Fig. 9

Reversible decondensation of chromatin in myelopoietic cells triggered by hypotonic conditions. a Live cell observation of granulocytes with Hoechst33342 stained DNA recorded by spinning disc LSM during repeated circles of normotonic (270 mOsm) and hypotonic (90 mOsm) conditions. The compacted chromatin rim surrounding large interior IC lacunae is seen under normotonic conditions (0 min). Within <1 min in hypotonic conditions a nuclear phenotype appears with (decondensed) chromatin expanding into the IC lacunae. This effect is reversible upon restoring normotonic conditions (2 min) and can be repeated over several cycles (7 and 8 min). Scale bar 10 µm. b comparison of representative myelopoietic cell nuclei seen under normotonic (upper panel) and hypotonic conditions (lower panel) in osmium ammine B stained TEM sections. Inset magnifications of hypotonic TEM sections demonstrate a similar, rather even distribution of (decondensed) chromatin throughout the nucleus in all cell types, with loss of larger IC channels and IC lacunae, in particular evident in monocytes and granulocytes. Scale bars 2 µm; insets 0.5 µm. c left panel: Different staining intensities after simultaneous DNA staining of granulocytes fixed after 30 s incubation in hypotonic conditions with DAPI (red) and 7-AAD (green). 7-AAD (high affinity to GC-rich regions) denotes the lobe interior while DAPI (high affinity to AT-rich regions) strongly stains the peripheral rim. This radial divergence of the two dyes illustrates a preferential expansion of GC enriched (gene dense) DNA segments toward the nuclear interior. z-projections of 400 nm axial distance are shown. Right panel 3D distance measurements of DAPI (red) and 7-AAD (green) signals to the nuclear border of granulocyte lobes confirm their significantly distinct radial distribution (p < 0.001, assessed by Mann–Whitney rank sum test). The ordinate denotes the normalized sum of voxel intensities for a respective fluorochrome, the abscissa the relative distance to the nuclear border. n number of analyzed nuclei; error bars standard error of means