1. Volume 31, Issue 3, Pages 305-318 (November 2014)
RuvB-like ATPases Function in Chromatin Decondensation at the End of Mitosis 
Adriana Magalska, Anna Katharina Schellhaus, Daniel Moreno-Andrés, Fabio Zanini, Allana Schooley, Ruchika Sachdev, Heinz Schwarz, Johannes Madlung, Wolfram Antonin 
Developmental Cell 
Volume 31, Issue 3, Pages (November 2014)
DOI: /j.devcel
Copyright © 2014 Elsevier Inc. Terms and Conditions

2. Developmental Cell 2014 31, 305-318DOI: (10.1016/j.devcel.2014.09.001)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1
Reconstitution of Chromatin Decondensation in Xenopus Egg Extracts
(A) Time course of the in vitro decondensation reaction. Mitotic chromatin clusters from HeLa cells were incubated with postmitotic Xenopus egg extracts for the indicated time. Samples were fixed with 4% PFA and 0.5% glutaraldehyde, stained with DAPI, and analyzed by confocal microscopy. For quantification of the decondensation reaction, the smoothness of the boundary of the chromatin (light gray) and the homogeneity of DAPI staining (dark gray) were analyzed. The means (± SEM) of three independent experiments are shown, each including at least ten chromatin substrates for each time point, ∗∗∗p < by one-way ANOVA, Dunnett’s C post hoc test. rel, relative.
(B) Mitotic chromatin clusters from HeLa cells were incubated for 120 min with CSF-arrested Xenopus egg extracts in the absence or presence of 1 mM CaCl2, which induces mitotic exit. Samples were fixed, and the decondensation reaction was quantified as in (A). The means (±SEM) of three independent experiments are shown, each including at least ten chromatin substrates, ∗∗∗p < by Mann-Whitney test.
Scale bars, 5 μm. See also Figure S1.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

4. Figure 2 Decondensing Chromatin Assembles into Functional Nuclei
(A) Mitotic chromatin clusters from HeLa cells were incubated with Xenopus egg extracts for the indicated time and fixed with 4% PFA. Immunofluorescence shows histone H3 serine 10 phosphorylation (H3P, upper panel), nuclear pore complexes (NPC, middle panel), and chromatin (DAPI).
(B) For visualization of nuclear envelope reformation, HeLa mitotic chromatin substrates and DiIC18 (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate)-labeled membranes (upper panel) were added to the egg extracts or the buffer control. Samples were fixed at indicated time points with 4% PFA and 0.5% glutaraldehyde, stained with DAPI (lower panel), and analyzed by confocal microscopy.
(C) Chromatin decondensation using HeLa mitotic chromatin was analyzed by transmission electron microscopy. Samples were fixed at indicated time points with 4% PFA and 2.5% glutaraldehyde, postfixed in 1% OsO4, and stained with 1% uranyl acetate. After embedding in Epon, ultrathin sections (50–70 nm) were stained with uranyl acetate and lead citrate and viewed with a Philips CM10 microscope.
(D) HeLa mitotic chromatin was decondensed for 120 min. An enhanced green fluorescent protein (EGFP)-fused import substrate (left column) or a shuttling substrate containing a nuclear localization signal and a nuclear export signal (middle and right column) was added. Nuclear export was inhibited by the addition of 300 nM leptomycin B. Samples were stained with DAPI and analyzed by confocal microscopy. The weighted average percentage of two independent experiments, each including at least 100 randomly chosen chromatin substrates, is shown. Diamonds indicate data points of the individual experiments.
Scale bars, 5 μm.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

5. Figure 3 Chromatin Decondensation Requires ATP and GTP Hydrolysis
HeLa mitotic chromatin was decondensed in the presence of 10 mM ATPγS, 10 mM GTPγS, or buffer control (CTRL). Samples were fixed with 4% PFA and 0.5% glutaraldehyde at indicated time points, analyzed, and quantified. The means (±SEM) of three independent experiments are shown, each including at least ten chromatin substrates for each time point, ∗∗∗p < by one-way ANOVA, Dunnett’s C post hoc test. rel, relative. Scale bar, 5 μm. See also Figure S2.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

6. Figure 4 Chromatin Decondensation Requires RuvB-like ATPases
(A) Xenopus egg extracts were fractionated by differential ammonium sulfate precipitation, ion exchange, and size exclusion chromatography (see fractionation scheme on the left with the fractions showing decondensation activity in black) and were tested for the state of chromatin decondensation on HeLa mitotic chromatin after 120 min. For ion exchange and size exclusion fractions, reactions were performed in the presence of fraction B of the ammonium sulfate precipitation. The lower panels show the distribution of RuvBL1 and RuvBL2 in fractions analyzed by western blotting. Representative quantification and western blot analysis of one fractionation experiment is shown. FT, flowthrough. rel, relative.
(B) Chromatin decondensation on HeLa mitotic chromatin was performed for 120 min in the presence of 4 mg/ml affinity-purified IgG against RuvBL1, RuvBL2, or control IgGs.
(C) Western blot of untreated (UNTR), mock, and RuvBL1/2-depleted extracts, the latter two generated by two passages over control IgG- or anti-RuvBL1 or anti-RuvBL2 IgG-bound beads, respectively. NPM2 serves as a control protein unaffected by this treatment.
(D) Mock or RuvBL1/2-depleted extracts supplemented with buffer or purified recombinant RuvBL1-RuvBL2 complex (0.04 μg/μl to match the endogenous concentration) were tested for chromatin decondensation on HeLa mitotic chromatin (120 min time point).
In (B) and (D), the means (±SEM) of three independent experiments are shown, each including at least 20 chromatin substrates. ∗∗∗p < by one-way ANOVA, Dunnett’s C post hoc test. Scale bars, 5 μm. See also Figure S3.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

7. Figure 5
RuvBL1 or RuvBL2 Alone Is Sufficient to Support Chromatin Decondensation and Require ATPase Activity
(A) RuvBL1/2-depleted extracts (generated by two passages over anti-RuvBL1 or anti-RuvBL2 IgG-bound beads, respectively) were supplemented with purified recombinant RuvBL1 or RuvBL2 (0.02 μg/μl to match the endogenous concentration) and tested for chromatin decondensation on HeLa mitotic chromatin. rel, relative.
(B) Chromatin decondensation was analyzed in RuvBL1/2-depleted extracts (generated by consecutive passage over anti-RuvBL1 and anti-RuvBL2 IgG-bound beads) supplemented with ATPase-deficient mutant versions of the RuvBL1, RuvBL2, or the RuvBL1-RuvBL2 complex (RuvBL1 D302N and RuvBL2 D298N) matching the endogenous concentration. WT, wild-type.
(C) Chromatin decondensation in the presence of 40-fold excess compared to endogenous concentrations of recombinant wild-type RuvBL1, RuvBL2, or the RuvBL1-RuvBL2 complex or ATPase-deficient mutants of the respective proteins.
Samples were analyzed after 120 min. The means (±SEM) of three independent experiments are shown, each including at least 20 chromatin substrates. ∗∗∗p < by two-way ANOVA, Sidlak post hoc test for (A) and (B) and by one-way ANOVA, Dunnett’s C post hoc test for (C). WT, wild-type. Scale bars, 5 μm. See also Figure S4.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

8. Figure 6 RuvBL1 and RuvBL2 Localize to the Decondensing Chromatin
(A) HeLa mitotic chromatin was incubated with extracts for the indicated time. RuvBL1/2-depleted extracts (generated by consecutive passage over anti-RuvBL1 and anti-RuvBL2 IgG-bound beads) were supplemented with buffer, recombinant RuvBL1-RuvBL2 complex, RuvBL1, or RuvBL2 or ATPase-deficient versions of the proteins (matching the endogenous concentrations) and used in the decondensation reaction for 120 min. Samples were fixed with 4% PFA and processed for immunofluorescence, or chromatin was reisolated and analyzed by western blot (histone H2B shows equal chromatin loading). Scale bars, 5 μm.
(B) HeLa mitotic chromatin incubated as in (A) was reisolated and probed for the presence of Mel28/ELYS, the condensing I and II complex (CAP-G or CAP-D3 antibodies, respectively), topoisomerase IIα, the chromokinesin KIF4A, and Repo-MAN. Please note that during the reisolation procedure, rapid rebinding of Xenopus proteins to chromatin and/or their exchange with the HeLa proteins occurs so that they can be detected already at t = 0.
See also Figure S5.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

9. Figure 7
RuvB-like ATPases Are Specifically Required for Chromatin Decondensation during Mitotic Exit
Pronuclei were assembled on Xenopus sperm chromatin in mock-treated or RuvBL1/2-depleted extracts (using anti-RuvBL1 or anti-RuvBL2 antibodies). After 120 min, samples were fixed with 4% PFA and 0.5% glutaraldehyde and analyzed for membrane staining (DiIC18, upper panel) or for nuclear pore complexes (NPC, lower panel) by immunofluorescence with the antibody mAB414. Chromatin was stained with DAPI. Right panel shows the quantitation of chromatin substrates with closed nuclear envelopes as weighted average percentage of two independent experiments, each including at least 100 chromatin substrates. Diamonds indicate data points of the individual experiments. Scale bar, 5 μm. See also Figure S6.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions