Dynamics of Nuclear Envelope Assembly Induced by Hypotonic Shock
As was shown previously, hypotonic treatment of living mitotic cell results in formation of numerous micronuclei formed by individual chromosomes (Zatsepina et al., 1982). These micronuclei reveal some peculiarities in nuclear envelope structure: chromatin-free zones, reduced nuclear pore density etc (Vladimirskaya et al., 1998).
In present work we have analyzed the distribution of some nuclear envelope proteins at different phases of micronucleation. In control metaphase cells chromosomes do not bind with nuclear envelope proteins, but hypotonic treatment induces such interaction. We observed two types of metaphase cells with chromosomes labeled or unlabeled with antibodies to lamins A and B and LAP2B. These proteins display punctate distribution on the chromosome surface. Amount of metaphase cells with chromosomes, positively stained by antibodies to lamin A corresponds to the number of micronucleated cells. In contrast, LAP2A homogeneously distributes on the surface in all metaphase cells. Extraction of living cells with 0,5% Triton X-100 in hypotonic medium removes major part of these proteins from chromosomes and LAP2A remains only as discrete spots on chromosome surface. Upon reversal of cells to normotonic medium two types of staining with antibodies to lamins and LAP2B also are observed, while LAP2A still displays chromosomal localization in all metaphase cells. All described proteins display punctate distribution on the chromosome surface. It could be supposed that the character of interaction of nuclear envelope precursor proteins with chromosomes in this experimental system is determined by proteins other than LAP2A, despite its early recrutiment to chromosome surface. The study of dynamics of assembly of lamins A, B and LAP2B during micronucleation demonstrates that in contract to soluble lamins, lamin B and LAP2B display delayed formation of even layer at the surface of micronuclei. LAP2A also localizes at the nuclear envelope of micronuclei and only after 6h of isotonic treatment acquires interphase type of staining.
Kurchashova S. Yu., Gulak P.V., Kireyev I.I., *Hozak P., Poliakov V. Yu
A.N. Belozersky institute of Physico-Chemical Biology,