen et al., 2008; Sartori et
al., 2007). Both MRN and CtIP can associate with the BRCA1/BARD1 heterodimer to
regulate the G2 DNA damage checkpoint (Greenberg et al., 2006; Wang et al., 2000; Yu and
Chen, 2004). The tumor suppression function of BRCA1 is thought to reflect its role in
homology-dependent repair of double-strand breaks. Nonetheless, BRCA1 also regulates
mitotic spindle assembly downstream of the Ran GTPase (Joukov et al., 2006).

Here, we have used Xenopus egg extracts and mammalian cells to support a model in which
the MRN-CtIP pathway contributes to Ran-dependent mitotic spindle assembly and
metaphase chromosome alignment by recruiting or stabilizing RCC1 chromosome
association.

Results
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The MRN complex is essential for metaphase chromosome alignment in both CSF-arrested
and cycled Xenopus egg extracts
To assess whether the MRN complex regulates mitotic spindle functions, we examined the
consequences of quantitative MRN depletion on mitotic spindle assembly and metaphase
chromosome alignment using a specific anti-Mre11 antibody (Costanzo et al., 2001; Dupre
et al., 2006; Dupre et al., 2008) in Xenopus egg extracts. We confirmed by immunoblotting
that Mre11 was efficiently removed from CSF (cytostatic factor) – arrested (M-phase)
extracts following immunodepletion (Figure 1B). We previously reported that Mre11
depletion results in the co-depletion of its binding partners Rad50 and Nbs1 (Costanzo et al.,
2001; Dupre et al., 2008). Following addition of sperm nuclei to M-phase extracts,
condensed mitotic chromosomes are formed. As anticipated, Mre11 was associated with
mitotic chromosomes in control and mock-depleted extracts but not in MRN-depleted
extracts (Figure 1C). Depletion of MRN did not yield obvious effects on mitotic
chromosome morphology or kinetochore assembly in Xenopus egg extracts (Figure 1C).

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Mre11 depletion from interphase extracts prior to DNA replication results in the
accumulation of low levels of DSBs (Costanzo et al., 2001), which could adversely affect
spindle assembly. To circumvent this potential problem, we first assessed the consequences
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of MRN depletion from CSF-arrested, non-cycled M-phase egg extracts, which support
spindle assembly and metaphase chromosome alignment independently of DNA replication.
Undepleted, mock-depleted, and Mre11-depleted CSF extracts were supplemented with
demembranated sperm nuclei and spindle formation was monitored in the presence of
rhodamine-labeled tubulin. Mock-depleted and undepleted extracts yielded predominantly
bipolar spindles with chromosomes properly aligned at the metaphase plate. Depletion of the
MRN complex did not affect centrosome-dependent microtubule nucleation or spindle
assembly; however, it did induce misaligned chromosomes in a majority of bipolar spindles
(Figure 1, D and E). Significantly, the latter defect could be partially rescued by
supplementing Mre11-depleted extracts with purified recombinant MRN complexes (Figure
1, D and E). Longer incubation times did not alter the proportion of bipolar spindles with
properly aligned chromosomes. These results suggest that the MRN complex is essential for
proper chromosome alignment during mitotic spindle assembly, but not for centrosome-
dependent microtubule nucleation or bipolar-spindle formation, in Xenopus egg extracts.

Similar metaphase chromosome alignment defects were observed in “cycled” egg extracts
(Figure S1, A and C), in which sperm chromatin had undergone DNA replication and
arrested at the subsequent M phase. To strengthen these results in cycled egg extracts, we
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sought to inhibit the MRN activity through independent means by addition of a different
neutralizing anti-Mre11 antibody. CSF-arrested egg extracts were cycled through interphase
to generate duplicated sister chromatids and were subsequently trapped in M phase upon
addition of an aliquot of egg extract containing both active Cdk1 and CSF, allowing the
accumulation of M-phase structures. Mre11 antibody was added after DNA replication to
exclude possible defects caused by the lack of MRN activity during S-phase. Addition of
this neutralizing anti-Mre11 antibody yielded spindles with misaligned chromosomes
(Figure S1, B and C), in a manner similar to MRN immunodepletion. Chromosome
alignment was also disrupted by addition of mirin, a small-molecule inhibitor of MRN
(Dupre et al., 2008; Rass et al., 2009; Roques et al., 2009), to cycled egg extracts (Figure S1,
B and C). Collectively, these results further support a role for the MRN complex in
metaphase chromosome alignment in egg extracts and exclude the possibility that the
chromosome misalignment phenotype is caused by co-depletion of other components
associated with the MRN complex.

The CtIP protein, a binding partner of the MRN complex, is required for metaphase
chromosome alignment in Xenopus egg extracts
The BRCA1/BARD1 heterodimer has been shown to be required for mitotic spindle
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assembly downstream of Ran GTPase and for the accumulation of TPX2 on spindle poles in
cycled egg extracts and human cells (Joukov et al., 2006). Since MRN interacts with
BRCA1 (Chen et al., 2008), we examined mitotic spindle assembly and chromosome
alignment in BRCA1-depleted CSF-arrested egg extracts. BRCA1 depletion did not
significantly affect bipolar spindle morphology as seen by immunofluorescence microscopy.
However, BRCA1 depletion resulted in a higher frequency of misaligned chromosomes than
in control spindles (Figure 2, A, C, and J). This phenotype is similar to what we observe in
Mre11-depleted uncycled egg extracts (Figure 2, B and J), although with fewer bipolar and
misaligned structures (Figure 2J). Notably, BRCA1 depletion was carried out in M-phase
arrested extracts, ruling out the possibility that this phenotype results from defective S-phase
in the absence of BRCA1.

The CtIP protein is required for a subset of MRN functions, including the resection of DSBs
to generate 3′ ssDNA ends suitable for HDR (Mimitou and Symington, 2011; Sartori et al.,

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2007) and DNA damage-dependent MRN-BRCA1 interaction (Chen et al., 2008). To


understand how MRN regulates metaphase chromosome alignment, we quantitatively
depleted CtIP from CSF egg extracts using a monoclonal antibody (Peterson et al., 2011)
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(Figure 2F). CtIP depletion yielded bipolar spindles with misaligned chromosomes (Figure
2, G and J). Addition of purified wild-type recombinant CtIP protein to CtIP-depleted
extracts restored metaphase chromosome alignment to control levels (Figure 2, H and J),
indicating that the MRN-CtIP pathway regulates proper chromosome alignment on mitotic
spindles. CtIP is found in a complex with BRCA1 and MRN and it may tether or coordinate
MRN and BRCA1 activities in the initial steps of homologous recombination (Greenberg et
al., 2006). The physical interaction between CtIP and BRCA1 is dependent on
phosphorylation of CtIP at Ser327 (human) (Yu and Chen, 2004) or Ser328 (Xenopus)
(Peterson et al., 2011). To determine whether the CtIP-BRCA1 interaction is required for
CtIP function in mitosis, we supplemented CtIP-depleted extracts with purified recombinant
Xenopus CtIP protein that cannot be phosphorylated at S328 (CtIPS328A) and does not
interact with BRCA1 in S- and M-phase egg extracts (data not shown). Significantly,
CtIPS328A rescued the chromosome misalignment phenotype to the same extent as wild-type
CtIP (Figure 2, I and J), strongly suggesting that the CtIP-BRCA1 interaction is dispensable
for proper mitotic chromosome alignment. Nonetheless, our experiments show that MRN,
CtIP, and BRCA1 are each required for the assembly of mitotic spindles with properly
aligned chromosomes. Our data further suggest that the chromosome alignment defects
observed in the absence of these proteins are not a consequence of aberrant DNA
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replication.

A recent study showed that ATM phosphorylation of Bub1 is essential for the mitotic
checkpoint (Yang et al., 2011). Since the MRN complex is required to activate ATM upon
DNA damage (Costanzo et al., 2004; Falck et al., 2005; Uziel et al., 2003), we tested the
impact of ATM inhibition on mitotic chromosome alignment. In contrast to MRN inhibition,
inhibition of ATM by the small molecule KU55933 (10 μM) did not affect mitotic spindle
assembly or metaphase chromosome alignment in egg extracts (Figure 2, D, E, and J).

The MRN complex is essential for normal mitotic progression in mammalian cells
We next assessed whether the mitotic defects seen in MRN-deficient cell-free extracts were
also observed in mammalian cells, a setting in which metaphase chromosome alignment is
less reliant on the Ran-GTP gradient compared to Xenopus egg extracts (O’Connell et al.,
2009) (Figure 1A). Our previous studies indicate that Mre11 associates with M-phase
chromatin (Peterson et al., 2011). Here we confirm by indirect immunofluorescence analysis
that Mre11 is associated with mitotic chromosomes in the absence of DNA damage (Figure
3A).
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MRN is essential for cell growth and sustained down-regulation of Mre11 yields complex
pleiotropic phenotypes (Yamaguchi-Iwai et al., 1999). Therefore, we initially used mirin to
achieve acute Mre11 inhibition. HeLa cells expressing Histone H2B-EYFP were treated
with mirin (25 or 50 μM), and mitotic progression was immediately monitored using live
cell imaging. Although mirin-treated cells were able to congress chromosomes to the
metaphase plate with similar kinetics as untreated cells, a significant fraction of mirin-
treated cells paused for extended periods of time in a metaphase-like stage without anaphase
onset (Figure 3, B and C, and Movie S1 and S2). A delay in anaphase onset could reflect
defects in stable kinetochore-microtubule attachment. To test this possibility, we first
assayed for the presence of cold-stable microtubule bundles, which arise upon microtubule
capture of kinetochores, in fixed cells and observed no obvious difference in the amounts of
cold-stable kinetochore fibers between control and mirin-treated cells (Figure S2). Next, we
used indirect immunofluorescence to monitor BubR1, a mitotic checkpoint protein whose
kinetochore localization is substantially reduced once microtubules are attached and tension

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generated between sister kinetochores (Chan et al., 1998; Cheng et al., 2011; Guo et al.,
2012). In control cells, BubR1 level became reduced at kinetochores once chromosomes
were aligned at the metaphase plate (Figure 3, D and E). In contrast, BubR1 was present on
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at least two kinetochores per metaphase plate in mirin-treated cells (Figure 3, D and E),
indicating a defect in stable kinetochore-microtubule attachment. These results suggest that
inhibition of MRN function in mammalian cells results in kinetochore-microtubule
attachment defects, leading to mitotic checkpoint activation and anaphase onset delay.

To confirm that the mitotic delay was due to specific targeting of Mre11 by mirin, we used
inducible shRNAs to deplete Mre11 levels in mammalian cells (Figure 4A). Western blot
analysis of stable cell lines expressing Mre11-specific shRNAs identified two shRNAs that
significantly down-regulate Mre11 protein levels within 24 hrs after doxycycline treatment
(Figure 4B). Importantly, shRNA-mediated Mre11 depletion following S-phase elicited a
substantial delay in metaphase, but not in prometaphase, similar to mirin treatment (Figure
4, C and D). This result supports a critical role for the MRN complex in normal mitotic
progression of mammalian cells.

The MRN complex is essential for establishing the RanGTP gradient around mitotic
chromosomes
As noted above, the mechanisms of chromosome-microtubule interaction and metaphase
chromosome alignment differ between Xenopus egg extracts and mammalian cells. Whereas
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RanGTP-dependent “self-assembly” plays a major role in achieving chromosome alignment


in Xenopus egg extracts, mammalian cells mainly use a “search and capture” mechanism to
align chromosomes at the metaphase plate that still relies, to a significant but lesser extent,
on chromosomal RanGTP (Gadde and Heald, 2004; Walczak and Heald, 2008). Therefore,
we sought to investigate whether the chromosome misalignment phenotype observed in
Xenopus egg extracts and the kinetochore-microtubule attachment defects in mammalian
cells reflect a common role for the MRN pathway in regulating the RanGTP pathway. To
test this hypothesis, we analyzed the effects of Mre11 inhibition on microtubule nucleation
and the assembly of spindle-like structures in the absence of both centrosomes and
kinetochores using DNA-coated beads in CSF-arrested egg extracts. In this “artificial
chromosomes” system, microtubules polymerize around DNA beads, instead of nucleating
from centrosomes, and are sorted into bipolar structures by microtubule motors (Walczak et
al., 1998). In control egg extracts, spindle-like structures assemble around beads coated with
plasmid DNA (Figure 5, A and B). In contrast, inhibition of Mre11 by immunodepletion,
neutralizing antibodies, or mirin all resulted in a reduced frequency of normal spindle
assembly and an increased frequency of abnormal mitotic structures with reduced
microtubules (Figure 5, A and B), although the distribution between aberrant spindles and
structures with reduced microtubule polymerization was variable (Figure 5B). These data
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indicate that inhibition of the MRN complex is sufficient to impair chromatin-dependent


microtubule stabilization, as well as bipolar microtubule structure assembly, in egg extracts
without centrosomes and kinetochores, supporting the hypothesis that MRN regulates the
RanGTP pathway.

To test how MRN inhibition affects the gradient of RanGTP, we employed a fluorescence
resonance energy transfer (FRET) biosensor, Rango (Ran-regulated importin-β cargo) that
contains the importin-β-binding domain flanked by EYFP and CFP (Kalab et al., 2006).
HeLa cells were treated with 50 μM mirin following transfection of Rango. As anticipated,
FRET was highest across the metaphase plate in control untreated cells, and gradually
decreased toward the spindle poles (Figure 5, C and D), confirming the presence of a
RanGTP gradient (Kalab et al., 2006). In contrast, FRET was more uniform when plotted
across the spindle equator in mirin-treated cells (Figure 5, E and F). These data show that
MRN inhibition results in a loss of the RanGTP gradient during mitosis.

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MRN inhibition results in RCC1 dissociation from mitotic chromosomes


Chromatin-associated RCC1, a guanine nucleotide exchange factor for Ran GTPase, is
critical for the generation of the RanGTP gradient that regulates mitotic spindle assembly
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(Li et al., 2003). RCC1 binds to chromatin through interactions with both histone and DNA
(Makde et al., 2010) that entail a conformational switch within RCC1 (Hao and Macara,
2008). Since MRN associates with mitotic chromosomes (Figure 1C and 3A), we tested
whether MRN modulates RCC1 conformation. We used a FRET-based RCC1 biosensor
(CFP-RCC1-YFP) (Hao and Macara, 2008) to monitor RCC1 conformational changes upon
MRN inhibition by mirin. In HeLa cells were transfected with CFP-RCC1-YFP, we
immediately visualize the RCC1 conformation change upon treatment of mirin. Notably,
FRET was significantly reduced (p < 0.05) (Figures S3, A – E), indicating that RCC1
conformation was affected. To gain insights into the mechanism of RCC1’s regulation by
Mre11, we examined Mre11 immunoprecipitates from CSF-arrested egg extracts for the
presence of RCC1. This showed that endogenous Mre11 and RCC1 associate in extracts
(Figure S3F). Conversely, Mre11 was also present in RCC1 immunoprecipitates from CSF
egg extracts (Figure S3F). These data collectively indicate that MRN chromatin association
is important for the stable binding of RCC1 to chromatin.

To test whether MRN inhibition directly affects RCC1 association with mitotic
chromosomes, we next followed chromosome-associated CFP-RCC1-YFP by live cell
imaging (Figures 6A and S4A). Quantification of fluorescence intensity revealed that there
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was a significant reduction in chromosome-associated RCC1 in mirin-treated, but not


control, mitotic cells (Figure 6B). In contrast to mitotic cells, mirin treatment did not affect
the intensity of DNA-bound RCC1 in interphase cells (Figure S4).

To further confirm the hypothesis that inhibition of MRN affects RCC1 association with
mitotic chromosomes, we isolated mitotic chromosomes from CSF-arrested egg extracts and
compared chromatin-bound Mre11 and RCC1. The levels of Mre11 and RCC1 associated
with mitotic chromosomes were substantially reduced in extracts treated with mirin and in
Mre11-depleted egg extracts (Figure 6C). We conclude that inhibition of MRN impairs the
association of RCC1 with mitotic chromosomes.

We reasoned that if MRN regulates RCC1 chromatin association, which in turn establishes a
RanGTP gradient, addition of active Ran (RanGTP form) should bypass the requirement for
MRN in spindle microtubule assembly. We examined the stimulation of microtubule
formation and spindle assembly by the constitutively active GTPase Ran. An allele of Ran
with a mutation in the effector domain, RanL43E, was able to stimulate the formation of
bipolar microtubule structures that resembled mitotic spindles (Figure 7A), as previous
reported (Wilde and Zheng, 1999). Moreover, inhibition of MRN by Mre11 depletion or
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mirin treatment did not affect the formation of bipolar microtubule structures induced by
RanL43E (Figure 7, B – C), confirming that MRN plays a role in mitotic spindle assembly
upstream of Ran, consistent with its interaction with RCC1.

Discussion
Increasing evidence suggests that proteins involved in DNA transactions in interphase can
play additional roles in mitosis. For example, the origin recognition complex, a key
component of the pre-replicative complex which marks the sites of replication origins, also
regulate centrioles, centrosomes, and sister chromatid cohesion (Hemerly et al., 2009;
Shimada and Gasser, 2007). Conversely, mitotic kinases can modulate genomic stability
during interphase (Takaki et al., 2008). Our results reveal a critical role for the MRN-CtIP
pathway in regulating proper chromosome alignment and kinetochore-microtubule
attachment in mitosis. Consistent with functions outside of the DNA damage response, the

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MRN complex is present on undamaged chromosomes in interphase (Maser et al., 2001;


Peterson et al., 2011) and in mitosis (Giunta and Jackson, 2011; Peterson et al., 2011)
(Figures 1C and 3A). Importantly, four independent methods of inhibiting the MRN-CtIP
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pathway (Mre11 depletion, Mre11 neutralization, Mre11 inhibition by mirin, and CtIP
depletion) yielded very comparable spindle phenotypes. The chromosome alignment defects
in Mre11-depleted egg extracts can be partially rescued by addition of purified recombinant
MRN complex (Figure 1D). The requirement for MRN and CtIP in proper alignment of
mitotic chromosomes at the metaphase plate may account, at least in part, for the
chromosome and genomic instability observed in cells deficient for these factors. Of note,
MRN and CtIP are expressed at significant levels in G2/M (Yu and Chen, 2004) and Rad50
is structurally related to SMC proteins, a family of proteins that comprises cohesions and
condensins, which are critical regulators of chromosome structure (D’Amours and Jackson,
2002; Stracker et al., 2004). Moreover, mutations in the MRN pathway in flies result in
telomere defects that activate the mitotic checkpoint (Musaro et al., 2008). In budding yeast,
Mre11 interacts physically with the Spc24 kinetochore components (Cho et al., 1998) and
also shows genetic interactions with the mitotic checkpoint components Bub1, Bud3, Mad2
and Mad3 (Myung et al., 2004).

Our data strongly suggest that the MRN-CtIP pathway regulates mitotic spindle assembly
and metaphase chromosome alignment by regulating RCC1 chromatin association and the
subsequent establishment of a RanGTP gradient (Figure 7D). Chromatin-bound RCC1 is
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essential to establish a spherical Ran-GTP gradient that emanates from chromatin, thereby
promoting chromosome-directed spindle assembly in Xenopus egg extracts and facilitating
stable kinetochore-microtubule attachment in mammalian cells. While RCC1 binds
nucleosomes (England et al., 2010; Makde et al., 2010), additional factors are likely to affect
RCC1 chromatin association. The MRN pathway is able to regulate chromatin structure in at
least two ways: by recruiting ATM, which phosphorylates multiple chromatin proteins
(Matsuoka et al., 2007) and by generating single-strand DNA at sites of DNA damage
(Mimitou and Symington, 2011; Williams et al., 2007). It is therefore conceivable that loss
of chromatin-bound RCC1 upon MRN down-regulation is due to defects in chromatin
organization. Our study unravels a novel function for the MRN-CtIP DNA repair complex in
mitotic spindle assembly and a potentially important contribution to proper chromosome
segregation. This unsuspected role could account for the essential role of MRN and CtIP in
genome stability maintenance.

Experimental Procedures
Xenopus egg extracts
CSF-arrested extracts were prepared from unfertilized Xenopus eggs as previously described
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(Murray, 1991). For immunodepletion, 100 μg of affinity-purified MRE11, CtIP, BRCA1


antibodies or nonimmune rabbit IgG were bound to 100 μl Dynal beads protein A. CSF egg
extracts (100 μl) were added for 1 hr at 4°C. For antibody addition experiments, affinity-
purified antibodies or nonimmune IgG were added to CSF egg extracts at 100 μg/ml.
Purified recombinant MRN complex used in reconstitution experiments was a generous gift
from Dr. Tanya Paull (University of Texas at Austin). The RanL43E construct was kindly
provided by Dr. Yixian Zheng (Carnegie Institution of Washington).

Demembranated sperm were added to a portion of each extract and rhodamine-labeled


bovine brain tubulin was added at 1 μl/300 μl of extract, and exit from metaphase arrest was
induced by addition of Ca++. Cell cycle progress of egg extracts was followed by
fluorescence microscopic examination of 1 μl aliquots squashed under a coverslip. 80 min
after exiting from metaphase, one half volume of the appropriate egg extracts was added and
incubated for additional 60–120 min. M-phase structures accumulating in egg extracts were

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Rozier et al. Page 8

scored in squashed samples. Bipolar spindles with all chromosomes aligned at the spindle
equator were scored as bipolar-aligned, while bipolar spindles with scattered chromosomes
were counted as bipolar-misaligned. Monopolar and other mitotic structures were scored as
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other. Image acquisition and data analysis were performed at room temperature using an
Olympus IX81 inverted microscope with an oil immersion 60× Plan Apo objective lens
(N.A. = 1.42), a Cooke Sensicam QE Monochrome CCD, and the Slidebook software
package.

Immunoprecipitation and Immunoblotting Analysis


Affinity purified antibodies were coupled to Dynal beads and the beads were washed twice.
The beads were then incubated at 4°C for 1h with egg extracts that had been incubated at
room temperature for 30 min with or without sperm nuclei. After mixing, the beads were
washed twice with PBS buffer, twice with PBS plus 0.5 M NaCl. The immunoprecipitates
were then solubilized in SDS-sample buffer and subjected to immunoblot analysis.

Immunoblots were blocked with TBST (20 mM Tris·HCl, pH 7.6, 150 mM NaCl, 0.1%
Tween) containing nonfat dry milk, and then probed with affinity purified primary
antibodies in TBST. Primary antibodies were visualized using a horseradish peroxidase-
labeled goat anti-rabbit IgG secondary antibodies and ECL.

Tissue culture and Drug Treatment


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HeLa cells stably expressing H2B-EYFP were cultured in DMEM with 10% FBS and 1%
penicillin-streptavidin at 37°C in 5% CO2. Mirin was added to a final concentration of 25 or
50 μM.

To generate cell lines expressing inducible MRE11 shRNA, shRNA vectors were co-
expressed with helper viruses to obtain control and MRE11 Lenti-shRNA viruses. HeLa
cells stably expressing H2B-EYFP were supplemented with 10 μg/ml polybrene and spin
(1000 rpm) infected with 1 ml viruses at 30–50% confluency. Post 48 hrs infection, cells
were passed into medium with puromycin (2 μg/ml) to select stable cell lines. The shRNA
expression, as well as RFP co-expression, was induced by addition of doxycycline (1 μg/
ml). Cells were then either lysed to assess the MRE11 protein level with immunoblotting
analysis after 24 hrs or subjected to live cell imaging to follow the first mitotic progression.

Immunofluorescence Microscopy and live Cell Imaging


For indirect immunofluorescence, cells grown on poly-L-lysine-coated coverslips were
washed once with microtubule stabilizing buffer (MTSB: 100 mM Pipes, 1 mM EGTA, 1
mM MgSO4, and 30% of glycerol), extracted with 0.5% Triton X-100 in MTSB for 5 min,
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fixed with 4% formaldehyde in MTSB or methanol for 10 min, and blocked in TBS
containing 0.5% Tween-20 and 1% BSA (Sigma) for 1 h. Coverslips were subjected to
primary antibodies diluted in blocking buffer for 1 h, and to secondary antibodies (Jackson
ImmunoResearch Laboratories). Image acquisition and data analysis were performed at
room temperature using an inverted microscope (IX81; Olympus) with a 60× NA 1.42 plan
Apo oil immersion objective lens (Olympus), a monochrome CCD camera (Sensicam QE;
Cooke), and the Slidebook software package (Olympus). All images in each experiment
were collected on the same day using identical exposure time.

For live-cell imaging, HeLa cells stably expressing H2B-EYFP were plated onto 35 mm
glass bottom dishes (MatTek). Images were acquired using a 40× NA dry objective lens
every 4 min with a 37°C chamber and were processed using Slidebook software. All
statistical significance of quantification experiments was verified by Student’s t-test using
Microsoft Excel software.

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Rozier et al. Page 9

FRET
The Rango and CFP-RCC1-YFP sensors were created and kindly provided by the
laboratories of K. Weis and R. Heald (University of California at Berkeley) and I. Macara
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(University of Virginia), respectively. The distribution of the RanGTP gradient in live cells
was visualized with the FRET biosensor Rango (Kalab et al., 2006). The conformation
change of chromatin-associated RCC1 in live cells was visualized with CFP-RCC1-YFP
(Hao and Macara, 2008). 48 hours after plasmid transfection, cells were treated with mirin
or not. Images were immediately obtained in the CFP, YFP, and FRET channels (CFP
excitation/YFP emission) using an inverted Nikon AIR MP confocol microscope with a 60×
NA 1.49 lens. Images were collected under identical conditions and analyzed using the NIS
Elements Software (Nikon).

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Acknowledgments
We thank Drs. Tanya Paull, Karsten Weis, Rebecca Heald, Ian Macara, and Yixian Zheng for reagents. We thank
Chenshu Liu for help with Lentivirus stable cell line productions. We thank Theresa Swayne and Adam White at
Herbert Irving Comprehensive Cancer Center Shared Resources for technical support on FRET analysis. We thank
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all members of the Mao laboratory for stimulating discussion.

This work has been supported by a start-up fund from the Department of Pathology and Cell Biology of Columbia
University College of Physicians and Surgeons to Y. M. and grant (CA92245) from the National Institutes of
Health to J.G. Y. M. is a recipient of Irma T. Hirschl/Monique Weill-Caulier Trusts Research Award.

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Highlights
• The MRN-CtIP pathway is required for metaphase chromosome alignment in
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egg extracts
• MRN inhibition interferes with spindle assembly around DNA-coated beads
• MRN inhibition in cells causes a metaphase delay and disrupts the RanGTP
gradient
• The MRN complex regulates the stable association of RCC1 to chromatin
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Rozier et al. Page 14
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Figure 1. The MRN complex is essential for metaphase chromosome alignment in Xenopus egg
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extracts
(A) Mechanisms of mitotic spindle assembly and metaphase chromosome alignment.
Through the “search and capture” mechanism, centrosomally nucleated microtubules form
bipolar spindles and align all chromosomes at the metaphase plate. Alternatively,
microtubules are nucleated in the vicinity of chromatin due to a surrounded RanGTP
gradient (pink oval), “microtubule self-organization”, which assists metaphase chromosome
alignment. Comparing to mammalian cells, the RanGTP gradient covers a larger area around
the metaphase plate in Xenopus egg extracts (Kalab et al., 2006).
(B) Immunoblot of immunodepletion of CSF egg extracts. CSF-arrested Xenopus egg
extracts were immunodepleted of MRE11 using a specific antibody. 1 μl of mock depleted
and MRE11 depleted extracts was analyzed for MRE11.
(C) MRE11 associates with mitotic chromosomes in Xenopus egg extracts.
Immunofluorescence detection of BubR1 (a kinetochore marker) and Mre11 in mock- or
Mre11-depleted CSF-arrested egg extracts upon addition of sperm nuclei. DNA was
visualized with DAPI.
(D) Metaphase spindles assembled in uncycled egg extracts. Extracts were mock depleted or
MRE11 depleted, and supplemented with buffer or purified recombinant MRN complexes
(2.5 ng/μl final concentration). DAPI – green and microtubule – magenta.
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(E) Quantification of structures formed from sperm nuclei in uncycled (D) egg extracts as
indicated. At least 50 mitotic structures were scored for each extract. Data are presented
from one representative experiment. Three independent depletion experiments revealed
similar results. Mitotic structures were scored as bipolar spindles with chromosome aligned
at the metaphase plate, bipolar spindles with misaligned chromosomes, and others including
monopolar spindles, half spindles, and chromosomes associated with irregular microtubule
organizations.
See also Figure S1.

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Figure 2. MRN, CtIP, and BRCA1 proteins are all required for proper metaphase chromosome
alignment in Xenopus egg extracts
(A – E, G – I) Metaphase spindles assembled in uncycled egg extracts. Extracts were mock-
depleted (A), MRE11-depleted (B), BRCA1-depleted (C), supplemented with DMSO (D) or
10 μM KU55933 (E), CtIP depleted (G), CtIP depleted and supplemented with wild-type
recombinant CtIP (H) or CtIP S327A mutant (I) as indicated. DAPI – green and microtubule
– magenta.
(F) Immunoblot of immunodepletion of CSF egg extracts. CSF-arrested egg extracts were
immunodepleted of CtIP using a specific antibody. 1 μl of mock depleted and CtIP depleted
extracts was analyzed for CtIP and MRE11.
(J) Quantification of structures formed from sperm nuclei in uncycled egg extracts as
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indicated. At least 50 mitotic structures were scored for each extract. Data are presented
from one representative experiment. Three independent depletion experiments revealed
similar results.

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Figure 3. Inhibition of MRN results in prolonged metaphase in mammalian cells


(A) The MRN complex is associated with mitotic chromosomes in mammalian cells.
Indirect immunofluorescence images of metaphase cells acquired using antibodies against
CENP-A (a centromere/kinetochore marker), MRE11, and γ-H2AX, as indicated.
(B) Cells treated with mirin have a prolonged metaphase-like stage. Representative still
frames of live cell microscopy of H2B-EYFP HeLa cells treated with or without 25 μM
mirin.
(C) Time spent in metaphase from cells aligned all chromosomes to anaphase onset of
transient transfected H2B-EYFP HeLa cells treated with or without mirin (25 and 50 μM as
indicated). Each dot represents a single cell. The numbers of cells filmed, the mean time ±
SD, and the p value are presented. See also Movie S1 and S2.
(D) BubR1 and ACA were observed by indirect immunofluorescence with specific
antibodies.In merge: BubR1, green; ACA, red; and chromatin was visualized with DAPI,
blue.
(E) Indirect immunofluorescence showing the number of kinetochores with high level
BubR1 per metaphase cell (more than 30 cells counted for each condition).
See also Figure S2.
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Figure 4. Reducing the expression level of MRE11 causes a metaphase delay in mammalian
cultured cells
(A) Schematic representation of Lenti-shRNA virus production, transfection, control or
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MRE11 shRNA induction protocol.


(B) Cell lysates were probed with MRE11 and tubulin antibodies 24 hr after control or
MRE11 shRNA induction by addition of doxycycline as indicated.
(C) Time spent in prometaphase and metaphase in H2B-EYFP cells stably transfected with
control or MRE11 Lenti-shRNA viruses following addition of doxycycline. Each dot
represents a single cell. The mean time ± SD and the p value are presented. We should note
that some cell entering mitosis early may still have high levels of MRE11; therefore, the
MRE11-depletion effect on the time in mitosis for those cells showed here is probably
underrepresented.
(D) Stills of live-cell imaging showing a cell expressing MRE11 shRNA as determined by
co-expressing of RFP.
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Figure 5. MRN inhibition disrupts the RanGTP gradient during metaphase


(A) MRN inhibition reduces the fidelity of mitotic structures assembled around DNA beads
in Xenopus egg extracts. Microtubule structures form around DNA beads in control CSF-
arrested egg extracts or egg extracts depleted of MRE11 or supplemented with MRE11
antibody (Ab) or mirin (50 μM).
(B) Quantification of the effect of MRE11 inhibition on bead spindle assembly in one
representative experiment. At least 50 mitotic structures were scored for each extract. Three
independent depletion experiments revealed similar results. The bead structures include
three categories: normal spindles, abnormal structures, and reduced microtubules as shown
in (A).
(C and E) DIC, CFP, and FRET/CFP of Rango intensities in a control metaphase cell (C)
and a metaphase cell treated with 50 μM mirin (E).
(D and F) Linear intensity plots of the cells in (C) and (E), respectively, from one spindle
pole to the other with the metaphase plate designated as 0 μm (blue line). All imaged cells
(n = 5), including the one plotted as the grey line in (F), showed similar phenotypes, though
the IFRET/ICFP ratio differs among individual cells.
See also Figure S3.
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Figure 6. MRN inhibition results in a reduction of RCC1 binding to chromatin


(A) Stills of live cell imaging showing metaphase cells expressing RCC1-YFP upon
treatment of mirin or not as indicated. Cells were arrested in metaphase upon treatment of
MG132. See also Figure S4.
(B) Quantitation of the normalized integrated intensity of chromatin-associated RCC1-YFP
signals. At least 10 cells quantified for each bar were shown. Error bars represent standard
error (p < 0.05).
(C) Inhibition of MRE11 reduces chromatin associated RCC1 in egg extracts. Mitotic
chromosomes were isolated from CSF-arrested egg extracts supplemented with mirin (left
panels) or immunodepleted of MRE11 (right panels). The chromosomal fractions were
blotted for MRE11, RCC1, and Histone H3 proteins as indicated.
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Figure 7. MRN function is not essential for Ran-dependent microtubule nucleation


(A – C) Induction of bipolar microtubule structures in Xenopus egg extracts supplemented
with 25 μM GST-RanL43E, a constitutively active form of Ran. A typical bipolar
microtubule structure is shown in the inset of each panel.
(D) A model for the regulation of spindle assembly and metaphase chromosome alignment
by the MRN-CtIP pathway. MRN (shown as a hypothetical ring) is present on undamaged
chromosomes, interacts with RCC1 and stabilizes its interaction with chromatin. Stable
chromatin association of RCC1 results in the generation of a RanGTP gradient yielding
stable kinetochore-microtubules attachment (left). Upon inhibition of the MRN-CtIP
pathway, RCC1 association to chromatin is reduced and a RanGTP gradient fails to develop.
Consistent with previous reports (Kalab et al., 2006), disruption of the RanGTP gradient
impairs metaphase chromosome alignment in Xenopus egg extracts, but has less severe
consequences (metaphase-like arrest) in mammalian cells.
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