A model for how condensin II might contribute to the formation of chromosome territories in diploid cells.

<p>(A) After mitosis, decondensed chromosomes adopt the Rabl configuration, in which the centromeric regions (circles) of all chromosomes cluster near the nuclear periphery and the telomeres (stars) cluster near the opposite side of the nucleus. This configuration reflects the orientation of chromosomes in previous anaphase. In <i>Drosophila</i> diploid cells, homologous chromosomes pair along their lengths. For simplicity, only one arm of each chromosome is drawn here. (B) According to the model proposed by Bauer at al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002939#pgen.1002939-Bauer1" target="_blank">[3]</a>, condensin II promotes axial compaction of the chromosome arms. Coincidentally, centromere clustering is dissolved, allowing heterochromatic regions (represented by the centromeres and telomeres) to slide along the nuclear periphery. As a natural consequence of these two conditions, each pair of homologous chromosomes is sequestered into a discrete chromosome territory. Intrachromosomal folding mediated by condensin II helps disrupt interchromosomal interactions, thereby promoting unpairing of homologous chromosomes and antagonizing transvection. The basic idea presented here could potentially be applied to diploid cells in other organisms where the pairing of homologous chromosomes is not necessarily observed.</p

Condensins I and II are both present in NSCs.

<p>(<b>A</b>) Schematic representation of NSC division and neural differentiation. NSCs possess radial fibers along apico-basal polarity and exhibit cell cycle-dependent nuclear movement (i.e., interkinetic nuclear migration). LV, VZ and CP indicate the lateral ventricle, the ventricular zone and the cortical plate, respectively. (<b>B</b>) Subunit composition of the condensin complexes. Both complexes share the same pair of SMC core subunits (SMC2 and SMC4). Condensin I possesses three non-SMC subunits (CAP-D2, CAP-G and CAP-H) whereas condensin II has a distinct set of non-SMC subunits (CAP-D3, CAP-G2 and CAP-H2). (<b>C</b>) Frozen sections of embryonic brains were immunolabeled with antibodies against SOX2 (an NSC marker) and condensin subunits. Areas containing the VZ were labeled with antibodies against SOX2 and SMC2 (upper panels) or against CAP-H and CAP-H2 (middle panels). An area containing the CP was labeled with an SMC2 antibody. DNA was counterstained with Hoechst (lower panes). The data shown are from a single representative experiment out of three repeats. Sections from three different embryos were used. Bar, 20 µm. (<b>D</b>) Brain extracts prepared from C57BL/6J embryos at E12.5 are subjected to immunoprecipitation assay with antibodies against condensin subunits and control IgG as indicated on the top. The precipitates (P) along with whole cell extracts (W; 5% of input) and non-precipitated supernatants (S; 5% of input) were analyzed by western blotting. (<b>E</b>) Metaphase spreads were prepared from NSC cultures and immunolabeled with specific antibodies against CAP-H and CAP-H2. DNA was counterstained with Hoechst. Maximum intensity projections are shown. The right four panels indicate close-up views of a representative chromosome (indicated by the yellow rectangles in the left panels). The perimeter of the chromosome is indicated by the dotted lines. The data shown are from a single representative experiment out of two repeats. Bars, 5 µm.</p

Overlapping and Non-overlapping Functions of Condensins I and II in Neural Stem Cell Divisions

<div><p>During development of the cerebral cortex, neural stem cells (NSCs) divide symmetrically to proliferate and asymmetrically to generate neurons. Although faithful segregation of mitotic chromosomes is critical for NSC divisions, its fundamental mechanism remains unclear. A class of evolutionarily conserved protein complexes, known as condensins, is thought to be central to chromosome assembly and segregation among eukaryotes. Here we report the first comprehensive genetic study of mammalian condensins, demonstrating that two different types of condensin complexes (condensins I and II) are both essential for NSC divisions and survival in mice. Simultaneous depletion of both condensins leads to severe defects in chromosome assembly and segregation, which in turn cause DNA damage and trigger p53-induced apoptosis. Individual depletions of condensins I and II lead to slower loss of NSCs compared to simultaneous depletion, but they display distinct mitotic defects: chromosome missegregation was observed more prominently in NSCs depleted of condensin II, whereas mitotic delays were detectable only in condensin I-depleted NSCs. Remarkably, NSCs depleted of condensin II display hyperclustering of pericentric heterochromatin and nucleoli, indicating that condensin II, but not condensin I, plays a critical role in establishing interphase nuclear architecture. Intriguingly, these defects are taken over to postmitotic neurons. Our results demonstrate that condensins I and II have overlapping and non-overlapping functions in NSCs, and also provide evolutionary insight into intricate balancing acts of the two condensin complexes.</p></div

Condensin II prevents hyperclustering of chromocenters in the interphase nucleus in NSCs.

<p>(<b>A</b>) Shown here is a schematic view of <i>M. musculus</i> chromosomes and an interphase nucleus. Pericentric heterochromatin derived from non-homologous chromosomes associate with each other to form clusters within the interphase nucleus. These structures are referred to as chromocenters. (<b>B</b>) Frozen sections of embryonic brains at E16.5 were immunolabeled with an antibody against histone H3 trimethylated at lysine 9 (H3K9me3) along with Hoechst staining. Shown on the top are wide areas containing the VZ. The lower panels indicate close-up views of the nuclei indicated by the white rectangles. The data shown are from a single representative experiment out of three repeats. Sections from three different embryos of each genotype were analyzed. Maximum intensity projections are shown. Bars, 10 µm. The numbers of chromocenters per nucleus in the VZ were measured and plotted. Data were obtained from 50 nuclei in the VZ. *** <i>P</i><0.001 (<i>t</i>-test with a Holm correction for multiple comparisons). (<b>C</b>) Frozen sections of embryonic brains at E16.5 were immunolabeled with an antibody against nucleolin along with Hoechst staining. Wide and close-up views are shown as in (<b>B</b>). The data shown are from a single representative experiment out of three repeats. Sections from three different embryos of each genotype were analyzed. Maximum intensity projections are shown. Bars, 10 µm. The numbers of nucleoli per nucleus in the VZ were measured and plotted. Data were obtained analyzed as in (<b>B</b>).</p

Functional diversity of condensins I and II in evolution and during brain development.

<p>(<b>A</b>) Requirements of condensins I and II for mitotic cell divisions differ among different model organisms. (<b>B</b>) Summary of defective phenotypes observed in NSCs depleted of condensin I, condensin II or both. See the text for details.</p

Condensins I and II are both required for proper development of the cerebral cortex.

<p>(<b>A</b>) Genotypes of mice used in the current study. The heterozygotes of <i>Ncaph</i> or <i>Ncaph2</i> mutants in the <i>NesCre</i> background were used as a control (Ctrl). (<b>B</b>) Frozen sections of embryonic brains from cKO mice at E13.5 or E16.5 were immunolabeled with cell-type specific markers: SOX2 for NSCs; TBR1 for deep-layer neurons; BRN2 for upper-layer neurons. DNA was counterstained with Hoechst in the upper and middle panels. The data shown are from a single representative experiment out of three repeats. Sections from at least two different embryos of each genotype were analyzed. Bar, 100 µm. (<b>C</b>) The numbers of SOX2-positive cells were measured in the dorsal area of cortices in control and cKO mice. Data were obtained from four independent sections from two different embryos and normalized to the mean number of NSCs in control mice as 100%. Bars indicate the mean and SD. * <i>P</i><0.05, *** <i>P</i><0.001 (<i>t</i>-test with a Holm correction for multiple comparisons). (<b>D</b>) The numbers of cells present in the CP were measured. Data were obtained and analyzed as in (<b>C</b>). (<b>E</b>) The ratios of TBR1-positive, BRN2-positive, and double-positive (DP) cells were measured in the CP at E16.5. Data were obtained from two different embryos (at least 600 neurons scored). *** <i>P</i><0.001 (Chi-squared test).</p

Condensins I and II differentially contribute to NSC divisions and survival.

<p>(<b>A</b>) Frozen sections of embryonic brains at E16.5 were immunolabeled with antibodies against SOX2 and Act-Casp3 (upper panels), antibodies against SOX2 and p53 (middle panels) or antibodies against SOX2 and 53BP1 (lower panels). The insets show close-ups of 53BP1-positive cells observed in cKO mice. The data shown are from a single representative experiment out of three repeats. Sections from three different embryos of each genotype were analyzed. Bars, 20 µm. The percentages of positive cells in the VZ were measured in the dorsal area of cortices at E16.5, and plotted in the right. Data were obtained from four independent sections from two different embryos. The bars indicate the mean and SD. *** <i>P</i><0.001 (<i>t</i>-test with a Holm correction for multiple comparisons). (<b>B</b>) One hour after injecting mice at E16.5 with BrdU, frozen sections were prepared and immunolabeled with antibodies against BrdU and PAX6, an NSC marker (upper panels). Alternatively, NSCs in mitosis were visualized by H3S10ph labeling (lower panels). The data shown are from a single representative experiment out of three repeats. Sections from two different embryos of each genotype were used for BrdU incorporation assay and from three different embryos of each genotype were labeled with H3S10ph antibody. Bar, 20 µm. The percentages of BrdU-positive or mitotic cells in the PAX6-positive population were measured and plotted in the right. All data were obtained from three independent sections. The bars indicate the mean and SD. ** <i>P</i><0.01, *** <i>P</i><0.001 (<i>t</i>-test with a Holm correction for multiple comparisons). (<b>C</b>) Prophase and prometaphase chromosomes on the apical surface of the VZ were immunolabeled with H3S10ph antibody, and their morphologies were analyzed by Hoechst staining. A large Hoechst-dense structure observed in <i>Ncaph2</i> cKO mice at prophase is shown by the arrow. Chromatin bridges in anaphase or postmitotic cells were observed only in <i>Ncaph2</i> cKO mice (postmetaphase panels). Tail-like chromatin structures in postmitotic cells were immunolabeled with an antibody against 53BP1. The data shown are from a single representative experiment out of three repeats. Sections from three different embryos of each genotype were analyzed. Maximum intensity projections are shown. Bars, 5 µm. (<b>D</b>) Mitotic stages were determined based on chromosome morphologies, and their distributions were plotted. Data were obtained from two different embryos (at least 98 mitotic chromosomes scored). *** <i>P</i><0.001 (Chi-squared test).</p

Defective chromosome segregation leads to DNA damage-induced apoptosis in NSCs depleted of both condensins.

<p>(<b>A</b>) Frozen sections of embryonic brains at E13.5 were immunolabeled with an antibody against an active form of caspase 3 (Act-Casp3), an indicator of apoptotic cell death, and counterstained with Hoechst. The data shown are from a single representative experiment out of three repeats. Sections from at least two different embryos of each genotype were analyzed. Bar, 50 µm. (<b>B</b>) Frozen sections were immunolabeled with antibodies against lamin B and p53. The insets show close-ups of p53-positive nuclei. The data shown are from a single representative experiment out of three repeats. Sections from at least two different embryos of each genotype were analyzed. Bar, 50 µm. (<b>C</b>) Real-time RT-PCR analysis was performed to determine mRNA levels of two p53 target genes (<i>Bax</i> and <i>Noxa</i>) in the cortices of cKO mice at E13.5. The mRNA levels were normalized to the internal control (<i>Gapdh</i> mRNA level). Data were obtained from four experiments and show the mean mRNA levels of Ctrl mice as 100%. Bars indicate the mean and SD. ** <i>P</i><0.01, *** <i>P</i><0.001 (<i>t</i>-test with a Holm correction for multiple comparisons). (<b>D</b>) Frozen sections were immunolabeled with a 53BP1 antibody to visualized damaged DNA foci in interphase nuclei. The insets show close-ups of 53BP1-positive nuclei. The panels shown are from a single representative experiment out of three repeats. Sections from at least two different embryos of each genotype were analyzed. Bar, 20 µm. The percentages of 53BP1-positive nuclei in the VZ were measured in the dorsal area of cortices, and plotted in the bottom. Data were obtained from four independent sections from two different embryos. The bars indicate the mean and SD. * <i>P</i><0.05, ** <i>P</i><0.01 (<i>t</i>-test with a Holm correction for multiple comparisons). (<b>E</b>) Shown are close-up images of cells with tail-like structures from the same experiments as (<b>D</b>). Bar, 10 µm. (<b>F</b>) Mitotic cells were identified as H3S10ph-positive cells on the apical surface of the VZ (middle panels), and their chromosome morphology was visualized with Hoechst stain (upper panels). Also shown are chromatin bridges in anaphase or postmitotic cells (lower panels) in the same region. The data shown are from a single representative experiment out of three repeats. Sections from at least two different embryos of each genotype were analyzed. Maximum intensity projections are shown. Bars, 5 µm.</p

Mathematica file for the project of "Elasticity control of entangled chromosomes: crosstalk between condensin complexes and nucleosomes "

Mathematica file used to do numerical calculations in "Elasticity control of entangled chromosomes:  crosstalk between condensin complexes and nucleosomes" </p

Infrared Spectroscopic Study on Hydration and Chiral Interaction of Temperature-Responsive Polymer with l‑Proline Moieties

We studied the hydration of a temperature-responsive polymer containing l-proline moieties (poly­(acryloyl-l-proline methyl ester), PAProM) by using infrared spectroscopy. Red shifts of ν­(C–H) bands and blue shifts of amide and ester carbonyl bands of PAProM during temperature-induced phase separation indicate that the alkyl, amide, and ester groups are partially dehydrated. The population of the amide carbonyls forming hydrogen bonds (H-bonds) with two water molecules decreased from 63 to 33%, while that of the ester carbonyls forming one H bonding decreased from 100 to 84%. We labeled the methyl groups of PAProM by introducing deuterium (poly­(acryloyl-l-proline methyl-<i>d</i>₃ ester, PAProMd₃) to clarify hydration change of the labeled groups. Red shifts of three ν­(C–D) bands appearing at 2000–2200 cm^–1 clearly showed that the methyl groups at the end of side chains also dehydrated as well as the alkyl groups on the main chain. As for the effects of additives, methanol raised the phase separation temperature (<i>T</i>_p) of PAProM. The IR spectra show that the average number of H bonds to the amide and ester carbonyls decreases with increasing methanol concentration and that the water molecules surrounding the alkyl groups of PAProM are replaced by methanol molecules. The increase in <i>T</i>_p suggests that the favorable effect of the latter is superior to the unfavorable effect of the former. On the other hand, malic acid (MA) reduced <i>T</i>_p of PAProM. Moreover, a chiral interaction occurs; that is, <i>T</i>_p was lower in the presence of d-isomer than l-isomer. The analysis of the amide band revealed that the d-isomer associates more effectively with the amide carbonyls of PAProM than the l-isomer does