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First published online 12 April 2007
doi: 10.1242/jcs.03440

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Experimental evidence for the influence of molecular crowding on nuclear architecture

Division of Molecular Genetics, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany

View larger version (101K): [in this window] [in a new window]   Fig. 1. Hypertonic incubation causes compaction of chromatin in living cells: MCF7 cells were incubated for 20 minutes with growth medium enriched with varying concentrations sucrose as indicated. The cells were fixed and stained with ToPro3 to observe the chromatin distribution by confocal laser scanning microscopy. The confocal sections demonstrate the progress in chromatin compaction with increasing load of sucrose. Neither chromosome bands nor individual chromosomes became discernible. Bar, 10 µm.

View larger version (53K): [in this window] [in a new window]   Fig. 2. Cells adapt to hypertonic medium after long-term incubation. Cultures of MCF7 and HeLa cells adapted within 4 days to 160 mM sucrose in the medium and were cultivated for a further 3 weeks at this load. Compared with compaction at 20 minutes (right; for MCF7 see Fig. 1), chromatin no longer appeared compacted in cells grown long-term in high sucrose medium. Bar, 10 µm.

View larger version (114K): [in this window] [in a new window]   Fig. 3. At high hypertonic load, the nuclear periphery re-organizes: (A) Co-staining of chromatin (red, ToPro3 in MCF7 and H2B-GFP in HeLa) and anti-LaminAC (green) in MCF7 (top row) and HeLa cells (bottom row). The lamin-rim stain allows us to demonstrate retraction of peripheral chromatin from the nuclear envelope, which occurred in addition to the compaction of chromatin upon incubation with 320 mM sucrose load, but not with 160 mM sucrose. (B) Co-staining of chromatin (red, ToPro3) with anti-SC35 (green) in MCF7 cells. Together with the retraction of chromatin in 320 mM sucrose medium, SC35-positive foci established in the outer border of peripheral chromatin. These peripheral speckles were smaller in size than those of the inner nucleus (top, left). In control cells (top, right) and cells after incubation in 160 mM sucrose (top, middle), peripheral speckles were never observed. Incubation with sorbitol (bottom, left) or NaCl (bottom, middle) to achieve osmotic loads equivalent to 320 mM sucrose (see Table 1) also induced chromatin compaction and formation of peripheral speckles. By contrast, 10 kDa dextran added in a weight/concentration comparable to 320 mM sucrose had no noticeable effect (bottom, right). Substances were added to the normal growth medium (DMEM) as indicated and cells incubated for 20 minutes prior to fixation. Bars, 10 µm.

View larger version (111K): [in this window] [in a new window]   Fig. 4. In contrast to osmotropes, `crowders' promote chromatin compaction, if they gain access to the cell. The chromatin distribution (ToPro3) in MCF7 nuclei was challenged by incubation with NaCl and/or dextran for 15 minutes in combination with digitonin permeabilization of the cells. The incubation sequence for each example is indicated. Digitonin treatment abolished chromatin compaction by NaCl (top row). In contrast to cells with intact membranes (see Fig. 3B), dextran induced chromatin compaction when cells were permeabilized (bottom row). Dextran still asserted this effect in cells where previous chromatin compaction upon hypertonic incubation with NaCl had been abolished because of digitonin permeabilization (right). Bar, 10 µm.

View larger version (62K): [in this window] [in a new window]   Fig. 5. Sucrose-induced chromatin compaction proceeds gradually with incubation time. Chromatin compaction during incubation with 320 mM sucrose medium was studied by live observation of GFP-tagged histone H2B in a HeLa cell nucleus. (A) Chromatin compaction became visible within seconds of hypertonic treatment, proceeded substantially over the first minute and slowly approached its steady state (pre, H2B-GFP signal of cell in DMEM before start of sucrose treatment; other labels show duration of sucrose incubation in seconds). (B) Upon re-incubation in normal growth medium, the chromatin distribution recovered to close to its initial state. In particular, the original dense domains appeared to be preserved (arrowheads). (C) Density analysis of the two image series in A and B (colors code for time points as indicated, images were corrected for gain and offset, and normalized to equal summed intensity to account for constant total amount of histones). Chromatin compaction in 320 mM sucrose is shown in the graph on the left and recovery in normal growth medium on the right. The density distributions are monophasic and no distinct intermediate or terminal states of chromatin compaction are revealed. Higher compaction states correlate with a larger width of distribution. Bar, 10 µm.

View larger version (225K): [in this window] [in a new window]   Fig. 6. Hypertonic shock causes a complex pattern of segregation of nuclear compounds. (A) Upon in-vivo incubation of MCF7 cells with 320 mM sucrose medium and subsequent preparation for electron microscopy, ultrathin sections show chromatin (ch) as distinct portions of homogeneous, dense granularity. Lacunas of fine fibrillar material (fm) comprise a major fraction of the interchromatin space. They frequently include body-like structures of very low contrast and spherical shape (sb). Between chromatin and the fine-fibrillar lacunas, granular and fibrillar parts were arranged in various ultrastructural units, among them interchromatin granule clusters (ig), and dense bodies (db). At the nuclear periphery, the peripheral chromatin retracted by several hundred nanometers giving rise to the peripheral layer (double-headed arrow), which predominantly comprises fibrillar material like that of the interchromatin space. Its interface with the retracted chromatin is defined by several structures, among them small clusters of interchromatin granules (ig). The nucleolus (no) is of compact shape and homogeneous granular composition. Association with perinucleolar chromatin appears reduced. (B) Treatment with 160 mM sucrose medium caused intermediate states of nuclear re-organization. Although chromatin (ch) compacted to portions of similar texture, and dense bodies (db) arose as with 320 mM sucrose load, fine-fibrillar material (fm) did not segregate and separate from other nucleoplasmic compounds as sharply, e.g. interchromatin granule clusters (ig). Furthermore, peripheral and perinucleolar chromatin still associated with the nuclear envelope and the nucleolus (no), respectively. (C) Incubation of cells with 10% dextran of 41 kDa size had no influence on nuclear ultrastructure. Like control preparations, chromatin (ch) remained dispersed and did not cluster into distinct portions. Fine-fibrillar material did not segregate from interchromatin granule clusters (ig) and chromatin. The nucleolus (no) is of normal, polymorphous appearance, differentially organized into fibrillar and granular parts, and associated with perinucleolar chromatin. Bars, 1 µm.

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