MANIPULACION Y ALMACENAMIENTO DE SUSTANCIAS QUIMICAS

Little is known about the mechanism of HCC formation. The increased osmolarity led to a loss of water from the cells and a concomitant decrease of the nuclear volume (Fig. 9). These changes are necessarily accompanied by a concomitant increase in the concentration of other cellular components. Two potential candidates, which may be able to trigger chromatin hypercondensation in these conditions, are cations and macromolecules.

7.3.1 Cations

An increase in the concentration of divalent cations may play an essential role since it could result in a neutralization of the negatively charged DNA backbone (Hansen, 2002; Horn and Peterson, 2002). In-vitro studies demonstrated that divalent cations (Mg2+_{, Ca}2+_{) applied on} stretched chromatin fibers lead to their condensation (Garcia-Ramirez et al., 1992; Hansen et al., 1989; Marko and Poirier, 2003; Poirier and Marko, 2003; Schwarz and Hansen, 1994). Measurements on the ion levels on condensed mitotic chromosomes revealed that only Na+_, K+_{, Mg}2+ _{and Ca}2+ _{were detected on mitotic chromosomes but no other ones. Increased} concentrations of Ca2+ _{were found along the axis of mitotic chromosomes (Strick et al.,} 2001). The authors concluded that their findings implicate that Na+_{, K}+_{, Mg}2+ _{and Ca}2+ _{play a} role in higher order chromosome structure through electrostatic neutralization (Strick et al., 2001). In accordance with this claim is the observation that formation of hypercondensed chromatin in cells with a permeabilized membrane was possible only if the concentration of divalent cations was raised in the surrounding medium (see 6.2.6). Monovalent cations were not sufficient to trigger a condensation effect in the concentrations tested. The pattern of hypercondensed chromatin induced by divalent cations in permeabilized cells was further different from the HCC pattern normally observed in intact cells. These results hint that the chromatin condensation effect observed for the standard hyper-osmotic treatment may differ from the effect obtained by DNA electrostatic neutralization mediated by increased concentrations of cations. Since not only ion concentrations but also the concentration of other nuclear components including proteins and macromolecules were increased in the hyper-tonic conditions, further factors could be considerable mediators of HCC formation.

7.3.2 Macromolecular crowding effects

A biophysical concept, recently adapted on nuclear architecture (Cook, 2002; Hancock, 2004a; Hancock, 2004b; Hancock, 2007) is macromolecular crowding (Zimmerman and Minton, 1993). This concept argues for a changing response of molecular interactions in

regard to the change of crowding conditions. Crowding is thereby defined as the occurrence of macromolecular molecules occupying volume and thereby decreasing the effective volume ingestible for other molecules. A concentration dependent response of molecular interactions to changes in macromolecular crowding is postulated to induce the formation/deformation of nuclear compartments built up by interacting molecules. The initially widespread molecules will form compact compartments in a crowded system (Cook, 2002; Hancock, 2004b), since a higher compaction leads to less excluded volume roamed by the molecules themselves and thereby provides a higher entropy level of the system in total. The application of this idea to the formation of nuclear compartments was first applied in the group of Ronald Hancock, which tested effects of macromolecular crowding on nuclear compartments like PML bodies and nucleoli (Hancock, 2004b). To decrease the crowding force, isolated nuclei were incubated in low ionic (=low osmotic) media, water was taken up by the nuclei and their volume increased. Since this treatment reduces the molecular crowding force (enlarged volume but constant molecular content) they expected and experimentally confirmed that PML bodies and nucleoli disappeared (Hancock, 2004b). When inert macromolecules (Polyethylene glycol), which enter the nuclear membrane were added subsequently, the reformation of PML bodies as well as of nucleoli was observed (Hancock, 2004b). In the light of the CT-IC model, also chromatin polymers could be considered as crowding macromolecules occupying nuclear volume, concomitantly leading to the formation of compartments in the interchromatin space. Furthermore chromatin itself could be affected by a crowded nuclear environment (Hancock, 2007) which may trigger the self-organization of CTs and the formation of densely packed 'heterochromatin', if one assumes molecular interactions between e.g. heterochromatic histone modifications and their binding partners. This argument may also provide a way to explain the observed polarity of chromatin arrangements in the nuclear volume, with compact ‘heterochromatic’ chromatin compartments found preferentially at the periphery or perinucleolar: The reason for this edge-affine topography may be that the effective excluded volume of interacting molecules is further minimized, if compartments were formed at physical borders like the nuclear membrane respectively nucleoli.

On the other hand, a significant decondensation of chromatin, expected under hypo-osmotic conditions (diminished crowding) was not observed on fluorescently labeled ~1 Mb chromatin domains (see 6.2.4). The effect may be too small or not existent at the level of single ~1 Mb chromatin domains. Nevertheless it was striking, that the overall chromatin appeared diffuser under hypo-osmotic conditions, matching the expectations of the nuclear crowding theory (Fig. 14). Following the opposite way by incubating cells in hyper-osmotic media (exactly as performed in this study), should - following the crowding theory - lead to increased crowding forces and concomitantly to a more contrasted compartmentalization in the nucleus. The

observation that chromatin hypercondenses in these conditions matches exactly this expectation. In conclusion, the data of the Hancock group and of the present study match the postulations of the crowding theory and accordingly provide support for a new biophysical approach in investigating and understanding the organization of nuclear architecture. It is worth considering that the organization of nuclear compartments just based on molecular crowding forces works with no demand for an additional structural organizer like the 'nuclear matrix' (Hancock, 2004b, see 7.6.2).