Chromosomes Progress to Metaphase in Multiple Discrete Steps via Global Compaction/Expansion Cycles

Abstract

Mammalian mitotic chromosome morphogenesis was analyzed by 4D live-cell and snapshot deconvolution fluorescence imaging. Prophase chromosomes, whose organization was previously unknown, are revealed to comprise co-oriented sister linear loop arrays displayed along a single, peripheral, regularly kinked topoisomerase II/cohesin/condensin II axis. Thereafter, rather than smooth, progressive compaction as generally envisioned, progression to metaphase is a discontinuous process involving chromosome expansion as well as compaction. At late prophase, dependent on topoisomerase II and with concomitant cohesin release, chromosomes expand, axes split and straighten, and chromatin loops transit to a radial disposition around now-central axes. Finally, chromosomes globally compact, giving the metaphase state. These patterns are consistent with the hypothesis that the molecular events of chromosome morphogenesis are governed by accumulation and release of chromosome stress, created by chromatin compaction and expansion. Chromosome state could evolve analogously throughout the cell cycle.

Figure 1. Stages of Mitotic Chromosome Morphogenesis in Living Muntjac and HeLa Cells

Living whole cells were imaged in 3D by epi-fluorescence microscopy from Early Prophase to Metaphase. Stages illustrated by single plane images (1x deconvolution). (A) Snapshots from living muntjac cells stained with Hoechst 33342. Yellow arrowheads indicate chromosome bends specific to Mid-prophase. (B) Selected images from time lapse analysis of a single HeLa H2B-EGFP cell. Colors as in (A). (C) Second Muntjac Mid-prophase nucleus as in (A). Dashed line indicates nuclear envelope. NEB = Nuclear envelope breakdown. Bars: (A) top row, (C) = 5 μm; (A) bottom row, (B) = 2 μm. See also Figure S1.

Figure 2. Chromosome Compaction/Expansion Cycles and Changes in Chromosome Width and Length

(A–F) Chromosome compaction/expansion. (A) 3D single cell time lapse imaging of a HeLa histone H2B-GFP cell. Successive stages illustrated by maximum intensity projections of 1.5 μm z-slices. (B, C) Distributions of pixel intensities at successive stages for: (B) entire 3D stacks plus corresponding maximally abundant pixel intensities (inset); and (C) 1.5 μm z-slices shown in (A). From Mid-prophase (red), chromatin expands during Late Prophase (progressive decrease in intensities; dark to light blue); chromatin compacts during Prometaphase (green) and Metaphase (purple) (progressive increase in intensities). (D) 3D rendering of a living muntjac Mid-prophase nucleus (Hoechst 33342). (E–F) Chromosome volumes of whole nuclei defined by Volocity software as number of voxels above an appropriate intensity threshold for: (E) the HeLa histone H2B-GFP nuclei in (A–C); and (F) living Hoechst 33342-stained muntjac nuclei at the indicated stages (colored as in (A). In both (E) and (F), volumes increase from Mid-prophase (red) to Late Prophase (light to dark blue) and then decrease from Prometaphase through Metaphase (green and purple). (G–I) Chromosome widths. (G): Widths defined from traces across chromosomes perpendicular to their lengths in single-plane images (turquoise bar). (H) Widths in the HeLa histone H2B-GFP nuclei of Panels (A–C, E). (I) Widths for muntjac chromosomes from nuclei analyzed in (F) (stages colored as in (A)). In both (H) and (I), chromosome width increases progressively from Mid-prophase to Prometaphase and beyond into Metaphase (also Figure 5F below). (J) Chromosome lengths were analyzed in 3D z-stacks by projecting the top and bottom halves of each nucleus onto a single xy plane, tracing of chromosome contours and summing the lengths in the two halves. (K) Contour lengths traced in spread muntjac chromosomes. (L) Lengths determined as in (J) for 3D stacks of the HeLa histone H2B-GFP nuclei in Panels A–C, E, H. (M) Lengths for the muntjac nuclei analyzed in 3D in (F) and (I) (left) and in spread preparations as in (K) (right). Bars = 5 μm (A, D, J, K); 2 μm (G). See also Figure S2

Figure 3. Mid-prophase Chromosomes Exhibit a Single TopoIIα Axis

(A) 3D epi-fluorescence images of fixed cells stained with DAPI and anti-TopoIIα. Single plane images from snapshots at the indicated stages (1x deconvolution). Top and middle rows: TopoIIα. Bottom row: TopoIIα overlaid on DAPI. TopoIIα signal is diffuse/disorganized at Early Prophase (left), forms a single narrow linear signal along Mid-prophase chromosomes (second from left) and splits region-by-region at Late Prophase (turquoise arrows). Prometaphase and Metaphase chromosomes (right) exhibit two parallel TopoIIα signals corresponding to sister chromatids. Dashed line indicates nuclear envelope. NEB = Nuclear envelope breakdown. Bars: top row = 5μm; middle and bottom rows = 2 μm. (B) 3D time lapse imaging of a pig LLC-Pk EGFP-TopoIIα cell reveals the same progression of TopoIIα signals as for muntjac in (A) (single plane images shown). Bar = 1μm.

Figure 4. A Regular Array of Loops Along a Single Kinked Peripherally-localized Axis Containing TopoIIα, Cohesin and Condensin, at Mid-prophase

(A) Two muntjac Mid-prophase chromosome segments stained with DAPI and anti-TopoIIα (top) are reconstructed as 3D images in PyMOL (bottom). A kinked TopoIIα axis (pink and green) lies peripherally to, and paranemically along one face of, the chromatin. Segments (1) and (2) illustrate more-kinky and less-kinky paths. The TopoIIα signal (≥ 95% of maximum pixel intensity) is shown alone; accompanied by an envelope (grey net) representing nearly total chromatin signal (≥ 60% of maximum pixel intensity); and with the envelope plus the highest density chromatin signal (≥ 95% of maximum pixel intensity). Turquoise arrows indicate a major bend observed in DNA and TopoIIα paths. Bars = 1 μm. (B) “Tube end views” of muntjac, HeLa and pig chromosome segments (arrows; Extended Experimental Procedures). For each cross-section, left and right columns show, respectively: position of the TopoIIα center (maximum intensity pixel) relative to a heat map of DNA or DNA contours at 95%, 80% and 50–60% intensity of the brightest pixel. The TopoIIα signal wanders from one side of the DNA signal to the other (see also (A)). Examples 1 and 2: muntjac segments as in (A). Examples 3 and 4: segments from pig and HeLa cells (DNA stained with Hoechst and DAPI respectively). Bars = 1 μm. (C) Immuno-staining of muntjac chromosomes for cohesin Rad21 reveals a peripheral axial signal analogous to that seen for TopoIIα (above). Bars = 5 μm (left) and 2 μm (middle). Right: “tube end views” as in (B). (D) Spread muntjac chromosomes double-stained for TopoIIα and Rad21. At Mid-prophase, Rad21 localizes along the TopoIIα axis, with differential abundance of the two molecules at different positions (top). At Late Prophase, Rad21 is lost while TopoIIα remains (bottom). Bar = 2 μm. (E) HeLa cell double-stained for TopoIIα and condensin II’s hCAP-H2. Condensin II localizes along the TopoII axis, with different abundancies of the two components at different positions. Bar = 2 μm. (F) A suitably spread muntjac Mid-prophase nucleus shows even arrays of chromatin (presumptively loops) along continuous TopoIIα axes. Arrow indicates region of duality potentially reflecting split sister units (enlarged portion at right). Bar = 5 μm. (G) Cartoon of meiotic versus mitotic Mid-prophase chromosomes. Chromatin loops (red, green) are in a topologically linear array along a single axis (black) which is straight in meiosis (top) or contorted/kinked in mitosis (bottom). See also Figures S3 and S4

Figure 5. Sister individualization and Radial Chromatin Disposition Emerge at Late Prophase

(A–F). Sister-chromatid chromatin individualizes during Late Prophase. (A) Basis for differential BrdU labeling of sister chromatids. (B) 3D imaging of BrdU-labeled muntjac chromosomes. (Top): Single plane images representative of successive stages. Red asterisk (right) marks a sister chromatid exchange. (Bottom): Cross-sections at the positions indicated by arrows at Top. (Left to Right): Symmetrical single unit at Mid-prophase transits to distinct side-by-side sister units at Metaphase. Bars = 1 μm. (C) Top: DAPI intensity profiles along the cross-sections indicated in B-Top. Bottom: DAPI intensity profiles (black dots) were fit to a pair of Gaussians corresponding to sister DNA units (red and blue; summed in turquoise). Curves are normalized such that the maximum peak height = 1; peak widths not normalized. (D) Two-Gaussian best-fit analysis ((C) bottom) yields two parameters that define sister relationships (text). (i) Δ Peak Height indicates sister individualization. Peak heights for each trace are normalized to a value of one for the highest of the two peaks (“Normalized Intensity” = “N.I.”), with Δ peak height expressed as a fraction of that value. (ii) Inter-Peak Distance defines sister chromatid separation. Chromosome widths defined from traces across chromosomes as in Figures 2G–I. (E) Mid-prophase chromosomes are fit by a single Gaussian, represented as Δ Peak Height = zero. Δ Peak Height increases during Late Prophase and remains essentially constant thereafter, indicating that sister individualization occurs during Late Prophase (colors in (F)). (F) Inter-sister (peak) distance is zero at Mid-prophase (circle) then increases progressively, in concert with increased chromosome width, from Late Prophase through Metaphase. Increased width at Late Prophase reflects sister individualization (E) and thereafter reflects increased center-to-center distance due to increased width of individualized sister units. Deviation for very widest chromosomes reflects a Metaphase/Anaphase transition stage. (G–L) Sister chromatin, intermingled and peripheral to a single TopoIIα axis at Mid-prophase, becomes radially localized around split TopoIIα axes concomitant with individualization during Late Prophase. (G) 2D single-plane images of muntjac chromosomes stained with TopoIIα (top) and merged (red) with DAPI signals (green)(bottom). Bar = 2 μm. (H) 3D cross-sections of the chromosomes at positions indicated by green arrows in (G-top). (I) Intensity profiles for traces across chromosomes in single planes along green arrows in (G-top) for TopoIIα (green) and DAPI (black). The highest TopoIIα peak and the maximum DAPI intensity were normalized to 1. DAPI profiles were fit by a pair of Gaussians (red and blue) corresponding to two sister units as in (D) and Figure S6. (J) “Tube end views” along two Late Prophase chromosome segments (staining as in (G)). Top: early axis splitting. Split, peripheral axis signals flank an unsplit signal with rotation of the split signal. Bottom: segment with complete splitting and sister axis signals located internally to the chromatin. (K) Relative positions of TopoIIα axis peak(s) (Panel I, green) and the peak(s) of Gaussian(s) representing chromosome unit(s) (Panel I, red and blue) at indicated stages. Each Mid-prophase case comprises a single peak of each type (DAPI and TopoIIα). At later stages, for each case, the distance between the two Gaussian peaks was subtracted from the distance between the two TopoIIα peaks and the difference divided by two. Values above zero imply localization of the TopoIIα signals outside of the two Gaussian signals (a peripheral axis/chromosome relationship) while values around zero correspond to overlap of the two types of signals (central axes and radially-distributed chromatin). At late Metaphase (largest widths), below-zero values represent a real tendency for the TopoIIα signals to be closer together than the centers of their sister units at the Metaphase/Anaphase transition stage seen also in (F). (L) 3D PyMOL reconstructions of representative segments from muntjac chromosomes at indicated stages. During Late Prophase: (i) axis split initially as bubbles; (ii) longer split regions are peripheral [arrow indicates junction between split and unsplit regions]; and finally (iii) split regions are located centrally within the chromatin. Thereafter, sister axes further separate as chromosomes widen. Bottom row: top and side views show that, at the end of Late Prophase through metaphase, sisters lie side-by-side with axes centrally located within each sister chromatid chromatin. TopoIIα signal is ≥ 95% of maximum pixel intensity; DAPI signal is ≥60% of maximum pixel intensity. Bar = 1 μm. See also Figures S5 and S6.

Figure 6. Topoisomerase II Activity is Required for Progression from Mid- to Late Prophase

(A) 3D time-lapse imaging of a HeLa H2B-GFP cell at Mid-prophase identified within a minute after ICRF-193 addition, illustrated by maximum projections of 1.5 μm z-slices (compare with Figure 2A for untreated cells). Bar = 5 μm. (B–D) Distribution of pixel intensities for the nucleus in (A). (B) For each entire 3D stack. (C) Maximum intensities from distributions in (B). (D) For the thick slices in (A). Compare results in (B–D) with results for untreated cells in Figures 2B, C. (E) Enlarged portions of single plane images from untreated cells (left) and treated cells (right) illustrate absence of morphological progression in treated cells. Bar = 2 μm. (F) 3D time-lapse imaging of Pig EGFP-TopoIIα cells in the absence (top) or presence (bottom) of ICRF-193 added at Mid-prophase as in (A). Bar = 5 μm (G) Enlarged images as in (E). Left: without ICRF-193, TopoII signals are single at Mid-prophase and split during Late Prophase (left; double arrowheads at t=15 min indicate split region). Right: in contrast, in the presence of ICRF-193, TopoIIα signals remain unchanged from Mid-prophase to a time corresponding to Late Prophase in untreated cells (right). Bar = 2 μm. See also Figure S7.

Figure 7. Chromosome Morphogenesis and Expansion/Compaction Stress Cycling

(A) Chromosomes progress from Prophase to Metaphase via three discrete intermediates. Left: Mid-prophase chromosomes comprise cooriented sister linear loop arrays (red and blue) organized along a single peripheral TopoIIα/cohesin/condensin-II axis (yellow). Middle: At Late Prophase, sister axes split and the loops of each sister become radially disposed around its respective axis, which has lost cohesin (*). This progression requires release of cohesin and of TopoII-senstive linkages, presumptively catenations. Right: At Metaphase, chromosomes retain the organization established at Late Prophase but have become shorter and fatter and have acquired new tethers (*). (B) During the stages in (A), chromosomes alternate between compact and expanded states, interpreted as comprising higher and lower potential energy (more and less stressed) states (pink and green, respectively). (C) Release of chromosome expansion stress will involve release of different types of constraining linkages, potentially in an autocatalytic process where some release tether(s) increases stress on remaining tethers, promoting their release (etc.).