1. The MRN-CtIP Pathway Is Required for Metaphase Chromosome Alignment
Lorene Rozier, Yige Guo, Shaun Peterson, Mai Sato, Richard Baer, Jean Gautier, Yinghui Mao 
Molecular Cell 
Volume 49, Issue 6, Pages (March 2013)
DOI: /j.molcel
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2. Molecular Cell 2013 49, 1097-1107DOI: (10.1016/j.molcel.2013.01.023)
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3. Figure 1
The MRN Complex Is Essential for Metaphase Chromosome Alignment in Xenopus Egg 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, that assists metaphase chromosome alignment. Compared to mammalian cells, the RanGTP gradient covers a larger area around the metaphase plate in Xenopus egg extracts (Kaláb et al., 2006).
(B) Immunoblot of immunodepletion of CSF egg extracts. CSF-arrested Xenopus egg extracts were immunodepleted of MRE11 using a specific antibody. Mock-depleted and MRE11-depleted extracts (1 μl ) were 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.
(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 chromosomes 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.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
MRN, CtIP, and BRCA1 Proteins Are All Required for Proper Metaphase Chromosome Alignment in Xenopus Egg Extracts
(A–E) Metaphase spindles assembled in uncycled egg extracts. Extracts were mock-depleted (A), MRE11-depleted (B), BRCA1-depleted (C), or supplemented with dimethyl sulfoxide (DMSO) (D) or 10 μM KU55933 (E). 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. Mock-depleted and CtIP-depleted extracts (1 μl) were analyzed for CtIP and MRE11.
(G–I) Metaphase spindles assembled in uncycled egg extracts. Extracts were CtIP depleted (G) or CtIP depleted and supplemented with wild-type recombinant CtIP (H) or CtIP S327A mutant (I) as indicated. DAPI = green and microtubule = magenta.
(J) Quantification of structures formed from sperm nuclei in uncycled 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.
Molecular Cell  , DOI: ( /j.molcel )
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5. 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) HeLa cells transfected with H2B-EYFP were treated with or without mirin (25 and 50 mM) 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 Movies S1 and S2.
(D) BubR1 and ACA were observed by indirect immunofluorescence with specific antibodies. In merge: BubR1, green; ACA, red; chromatin 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.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
Reducing the Expression Level of MRE11 Causes a Metaphase Delay in Mammalian Cultured Cells
(A) MRE11 shRNA was induced in cells transfected with lenti-shRNA viruses.
(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 cells 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 coexpression of RFP.
Molecular Cell  , DOI: ( /j.molcel )
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7. 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) DIC, CFP, and FRET/CFP of Rango intensities in a control metaphase cell.
(D) Linear intensity plot of the cell in (C) from one spindle pole to the other with the metaphase plate designated as 0 μm (blue line).
(E) DIC, CFP, and FRET/CFP of Rango intensities in a metaphase cell treated with 50 μM mirin.
(F) Linear intensity plots of the cell in (E) from one spindle pole to the other with the metaphase plate designated as 0 μm (blue line). All imaged cells in (D) and (F) (n = 5), including the cell plotted as the gray line, showed similar phenotypes, though the IFRET/ICFP ratio differs among individual cells.
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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8. 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 ten cells quantified for each bar were shown. Error bars represent SE (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.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
MRN Function Is Not Essential for Ran-Dependent Microtubule Nucleation
(A–C) Bipolar microtubule structures were induced in control egg extracts (A), MRE11-depleteed egg extracts (B), and egg extracts supplemented with 50 μM mirin (C). All egg extracts were 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-microtubule 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 (Kaláb 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.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions