Condensin II drives large-scale folding and spatial partitioning of interphase chromosomes in <i>Drosophila</i> nuclei

<div><p>Metazoan chromosomes are folded into discrete sub-nuclear domains, referred to as chromosome territories (CTs). The molecular mechanisms that underlie the formation and maintenance of CTs during the cell cycle remain largely unknown. Here, we have developed high-resolution chromosome paints to investigate CT organization in <i>Drosophila</i> cycling cells. We show that large-scale chromosome folding patterns and levels of chromosome intermixing are remarkably stable across various cell types. Our data also suggest that the nucleus scales to accommodate fluctuations in chromosome size throughout the cell cycle, which limits the degree of intermixing between neighboring CTs. Finally, we show that the cohesin and condensin complexes are required for different scales of chromosome folding, with condensin II being especially important for the size, shape, and level of intermixing between CTs in interphase. These findings suggest that large-scale chromosome folding driven by condensin II influences the extent to which chromosomes interact, which may have direct consequences for cell-type specific genome stability.</p></div

Direct Observation of a Bent Carbonyl Ligand in a 19-Electron Transition Metal Complex

The photochemistry of [CpRu­(CO)₂]₂ in P­(OMe)₃/CH₂Cl₂ solution has been studied using picosecond time-resolved infrared (TRIR) spectroscopy. Photolysis at 400 nm leads to the formation of 17-electron CpRu­(CO)₂^• radicals, which react on the picosecond time scale to form 19-electron CpRu­(CO)₂P­(OMe)₃^• adducts. The TRIR spectra of this adduct display an unusually low CO stretching frequency for the antisymmetric CO stretching mode, suggesting that one carbonyl ligand adopts a bent configuration to avoid a 19-electron count at the metal center. This spectral assignment is supported by analogous experiments on [CpFe­(CO)₂]₂ in the same solvent, combined with DFT studies on the structures of the 19-electron adducts. The DFT results predict a bent CO ligand in CpRu­(CO)₂P­(OMe)₃^•, whereas approximately linear Fe–C–O bond angles are predicted for CpFe­(CO)₂P­(OMe)₃^•. The observation of a bent CO ligand in the 19-electron ruthenium adduct is a surprising result, and it provides new insight into the solution-phase behavior of 19-electron complexes. TRIR spectra were also collected for [CpRu­(CO)₂]₂ in neat CH₂Cl₂, and it is interesting to note that no singly bridged [CpRu­(CO)]₂(μ-CO) photoproduct was observed to form following 400- or 267-nm excitation, despite previous observations of this species on longer time scales

Condensin II is sufficient to drive whole-chromosome separation.

<p>(A) Left: Oligopaints labeling chromosomes X (green), 2L (red), and 2R (cyan) on representative Kc167 cell nuclei depleted of Brown (control) or the condensin II regulator SLMB, or stably expressing a copper sulfate-inducible Cap-H2-GFP construct (OX). Dashed lines represent the nuclear edge. Scale bar equals 5 μm. n>500 cells per condition. Right: 3D renderings of segmented structures. (B) Dot plot showing nuclear volume normalized to control. Each dot represents the average of a biological (SLMB RNAi) or technical (Cap-H2 OX) replicate with n>500 cells. *p = 0.02; t-test. (C) Dot plot showing 2L Oligopaint volume normalized to control. Each dot represents the average of a biological (SLMB RNAi) or technical (Cap-H2 OX) replicate with n>500 cells. *p = 0.04; t-test. (D) Histogram showing the binned distribution of 2L shape from a single biological (SLMB RNAi) or technical (Cap-H2 OX) replicate with n>300 cells. Higher compacity values indicate a more spherical structure. These results were confirmed by at least two additional biological or technical replicates. ***p < 0.0001; Mann-Whitney test. (E) IF/FISH on representative Kc167 cell nuclei depleted of Brown (control) or SLMB, or stably expressing a copper sulfate-inducible Cap-H2-GFP construct (OX). Heterochromatin is labeled with anti-H3K9me2 antibody (green) and heterochromatin FISH probes (Het) labeling AATAT, AATAG, AACAC, 359, and dodeca in red. Dashed lines represent the nuclear edge. DNA (Hoechst stain) is shown in blue. Scale bar equals 5 μm. n>300 cells per condition. (F) Tukey box plot of the number of Het foci shown in (E), showing the mean (black line) and distribution (minus outliers). Data shown are from a single biological (SLMB RNAi) or technical (Cap-H2 OX) replicate (n>300 cells each). These results were confirmed by two additional biological or technical replicates, respectively. ***p < 0.0001; Mann-Whitney test. (G) Heatmap showing median CT overlap for a population of n>500 cells per condition, where overlap is shown as a percentage of the structures listed along the bottom. (H) Histogram showing X-2L CT overlap as a percent of X CT volume. Binned data from a single biological or technical replicate are shown (n>500 cells). These results were confirmed by at least two additional biological or technical replicates. ***p < 0.0001; Mann-Whitney test. (I) Heatmap showing pairwise contact frequencies for a population of n>500 cells per condition. The diagonal boxes are whole-chromosome pairing frequencies.</p

Ultrafast Studies of Stannane Activation by Triplet Organometallic Photoproducts

The activation of Sn–H bonds in tributylstannane by three triplet organometallic photoproducts (Fe­(CO)₄, CpCo­(CO), and CpV­(CO)₃) has been studied using picosecond time-resolved infrared spectroscopy. Consistent with previous studies of triplet reactivity, the results suggest that triplet intermediates coordinate weakly at best with the alkyl groups in the solvent, allowing them to rearrange to form Sn–H bond activated products at, or near, diffusion-limited rates. For CpV­(CO)₃, an alkyl-coordinated singlet is initially formed along with the unsolvated triplet photoproduct, allowing for direct observation of the slower rate of bond activation by the alkyl-coordinated singlet species. Electronic structure theory calculations are used to investigate the potential energy surfaces, as well as to consider whether an external heavy atom effect may be important in mediating the extent of nonadiabatic behavior as the Sn–H bond approaches the metal center. Interestingly, we find no evidence for an external heavy-atom effect in the calculated spin–orbit coupling values, and we offer an explanation for the results of these calculations. To our knowledge, this study represents the first ultrafast investigation into Sn–H bond activation by organometallic catalysts

<i>Drosophila</i> Kc167 cells form robust CTs.

<p>(A) Schematic representation of <i>D</i>. <i>melanogaster</i> karyotype. The unlabeled heterochromatin is depicted in white, while euchromatin is depicted in green (X-chromosome), pink (chromosome 2), and gray (chromosome 3). (B) Representative Kc167 cell nucleus with Oligopaints labeling chromosomes X (green), 2 (pink), and 3 (gray). Total DNA (Hoechst stain) is shown in blue. Scale bar equals 5 μm. (C) Tukey box plot showing structure volume as a fraction of nuclear volume. X-axis denotes the structure being measured. (D) Top: Representative nucleus of IF/FISH in Kc167 cells with Oligopaints labeling chromosomes X, 2, and 3 all in red (all CTs), and anti-H3K9me2 IF in cyan. Bottom: IF to euchromatin (H3K4me3) in red and heterochromatin (H3K9me2) in cyan. Scale bar equals 5 μm. (E) Left: Heatmap of pairwise contact frequencies for a population of n>500 Kc167 cells. The diagonal boxes are whole-chromosome pairing frequencies. Right: Heatmap of median CT overlap for a population of n>500 Kc167 cells. Overlap is shown as a percentage of CT volume. Values in the bottom half of the heatmap are normalized to the structures listed along the bottom, while the top half are normalized to the structures listed on the left. (F) Top: Representative images of Kc167 cells with whole chromosome Oligopaints, showing the varying degrees of CT intermixing found in the population. Scale bar equals 5 μm. Bottom: histogram showing CT overlap as a percent of CT volume. Overlap between chromosomes X and 2, and X and 3, are shown as a percent of chromosome X CT volume. Overlap between chromosomes 2 and 3 is shown as a percent of chromosome 2 CT volume.</p

Insights into the Photochemical Disproportionation of Transition Metal Dimers on the Picosecond Time Scale

The reactivity of five transition metal dimers toward photochemical, in-solvent-cage disproportionation has been investigated using picosecond time-resolved infrared spectroscopy. Previous ultrafast studies on [CpW­(CO)₃]₂ established the role of an in-cage disproportionation mechanism involving electron transfer between 17- and 19-electron radicals prior to diffusion out of the solvent cage. New results from time-resolved infrared studies reveal that the identity of the transition metal complex dictates whether the in-cage disproportionation mechanism can take place, as well as the more fundamental issue of whether 19-electron intermediates are able to form on the picosecond time scale. Significantly, the in-cage disproportionation mechanism observed previously for the tungsten dimer does not characterize the reactivity of four out of the five transition metal dimers in this study. The differences in the ability to form 19-electron intermediates are interpreted either in terms of differences in the 17/19-electron equilibrium or of differences in an energetic barrier to associative coordination of a Lewis base, whereas the case for the in-cage vs diffusive disproportionation mechanisms depends on whether the 19-electron reducing agent is genuinely characterized by 19-electron configuration at the metal center or if it is better described as an 18 + δ complex. These results help to better understand the factors that dictate mechanisms of radical disproportionation and carry implications for radical chain mechanisms

Chromosome arms form independent territories.

<p>(A) Schematic of chromosome arm-specific Oligopaints. (B) Representative Kc167 cell nucleus with Oligopaints labeling chromosomes X (green), 2L (red), 2R (cyan), 3L (yellow), and 3R (magenta). Total DNA (Hoechst stain) is shown in blue. Scale bar equals 5 μm. (C) Tukey box plot showing CT volumes as a fraction of nuclear volume. n>500 cells. (D) Heatmap showing median CT overlap for a population of n>500 Kc167 cells, where overlap is shown as a percentage of CT volume. Values in the bottom half of the heatmap are normalized to the structures listed along the bottom, while the top half are normalized to the structures listed on the left. (E) Heatmap showing pairwise contact frequencies for a population of n>500 Kc167 cells. The diagonal boxes represent whole-chromosome pairing frequencies. (F) Radial position of chromosomes in the nucleus determined by shell analysis with five shells of equal volume, where shell 1 is the closest to the nuclear periphery and shell 5 is the nuclear center. n>500 cells. ***p<0.0001, Mann-Whitney test. (G) Left: plot of DNA content in Kc167 cells after FACS. Right: Representative nuclei after FACS with Oligopaints labeling chromosomes X (green), 2L (red), and 2R (cyan). Dashed lines represent nuclear edge. Scale bar equals 5 μm. (H) Left: Tukey box plot showing nuclear volumes of G1, S, and G2 phased cells after FACS sorting, determined by Hoechst stain. Right: Tukey box plot showing CT volumes after FACS sorting. The data shown represent one technical replicate (n = 220–350 cells per cell cycle phase). These data were confirmed by two additional technical replicates. (I) Top: heatmaps showing median CT overlap for a population of n>200 FACS sorted Kc167 cells, where overlap is shown as a percentage of the structures listed along the bottom. Bottom: heatmaps showing pairwise contact frequencies for a population of n>200 FACS sorted Kc167 cells. (J) Histogram showing X-2L CT overlap as a percent of X CT volume after FACS. Binned data from a single technical replicate are shown (n>200 cells). These results were confirmed by at two additional technical replicates. (K) Scatter plot of nuclear volume (X-axis) versus 2L CT volume or X-2L overlap volume (Y-axis). Chromosome 2L volume data are shown in blue, while X-2L overlap data are shown in gray. n = 835 cells.</p

Ultrafast TRIR and DFT Studies of the Photochemical Dynamics of Co₄(CO)₁₂ in Solution

The photochemical rearrangement dynamics of Co₄(CO)₁₂ were studied using picosecond time-resolved infrared spectroscopy. In cyclohexane and CH₂Cl₂ solvents, monitoring the kinetics of absorptions in the bridging carbonyl region reveals the formation of two transient rearrangement intermediates, both of which revert to the parent complex on the picosecond time scale. Density functional theory calculations are used to identify the structures of the rearrangement products, which arise from cleavage of an apical–basal Co–Co bond. While the lifetimes of both species exhibit a solvent dependence, the experimental kinetics and density functional calculations suggest that these species do not form solvent-coordinated complexes with cyclohexane or CH₂Cl₂, and instead, the solvent effect is believed to arise from differences in polarity, with the more polar CH₂Cl₂ solvent stabilizing the rearrangement intermediates, relative to when cyclohexane is the solvent. Carbonyl dissociation products are also observed and investigated by DFT calculations. No fragmentation products, such as Co­(CO)₄ or Co₂(CO)₈, are observed to form on the picosecond time scale, suggesting that subsequent chemistry of this cluster will occur via the single carbonyl-loss products. The experimental and computational results of this study provide insight into the role and nature of bridging carbonyl intermediates formed upon photoexcitation, as well as the formation of carbonyl-loss products and the role of solvation of transient species. To our knowledge, this study represents the first investigation into the dynamics of an M₄L₁₂ complex on the ultrafast time scale

Reactivity of TEMPO toward 16- and 17-Electron Organometallic Reaction Intermediates: A Time-Resolved IR Study

The (2,2,6,6-tetramethylpiperidin-1-yl)­oxyl radical (TEMPO) has been employed for an extensive range of chemical applications, ranging from organometallic catalysis to serving as a structural probe in biological systems. As a ligand in an organometallic complex, TEMPO can exhibit several distinct coordination modes. Here we use ultrafast time-resolved infrared spectroscopy to study the reactivity of TEMPO toward coordinatively unsaturated 16- and 17-electron organometallic reaction intermediates. TEMPO coordinates to the metal centers of the 16-electron species CpCo­(CO) and Fe­(CO)₄, and to the 17-electron species CpFe­(CO)₂ and Mn­(CO)₅, via an associative mechanism with concomitant oxidation of the metal center. In these adducts, TEMPO thus behaves as an anionic ligand, characterized by a pyramidal geometry about the nitrogen center. Density functional theory calculations are used to facilitate interpretation of the spectra and to further explore the structures of the TEMPO adducts. To our knowledge, this study represents the first direct characterization of the mechanism of the reaction of TEMPO with coordinatively unsaturated organometallic complexes, providing valuable insight into its reactions with commonly encountered reaction intermediates. The similar reactivity of TEMPO toward each of the species studied suggests that these results can be considered representative of TEMPO’s reactivity toward all low-valent transition metal complexes

Heterochromatin clustering is dispensable for CT formation.

<p>(A) IF/FISH on representative Kc167 cell nuclei depleted of Brown (control), CAL1, or HP1a. Heterochromatin is labeled with anti-H3K9me2 antibody (green) and heterochromatin FISH probes (Het) labeling the AATAT, AATAG, AACAC, 359, and dodeca satellites in red. DNA (Hoechst stain) is shown in blue. Dashed lines represent the nuclear edge. Scale bar equals 5 μm. n>500 cells per condition. (B) Tukey box plot of the number of Het foci shown in (A). Data shown are from a single biological replicate (n>500 cells each). These results were confirmed by two additional biological replicates. ***p < 0.0001; Mann-Whitney test. (C) Left: Oligopaints labeling chromosomes X (green), 2L (red), and 2R (cyan) on representative Kc167 cell nuclei depleted of Brown (control), CAL1, or HP1a. Dashed lines represent the nuclear edge. Scale bar equals 5 μm. n>500 cells per condition. Right: 3D renderings of segmented structures. (D) Dot plot showing nuclear volume normalized to control. Each dot represents the average of a biological replicate with n>500 cells. The differences shown are not significant (t-test). (E) Dot plot showing 2L Oligopaint volume normalized to control. Each dot represents the average of a biological replicate with n>500 cells. The differences shown are not significant (t-test). (F) Histogram showing the binned distribution of 2L shape from a single biological replicate with n>500 cells. Higher compacity values indicate a more spherical structure. These results were confirmed by at least two additional biological replicates. The differences shown are not significant (Mann-Whitney test). (G) Heatmap showing median CT overlap for a population of n>500 cells per condition, where overlap is shown as a percentage of the structures listed along the bottom. (H) Histogram showing X-2L CT overlap as a percent of X CT volume. Binned data from a single biological replicate are shown (n>500 cells). These results were confirmed by at least two additional biological replicates. The differences shown are not significant (Mann-Whitney test). (I) Heatmap showing pairwise contact frequencies for a population of n>500 cells per condition. The diagonal boxes are whole-chromosome pairing frequencies.</p